WO2023241577A1 - 阳离子脂质化合物及其制备方法和应用 - Google Patents

阳离子脂质化合物及其制备方法和应用 Download PDF

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WO2023241577A1
WO2023241577A1 PCT/CN2023/099945 CN2023099945W WO2023241577A1 WO 2023241577 A1 WO2023241577 A1 WO 2023241577A1 CN 2023099945 W CN2023099945 W CN 2023099945W WO 2023241577 A1 WO2023241577 A1 WO 2023241577A1
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cationic lipid
drug
lnp
formula
lipid compound
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PCT/CN2023/099945
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French (fr)
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吴钪锃
许风伟
戴永超
刘雨
徐宇虹
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杭州高田生物医药有限公司
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Publication of WO2023241577A1 publication Critical patent/WO2023241577A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/04—Formation of amino groups in compounds containing carboxyl groups
    • C07C227/06—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/04—Formation of amino groups in compounds containing carboxyl groups
    • C07C227/06—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid
    • C07C227/08—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid by reaction of ammonia or amines with acids containing functional groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/14—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof
    • C07C227/16—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions not involving the amino or carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C315/00—Preparation of sulfones; Preparation of sulfoxides
    • C07C315/04—Preparation of sulfones; Preparation of sulfoxides by reactions not involving the formation of sulfone or sulfoxide groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C317/00—Sulfones; Sulfoxides
    • C07C317/44—Sulfones; Sulfoxides having sulfone or sulfoxide groups and carboxyl groups bound to the same carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/30—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group
    • C07C67/31—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by introduction of functional groups containing oxygen only in singly bound form
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/30—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group
    • C07C67/313—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by introduction of doubly bound oxygen containing functional groups, e.g. carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C07C69/67—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of saturated acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C07C69/67—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of saturated acids
    • C07C69/675—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of saturated acids of saturated hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C07C69/67—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of saturated acids
    • C07C69/716—Esters of keto-carboxylic acids or aldehydo-carboxylic acids

Definitions

  • the present application relates to the technical field of pharmaceutical carriers, and more specifically, to cationic lipid compounds and their preparation methods and applications.
  • Gene therapy is the introduction of target genes into patients to correct or compensate for diseases caused by defective and abnormal genes, so that the disease can be treated. It is a new technology born from the combination of modern medicine and molecular biology. As a new means of disease treatment, gene therapy has had some successful applications, and the scientific breakthroughs achieved will continue to promote the development of gene therapy into mainstream medical care.
  • Common genetic drugs include plasmid DNA (pDNA), antisense oligonucleotide (antisense ODN), small interfering RNA (siRNA), small hairpin RNA (shRNA) and messenger RNA (mRNA).
  • mRNA is a type of single-stranded ribonucleic acid that contains specific genetic information.
  • mRNA transmembrane or intracellular proteins
  • the key to gene therapy is to deliver genetic drugs to target cells in the body so that they can work.
  • foreign genes are directly introduced into the body, they will be degraded by nucleases in the body. Before entering the target cells, they will be degraded into small molecule nucleotides, thus losing their therapeutic effect. Therefore, the key to realizing gene therapy lies in constructing an efficient and safe gene delivery system.
  • Gene carriers must go through multiple complex processes when transporting genes: reaching target cells through blood circulation, cellular uptake, endosomal escape, intracellular movement, and carrier release of genetic materials.
  • the main obstacles are extracellular obstacles and complex blood environment. Intracellular barriers to lysosomal enzyme degradation. Therefore, how to find a good gene carrier so that the target gene can reach the target site and be effective is an urgent problem for gene carrier researchers.
  • viral vector system is a natural vector resource.
  • the viral genome has a simple structure, high transfection efficiency, and strong target cell specificity. However, it has poor guidance, low carrying capacity, and is immunogenic and potentially tumorigenic, making it difficult to achieve clinical application requirements. Therefore, non-viral vector systems that are diverse, non-immunogenic and easy to control production have attracted much attention in recent years. attention and have applications in many therapeutic areas.
  • Non-viral vector systems are mainly lipid vectors.
  • Lipid carriers usually contain the positive charge of cationic lipids and combine with negatively charged genetic drugs through electrostatic interaction, thereby concentrating and packaging the genetic material into smaller particles to form lipid nanoparticles (LNP).
  • LNP shows advantages that other types of liposomes cannot match in the preparation of gene delivery vectors and cell transfection.
  • the smaller particle size of the complex reduces the chance of being recognized, phagocytized, and cleared by macrophages in the body, and improves the in vivo bioavailability of the drug.
  • the smaller particle size of the complex makes it easier to penetrate from the vascular endothelial cell gap into the tumor parenchyma through penetration and retention effects, increasing drug accumulation in the tumor tissue.
  • positively charged liposomes are more likely to adsorb to the cell surface and enter the cells through mechanisms such as endocytosis, which greatly increases the transfection ability of the liposomes.
  • LNP has become the most widely used non-viral vector due to its simple structure, easy operation, and high biological safety.
  • the preparation process of most LNPs is complex and difficult to scale up. Therefore, how to provide a non-viral vector system with simple preparation method and good therapeutic effect has become an urgent problem to be solved.
  • this application provides cationic lipid compounds and their preparation methods and applications.
  • This type of cationic lipid compound has a positive charge and is easier to enter cells, thereby better delivering drugs and producing better therapeutic effects. It is also simple to synthesize and easy to prepare, and has practical application value.
  • this application provides cationic lipid compounds, adopting the following technical solutions:
  • Cationic lipid compounds said cationic lipid compounds include compounds represented by formula I;
  • n 1, 2, 3 or 4;
  • R 1 and R 2 are each independently selected from the secondary long-chain alkyl ester represented by formula II;
  • an amphiphilic derivative obtained by combining 3-((2-(dimethylamino)ethyl)(methyl)amino)propionic acid and a secondary long-chain alkyl ester is used as a carrier for genetic drugs
  • the cationic lipid component in the system has a positive charge, so it can better deliver gene drugs and achieve better gene therapy effects.
  • the synthesis steps of this type of cationic lipid compound are simple, the raw materials are easily available, and large-scale production can be achieved.
  • the cationic lipid compound contains a total of three ester groups.
  • the compound has the best loading and delivery effect on genetic drugs, thereby achieving the best therapeutic effect.
  • the cationic lipid compounds described in this application include compounds represented by Formula III, Formula IV, Formula V, Formula VI, Formula VII, Formula VIII or Formula IX.
  • this application provides a method for preparing a cationic lipid compound, adopting the following technical solution:
  • a method for preparing a cationic lipid compound which method includes the following steps:
  • the preparation method of the cationic lipid compound includes the following steps:
  • the intermediate product 8 undergoes an esterification reaction with a monovalent secondary alcohol compound to obtain the cationic lipid compound.
  • the raw material in step (1) is a bromoester compound.
  • it can be specifically selected according to the structure of the specific cationic lipid compound synthesized.
  • the bromoester compound includes ethyl 8-bromooctanoate, ethyl 6-bromocaproate or ethyl 4-bromobutyrate.
  • step (1) TBAI (tetrabutylammonium iodide) is used as a catalyst for the reaction.
  • the reaction solvent includes DMSO (dimethyl sulfoxide) solution.
  • the reaction condition is carried out at room temperature.
  • concentrated sulfuric acid provides an acidic environment for the reaction in step (2).
  • step (2) the reaction condition is carried out at room temperature.
  • intermediate product 3 is a symmetric ketone compound.
  • the reaction condition is carried out at room temperature.
  • intermediate product 4 is an alcohol compound.
  • the acid chloride compounds in step (4) are a general term for a class of compounds containing different numbers of carbon atoms and whose terminals are not substituted by halogen.
  • the acid chloride compounds can be synthesized according to the specific cationic lipids synthesized. The structure of the compound is specifically selected.
  • the acid chloride compound includes 3-chloropropionyl chloride, 2-chloroacetyl chloride, 4-chlorobutyryl chloride or 5-chlorovaleryl chloride.
  • the reaction solvent includes a DCM solution containing pyridine, wherein the concentration of pyridine is 73-134mM.
  • step (4) the reaction condition is carried out at room temperature.
  • the reaction solvent includes acetone solution.
  • the reaction conditions are carried out at 68-72°C.
  • the reaction temperature can be, for example, 68°C, 70°C, 72°C, 68-70°C, 68-72°C or 70-72°C, etc. .
  • the reaction solvent includes DCM solution.
  • step (6) the reaction condition is carried out at room temperature.
  • step (7) the reaction condition is carried out at room temperature.
  • step (8) DMAP (4-dimethylaminopyridine) and DCC (dicyclohexylcarbodiimide) are used as catalysts for the reaction.
  • the reaction solvent includes DCM solution.
  • step (8) the reaction condition is carried out at room temperature.
  • the monovalent secondary alcohol compound is a monovalent alcohol containing different numbers of carbon atoms, and the hydroxyl group is not at position 1.
  • it can be determined according to the specific synthesis method.
  • the structure of the cationic lipid compound is specifically selected.
  • the monovalent secondary alcohol compounds include 13-pentadecanol, 11-pentadecanol, 9-heptadecanol, and 7-decacanol. Pentaol or 9-ecosanol.
  • this application provides an LNP carrier, adopting the following technical solution:
  • An LNP carrier containing any one or a combination of at least two of the cationic lipid compounds described in the first aspect.
  • the cationic lipid compound in the LNP carrier may be a single cationic lipid compound or a combination of multiple cationic lipid compounds.
  • the mole fraction of the cationic lipid compound in the LNP carrier is 15% to 70%, for example, it can be 15%, 30%, 45%, 60%, 70%, 15%. ⁇ 30%, 15% ⁇ 45%, 15% ⁇ 60%, 15% ⁇ 70%, 30% ⁇ 45%, 30% ⁇ 60%, 30% ⁇ 70%, 45% ⁇ 60%, 45% ⁇ 70 % or 60% ⁇ 70%, etc.
  • This mole fraction is the molar ratio of lipid material in the LNP carrier, excluding contained drugs (such as nucleic acid drugs).
  • the cationic lipid compound in the LNP carrier when the cationic lipid compound in the LNP carrier is one kind, its mole fraction in the LNP carrier is 15% to 70% (for example, it can be 15%, 30%, 45%, 60%). %, 70%, 15% ⁇ 30%, 15% ⁇ 45%, 15% ⁇ 60%, 15% ⁇ 70%, 30% ⁇ 45%, 30% ⁇ 60%, 30% ⁇ 70%, 45% ⁇ 60%, 45% to 70% or 60% to 70%, etc.); when the cationic lipid compounds in the LNP carrier are a combination of multiple types, the total amount of the combination of multiple cationic lipid compounds is in the LNP carrier
  • the mole fraction in is 15% ⁇ 70% (for example, it can be 15%, 30%, 45%, 60%, 70%, 15% ⁇ 30%, 15% ⁇ 45%, 15% ⁇ 60%, 15% ⁇ 70%, 30% ⁇ 45%, 30% ⁇ 60%, 30% ⁇ 70%, 45% ⁇ 60%, 45% ⁇ 70% or 60% ⁇ 70%, etc.).
  • the above mole fraction is the molar ratio of lipid
  • the LNP carrier also includes phospholipid 5% to 30% (for example, it can be 5% to 20% or 8% to 30%) and cholesterol 15% to 65% in mole fraction.
  • This mole fraction is the molar ratio of lipid material in the LNP carrier, excluding contained drugs (such as nucleic acid drugs).
  • the LNP carrier also includes phospholipid 5% to 30% (for example, it can be 5% to 20% or 8% to 30%), cholesterol 15% to 65%, and polyethylene glycol lipid in molar fraction.
  • the content is 1.5% to 3% (for example, it can be 1% to 3% or 0.5% to 2%).
  • This mole fraction is the molar ratio of lipid material in the LNP carrier, excluding contained drugs (such as nucleic acid drugs).
  • the molar fraction of the phospholipid in the LNP carrier is 5% to 40%, for example, it can be 8%, 10%, 15%, 20%, 25%, 30%, 5% to 10%, 8 % ⁇ 10%, 5% ⁇ 15%, 8% ⁇ 15%, 5% ⁇ 20%, 8% ⁇ 20%, 5% ⁇ 25%, 8% ⁇ 25%, 5% ⁇ 30%, 8% ⁇ 30%, 10% ⁇ 15%, 10% ⁇ 20%, 10% ⁇ 25%, 10% ⁇ 30%, 15% ⁇ 20%, 15% ⁇ 25%, 15% ⁇ 30%, 20% ⁇ 25% , 20% ⁇ 30% or 25% ⁇ 30%, etc.
  • This mole fraction is the molar ratio of lipid material in the LNP carrier, excluding contained drugs (such as nucleic acid drugs).
  • the molar fraction of cholesterol in the LNP carrier is 10% to 65%, for example, it can be 10%, 15%, 30%, 45%, 65%, 15% to 30%, 15% to 45%. %, 15% to 65%, 30% to 45%, 30% to 65% or 45% to 65%, etc.
  • This mole fraction is the molar ratio of lipid material in the LNP carrier, excluding contained drugs (such as nucleic acid drugs).
  • the LNP carrier further includes a polyethylene glycol lipid in terms of mole fraction, wherein the mole fraction of the polyethylene glycol lipid in the LNP carrier is 0.5% to 3%, for example, it can be 1.5%, 2%, 2.5%, 3%, 0.5% ⁇ 2%, 1% ⁇ 2%, 1.5% ⁇ 2%, 0.5% ⁇ 2.5%, 1% ⁇ 2%, 1.5% ⁇ 2.5%, 0.5% ⁇ 3%, 1% ⁇ 2%, 1.5% ⁇ 3%, 2% ⁇ 2.5%, 1% ⁇ 3%, 2% ⁇ 3% or 2.5% ⁇ 3%, etc.
  • This mole fraction is the molar ratio of lipid material in the LNP, excluding contained drugs (such as nucleic acid drugs).
  • the LNP carrier includes 15% to 70% of a cationic lipid compound (for example, it can be 15%, 30%, 45%, 60%, 70%, 30% to 70%) on a molar fraction basis. %, 15% to 30%, 15% to 45%, 15% to 60%, 15% to 70%, 30% to 45%, 30% to 60%, 30% to 70%, 45% to 60%, 45% to 70% or 60% to 70%, etc.), phospholipid 5% to 30% (for example, it can be 8%, 10%, 15%, 20%, 25%, 30%, 5% to 10%, 8% ⁇ 10%, 5% ⁇ 15%, 8% ⁇ 15%, 5% ⁇ 20%, 8% ⁇ 20%, 5% ⁇ 25%, 8% ⁇ 25%, 5% ⁇ 30%, 8% ⁇ 30 %, 10% ⁇ 15%, 10% ⁇ 20%, 10% ⁇ 25%, 10% ⁇ 30%, 15% ⁇ 20%, 15% ⁇ 25%, 15% ⁇ 30%, 20% ⁇ 25%, 20% to 30% or 25% to 30%, etc.) and cholesterol 10% to 65% (for example,
  • the LNP carrier includes 15% to 70% of a cationic lipid compound on a molar fraction basis (for example, it can be 15%, 30%, 45%, 60%, 70%, 30% to 70%, 15 % ⁇ 30%, 15% ⁇ 45%, 15% ⁇ 60%, 15% ⁇ 70%, 30% ⁇ 45%, 30% ⁇ 60%, 30% ⁇ 70%, 45% ⁇ 60%, 45% ⁇ 70% or 60% to 70%, etc.), phospholipid 5% to 30% (for example, it can be 8%, 10%, 15%, 20%, 25%, 30%, 5% to 10%, 8% to 10% , 5% ⁇ 15%, 8% ⁇ 15%, 5% ⁇ 20%, 8% ⁇ 20%, 5% ⁇ 25%, 8% ⁇ 25%, 5% ⁇ 30%, 8% ⁇ 30%, 10 % ⁇ 15%, 10% ⁇ 20%, 10% ⁇ 25%, 10% ⁇ 30%, 15% ⁇ 20%, 15% ⁇ 25%, 15% ⁇ 30%, 20% ⁇ 25%, 20% ⁇ 30% or 25% to 30%, etc.) and cholesterol 10% to 65% (for example, it can be 15%,
  • this application provides a medicine using the following technical solution:
  • a medicine comprising the LNP carrier described in the third aspect.
  • the drugs also include nucleic acid drugs.
  • the drug is a genetic drug.
  • genetic drugs include, but are not limited to, plasmid DNA (pDNA), antisense oligonucleotides (antisense ODN), small interfering RNA (siRNA), small hairpin RNA (shRNA), and messenger RNA (mRNA).
  • pDNA plasmid DNA
  • antisense ODN antisense oligonucleotides
  • siRNA small interfering RNA
  • shRNA small hairpin RNA
  • mRNA messenger RNA
  • the drug is mRNA.
  • the drug is mRNA encoding the SARS-CoV-2 virus spike glycoprotein (S protein).
  • the molar ratio of the nitrogen content of the cationic lipid compound in the LNP carrier to the phosphorus content in the nucleic acid drug is (2-15):1, for example, it can be 2 :1, 3:1, 4:1, 5:1, 6:1, (2 ⁇ 3):1, (2 ⁇ 4):1, (2 ⁇ 5):1, (2 ⁇ 6):1 , (3 ⁇ 4):1, (3 ⁇ 5):1, (3 ⁇ 6):1, (4 ⁇ 5):1, (4 ⁇ 6):1 or (5 ⁇ 6):1, etc.
  • the nucleic acid drug (for example, the mRNA encoding SARS-CoV-2 virus spike glycoprotein) accounts for 1% to 10% of the mass fraction (drug loading amount) of the drug-loaded LNP, for example, it can be 1 % ⁇ 7%, 1% ⁇ 6%, 2% ⁇ 6%, 2% ⁇ 5%, or 3% ⁇ 5%, etc.
  • the ratio of the cationic lipid compound to the nucleic acid drug directly affects the encapsulation rate of the drug and the subsequent drug release effect.
  • the nitrogen content of the cationic lipid compound in the LNP carrier is related to the nucleic acid drug
  • the molar ratio of the phosphorus content in the drug is (2-15):1
  • the drug-loaded structure has better stability and has the best intracellular release efficiency.
  • the treatment effect is the best, and it can improve the utilization rate of raw materials and save production costs.
  • the mass fraction (drug loading) of nucleic acid drugs in drug-loaded LNPs is 3% to 10%, the drug encapsulation rate is better, and the utilization rate of raw materials can be improved and production costs can be saved. .
  • the particle size and particle size distribution of the drug affect the drug loading capacity and subsequent practical applications.
  • the average particle size of the drug is 30nm ⁇ 300nm, for example, it can be 30nm ⁇ 270nm, 30nm ⁇ 250nm, 30nm ⁇ 200nm, 50nm ⁇ 200nm, 50nm ⁇ 150nm, or 60nm ⁇ 120nm, etc.
  • the polydispersity (PDI) of the drug is less than 0.15, for example, it can be less than 0.13, less than 0.12, less than 0.11, less than 0.1, or less than 0.09, etc.
  • the polydispersity (PDI) of the drug is 0.02 to 0.25, for example, it can be 0.02 to 0.20, 0.05 to 0.20, 0.05 to 0.25, 0.05 to 0.1, or 0.05 to 0.25, etc. In some aspects of the application, the drug has a PDI of less than 0.1.
  • the drug includes an LNP carrier and mRNA (for example, mRNA encoding the SARS-CoV-2 virus spike glycoprotein), wherein the mRNA drug loading is 2-6% (wt.), and the LNP Carriers include 60 mol% to 70 mol% TM3 lipid compound, 7 mol% to 12 mol% phospholipid (eg HSPC), 20 mol% to 25 mol% cholesterol, and 1 mol% to 2 mol% DSPE-PEG2000.
  • the above-mentioned drugs have an average particle size between 150 nm and 200 nm.
  • the above-mentioned drugs have a particle size distribution PDI less than 0.1.
  • the encapsulation efficiency of the above-mentioned drugs is greater than 85%.
  • the above drugs have an N/P value of 5.5 to 6.5.
  • the drug includes an LNP carrier and a nucleic acid drug, wherein the mass ratio of the nucleic acid drug to the LNP carrier is 5% to 7%, and the LNP carrier includes 60 mol% to 70 mol% of TM3 lipid compound, 10 mol % ⁇ 15mol% phospholipid (eg HSPC), 15mol% ⁇ 25mol% cholesterol, and 1mol% ⁇ 2mol% DSPE-PEG2000.
  • the above-mentioned drugs have an average particle size between 50 nm and 100 nm.
  • the drugs described above have a PDI of less than 0.1 or less than 0.05.
  • the encapsulation efficiency of the above-mentioned drugs is greater than 80% or greater than 90%.
  • the drug includes an LNP carrier and a nucleic acid drug, wherein the mass ratio of the nucleic acid drug to the LNP carrier is 4% to 5%, and the LNP carrier includes 60 mol% to 70 mol% of TM3 lipid compound, 5 mol % ⁇ 8mol% phospholipid (eg HSPC), 25mol% ⁇ 35mol% cholesterol, and 1mol% ⁇ 2mol% DSPE-PEG2000.
  • the above-mentioned drugs have an average particle size between 100 nm and 150 nm.
  • the PDI of the above-mentioned drugs is less than 0.1.
  • the encapsulation efficiency of the above-mentioned drugs is greater than 80% or greater than 90%.
  • the drug includes an LNP carrier and a nucleic acid drug, wherein the mass ratio of the nucleic acid drug to the LNP carrier is 4% to 5%, and the LNP carrier includes 60 mol% to 70 mol% of TM3 lipid compound, 10 mol % to 15 mol% phospholipid (eg HSPC), 15 mol% to 25 mol% cholesterol, and 2 mol% to 3 mol% DSPE-PEG2000.
  • the above-mentioned drugs have an average particle size between 100 nm and 150 nm. In some aspects, the above-mentioned drugs have an average particle size between 100 nm and 150 nm.
  • the PDI of the above-mentioned drugs is less than 0.1. In some aspects, the encapsulation efficiency of the above-mentioned drugs is greater than 80% or greater than 90%.
  • the drug includes an LNP carrier and a nucleic acid drug, wherein the mass ratio of the nucleic acid drug to the LNP carrier is 2% to 3%, and the LNP carrier includes 25 mol% to 35 mol% of TM3 lipid compound, 5 mol % ⁇ 12mol% phospholipid (eg HSPC), 50mol% ⁇ 60mol% cholesterol, and 1mol% ⁇ 2mol% DSPE-PEG2000.
  • the above-mentioned drugs have an average particle size between 50 nm and 100 nm.
  • the PDI of the above-mentioned drugs is less than 0.1.
  • the encapsulation efficiency of the above-mentioned drugs is greater than 80% or greater than 90%.
  • the drug includes an LNP carrier and a nucleic acid drug, wherein the nucleic acid drug and the LNP
  • the mass ratio of the carrier is 5% to 6%, in which the LNP carrier contains 35mol% to 45mol% of TM3 lipid compounds, 12mol% to 18mol% of phospholipids (such as HSPC), 40mol% to 50mol% of cholesterol, and 0.5mol % ⁇ 1mol% of DSPE-PEG2000.
  • the above-mentioned drugs have an average particle size between 100 nm and 150 nm.
  • the PDI of the above-mentioned drugs is less than 0.1.
  • the encapsulation efficiency of the above-mentioned drugs is greater than 80% or greater than 90%.
  • the drug includes an LNP carrier and a nucleic acid drug (such as mRNA encoding SARS-CoV-2 virus spike glycoprotein), wherein the LNP carrier contains 60 mol% to 70 mol% of TM6 lipid compounds, 10 mol % to 15 mol% phospholipid (eg HSPC), 15 mol% to 25 mol% cholesterol, and 2 mol% to 3 mol% DSPE-PEG2000.
  • the above-mentioned drugs have an average particle size between 100 nm and 150 nm.
  • the above-mentioned drugs have a particle size distribution PDI less than 0.1.
  • the encapsulation efficiency of the above-mentioned drugs is greater than 90%.
  • the drugs described above have an N/P value of 5.5 to 6.2.
  • the drug includes an LNP carrier and a nucleic acid drug (such as mRNA encoding SARS-CoV-2 virus spike glycoprotein), wherein the N/P value is 5.5 to 6.2, and the LNP carrier contains 25 mol% ⁇ 35 mol% TM6 lipid compound, 5 mol% ⁇ 15 mol% phospholipid (eg HSPC), 55 mol% ⁇ 65 mol% cholesterol, and 1 mol% ⁇ 2 mol% DSPE-PEG2000.
  • the above-mentioned drugs have an average particle size between 150 nm and 200 nm.
  • the above-mentioned drugs have a particle size distribution PDI less than 0.1.
  • the encapsulation efficiency of the above-mentioned drugs is greater than 85%.
  • the drug includes an LNP carrier and a nucleic acid drug (such as mRNA encoding SARS-CoV-2 virus spike glycoprotein), wherein the N/P value is 3.8 to 4.2, and the LNP carrier contains 45 mol% ⁇ 55 mol% TM6 lipid compound, 5 mol% ⁇ 15 mol% phospholipid (eg HSPC), 35 mol% ⁇ 45 mol% cholesterol, and 1 mol% ⁇ 2 mol% DSPE-PEG2000.
  • the above-mentioned drugs have an average particle size between 50 nm and 100 nm.
  • the above-mentioned drugs have a particle size distribution PDI less than 0.1.
  • the encapsulation efficiency of the above-mentioned drugs is greater than 85%.
  • the drug includes an LNP carrier and a nucleic acid drug (such as mRNA encoding SARS-CoV-2 virus spike glycoprotein), wherein the N/P value is 5.5 to 6.2, and the LNP carrier contains 60 mol% ⁇ 70 mol% of TM7 lipid compound, 10 to 15 mol% of phospholipid (eg HSPC), 15 to 25 mol% of cholesterol, and 2 to 3 mol% of DSPE-PEG2000.
  • the above-mentioned drugs have an average particle size between 100 nm and 150 nm.
  • the above-mentioned drugs have a particle size distribution PDI less than 0.15.
  • the encapsulation efficiency of the above-mentioned drugs is greater than 70%.
  • the drug includes an LNP vector and a nucleic acid drug (e.g., encoding SARS-CoV- 2 viral spike glycoprotein mRNA), where the N/P value is 5.5 to 6.2, and the LNP carrier contains 25 mol% to 35 mol% of TM7 lipid compounds, 5 mol% to 15 mol% of phospholipids (such as HSPC), 55 mol% ⁇ 65 mol% cholesterol, and 1 mol% ⁇ 2 mol% DSPE-PEG2000.
  • the above-mentioned drugs have an average particle size between 50 nm and 1100 nm.
  • the above-mentioned drugs have a particle size distribution PDI less than 0.1.
  • the encapsulation efficiency of the above-mentioned drugs is greater than 95%.
  • the drug includes an LNP carrier and a nucleic acid drug (such as mRNA encoding SARS-CoV-2 virus spike glycoprotein), wherein the N/P value is 3.8 to 4.2, and the LNP carrier contains 45 mol% ⁇ 55 mol% TM7 lipid compound, 5 mol% - 15 mol% phospholipid (eg HSPC), 35 mol% - 45 mol% cholesterol, and 1 mol% - 2 mol% DSPE-PEG2000.
  • the above-mentioned drugs have an average particle size between 100 nm and 150 nm.
  • the above-mentioned drugs have a particle size distribution PDI less than 0.1.
  • the encapsulation efficiency of the above-mentioned drugs is greater than 90%.
  • the drugs in this application can be used to treat or prevent certain diseases, such as COVID infections, viral and bacterial infections, tumors, metabolic diseases, autoimmune diseases, cardiovascular diseases and genetic diseases.
  • certain diseases such as COVID infections, viral and bacterial infections, tumors, metabolic diseases, autoimmune diseases, cardiovascular diseases and genetic diseases.
  • this application provides a preparation method for the above-mentioned medicine, adopting the following technical solution:
  • a preparation method of medicine includes the following steps:
  • the lipid alcohol phase and the nucleic acid aqueous phase are mixed to prepare an intermediate drug product, which is dialyzed to obtain the drug.
  • the solvent used includes ethanol (eg absolute ethanol).
  • dialysis is performed using a dialysis device to remove solvent.
  • the preparation method of the present application further includes dispersing the LNP in a biocompatible injection buffer. .
  • this application provides a vaccine that adopts the following technical solution:
  • a vaccine comprising the LNP vector described in the third aspect.
  • this application provides the use of the cationic lipid compound described in the first aspect in the preparation of LNP carriers, drugs or vaccines, or the use of the LNP carrier described in the third aspect in the preparation of drugs and/or vaccines.
  • This application uses amphipathic derivatives obtained by combining 3-((2-(dimethylamino)ethyl)(methyl)amino)propionic acid and secondary long-chain alkyl esters as carriers of genetic drugs
  • the cationic lipid component in the system has a positive charge, which makes it easier to adsorb to the cell surface and enter the cell.
  • the efficiency of drug delivery is higher. In some aspects, it makes gene transfection more efficient and the therapeutic effect more effective. Good; the production raw materials are easy to obtain, the preparation method is simple, and the synthesis efficiency is high, creating conditions for large-scale production;
  • the LNP carrier prepared using the above cationic lipid compound has good drug loading and delivery efficiency.
  • the encapsulation rate can reach more than 88%, which is related to the fact that nucleic acid molecules can be efficiently expressed in the body after being made into drugs.
  • the protein can be used as a therapeutic drug to treat diseases, or as a vaccine to activate immunity, and has broad application prospects.
  • LNP carriers prepared using the above-mentioned cationic lipid compounds can accurately and efficiently enter cells and express related proteins to exert therapeutic effects.
  • Figure 1 is a schematic diagram of the synthetic route of the cationic lipid compound TM3 in this application.
  • Figure 2 is a picture of the hydrogen spectrum detection result of intermediate product 2 in Preparation Example 1 of the present application.
  • Figure 3 is a picture of the hydrogen spectrum detection result of intermediate product 3 in Preparation Example 1 of the present application.
  • Figure 4 is a picture of the hydrogen spectrum detection result of intermediate product 4 in Preparation Example 1 of the present application.
  • Figure 5 is a picture of the hydrogen spectrum detection result of intermediate product 5 in Preparation Example 1 of the present application.
  • Figure 6 is a picture of the hydrogen spectrum detection result of intermediate product 7 in Preparation Example 1 of the present application.
  • Figure 7 is a picture of the hydrogen spectrum detection results of the cationic lipid compound TM3 in Preparation Example 1 of the present application.
  • Figure 8 is a picture of the detection results of the protein expression of the drug TP1 in 293T cells in Example 1 of the present application.
  • Figure 9 is a picture of the detection results of the anti-S protein antibody titer in mouse serum in Example 2 of the present application.
  • Figure 10 is a cryo-electron microscope structural diagram of HG-06LNP in Example 3 of the present application.
  • Figure 11 is a cryo-electron microscope structural diagram of HG-23LNP in Example 3 of the present application.
  • Figure 12 is an image of HG-23LNP in Example 3 of the present application entering cells and accumulating in endosomes after being fluorescently labeled.
  • Figure 13 is a picture of the hydrogen spectrum detection results of the cationic lipid compound TM6 in the embodiment of the present application.
  • Figure 14 is a comparison of the results of cell transfection of multiple formulations of TM6 and TM7LNP.
  • Figure 15 is a comparison of the antibody levels induced after immunizing mice with multiple formulations of TM6 and TM7LNP.
  • the present application provides cationic lipid compounds, which include compounds represented by Formula I;
  • n 1, 2, 3 or 4;
  • R 1 and R 2 are each independently selected from the secondary long-chain alkyl ester represented by formula II;
  • n 1, 2, or 3.
  • n 2.
  • n1 in R1 has the same value as n1 in R2 .
  • the cationic lipid compound includes TM1 represented by Formula III, TM2 represented by Formula IV, TM3 represented by Formula V, TM4 represented by Formula VI or TM5 represented by Formula VII.
  • this application takes TM3 as an example to illustrate the preparation method of the cationic lipid compound.
  • the schematic diagram of its synthesis route is shown in Figure 1, and the specific process is as follows:
  • TLC detects the reaction progress. After the reaction is complete, add 90 to 110 mL of ice water to the system to quench, and use DCM to extract three times, 90 to 110 mL each time. Combine all organic phases, use 45 to 55 mL of saturated sodium bicarbonate to elute the organic phase, dry and concentrate, and then purify through a silica gel column to obtain intermediate product 4.
  • the LNP carrier includes 15% to 70% of cationic lipid compounds, 8% to 30% of phospholipids, 15% to 65% of cholesterol and polyethylene glycol lipids. 1.5% ⁇ 3%.
  • the present application also provides an LNP carrier.
  • the LNP carrier includes 15% to 70% of cationic lipid compounds, 8% to 40% of phospholipids, and 10% to 65% of cholesterol.
  • This application also provides a drug, which includes an LNP carrier and a nucleic acid drug, wherein the drug loading of the nucleic acid drug is between 1.0-10.0%.
  • the molar ratio of the nitrogen content of the cationic lipid compound in the LNP carrier to the phosphorus content in the nucleic acid drug is 2:1 to 15:1, 2:1 to 10:1, 2:1 to 6:1, Or 4:1 to 6:1.
  • the drug can be prepared by:
  • the drug can be prepared by:
  • the lipid alcohol phase and the nucleic acid aqueous phase are mixed at room temperature in a mixer at a volume ratio of 1:(2-5) to prepare a pharmaceutical intermediate, which is dialyzed through a dialysis device to remove the solvent and replace it with a solution containing 0.2
  • the medicine is obtained by using a Tris solution with a concentration of 18-22mM of sodium chloride and 4% to 6% sucrose.
  • the drug can be prepared by:
  • the lipid alcohol phase and the nucleic acid aqueous phase are mixed at room temperature in a mixer at a volume ratio of 1:(2-5) to prepare a pharmaceutical intermediate, which is dialyzed through a dialysis device to remove the solvent and replace it with a solution containing 0.2
  • the medicine is obtained by using a Tris solution with a concentration of 18-22mM of sodium chloride and 4% to 6% sucrose.
  • Embodiment 1 Cationic lipid compound, characterized in that the cationic lipid compound includes a compound represented by formula I;
  • n 1, 2, 3 or 4;
  • R 1 and R 2 are each independently selected from the secondary long-chain alkyl ester represented by formula II;
  • Embodiment 2 The cationic lipid compound according to Embodiment 1, characterized in that the cationic lipid compound includes a compound represented by Formula III, Formula IV, Formula V, Formula VI or Formula VII;
  • Embodiment 3 The preparation method of the cationic lipid compound as described in Example 1 or 2, characterized in that the preparation method of the cationic lipid compound includes the following steps:
  • Embodiment 4 An LNP carrier, characterized in that the LNP carrier contains any one or a combination of at least two of the cationic lipid compounds described in Embodiment 1 or 2.
  • Embodiment 5 The LNP carrier according to embodiment 4, characterized in that, in terms of mole fraction, the LNP The carrier includes 15% to 70% of cationic lipid compounds;
  • the LNP carrier also includes 8% to 30% of phospholipids, 5% to 65% of cholesterol and 1.5% to 3% of polyethylene glycol lipids.
  • Embodiment 6 A medicament, characterized in that the medicament includes the LNP carrier described in Embodiment 4 or 5.
  • Embodiment 7 The drug according to Embodiment 6, characterized in that the drug further includes nucleic acid drugs;
  • the molar ratio of the nitrogen content of the cationic lipid compound in the LNP carrier to the phosphorus content in the nucleic acid drug is (2-6):1.
  • Embodiment 8 The preparation method of the medicament as described in Embodiment 6 or 7, characterized in that the preparation method of the medicament includes the following steps:
  • the lipid alcohol phase and the nucleic acid aqueous phase are mixed to prepare an intermediate drug product, which is dialyzed to obtain the drug.
  • Embodiment 9 A vaccine, characterized in that the vaccine includes the LNP vector described in Embodiment 4 or 5.
  • Embodiment 10 Application of the cationic lipid compound as described in Example 1 or 2 in the preparation of LNP carriers, drugs or vaccines, or the use of the LNP carrier as described in Example 4 or 5 in the preparation of drugs and/or vaccines. .
  • This preparation example provides a cationic lipid compound TM3.
  • the structural formula of the cationic lipid compound TM3 is shown in Formula V.
  • the cationic lipid compound TM3 is prepared by the following method, and the synthesis route is shown in Figure 1:
  • TLC detects the reaction progress. After the reaction is complete, add 100 mL of ice water to the system to quench, and use DCM to extract three times, 100 mL each time. Combine all organic phases, use 50 mL of saturated sodium bicarbonate to elute the organic phase, dry and concentrate, and then purify through a silica gel column to obtain intermediate product 4. The hydrogen spectrum detection results of intermediate product 4 are shown in Figure 4.
  • Intermediate product 7 undergoes a hydrolysis reaction with LiOH in a mixed solution of ethanol and water (volume ratio 1:2) at room temperature to generate intermediate product 8:
  • This preparation example provides a drug TP1, in which the nucleic acid drug is mRNA encoding the SARS-CoV-2 virus spike glycoprotein (S protein), and is prepared by the following method:
  • the intermediate product is passed through a dialysis device to remove ethanol and citric acid, and is replaced with a 20 mM Tris solution containing 0.4% sodium chloride and 5% sucrose to prepare drug TP1.
  • the drug TP1 prepared in Preparation Example 2 was subjected to performance testing, including particle size detection, dispersion degree detection and encapsulation efficiency detection.
  • a laser scattering particle size analyzer (PCS, Dandong BeNano 180 Zeta pro laser particle size analyzer) was used to measure the size and degree of dispersion (PDI) of nanoparticles.
  • PCS Dandong BeNano 180 Zeta pro laser particle size analyzer
  • PDI size and degree of dispersion
  • a 671nm solid laser was used as the incident light.
  • the dynamic light scattering test was performed at 25°C. , the scattered light path adopts 173° backscatter detection. The average value of three consecutive tests was used as the test data.
  • Encapsulation efficiency detection adopts fluorescence quantitative detection method, using Quant-it TM RiboGreen RNA Assay Kit as the detection kit. Add Triton to release the nucleic acid in the preparation after demulsification, add the specific nucleic acid dye ribogreen, and use the ALLSHENG Feyond-A300 microplate reader to set the excitation light wavelength to 470nm and the emission light wavelength to 525nm, detect the absorbance value of the sample, and calculate the total nucleic acid through the standard curve.
  • Encapsulation rate (total nucleic acid content - free nucleic acid content)/total nucleic acid content ⁇ 100%.
  • the drug TP1 prepared according to the formula and method of Preparation Example 2 has an average particle size of 169 nm and a PDI of 0.07. It has suitable particle size and uniform particle size distribution, and also has a high The encapsulation rate shows that it has good drug loading capacity, creating conditions for subsequent practical applications.
  • This preparation example provides the preparation of seven drugs, among which the nucleic acid drug is mRNA encoding luciferase. Specific lipid prescription information is shown in Table 3.
  • Millipore 0.22um sterile needle filter
  • Ribogreen detection reagent (thermo) was used to detect the mRNA loading and encapsulation efficiency of the filtered mRNA-LNP.
  • PCS detector to detect the zeta average particle size of the filtered mRNA-LNP. The test results are shown in Table 4.
  • Cryo-electron microscopy Take 2.5 ⁇ L LNP solution and drop it on the grid (Quantifoil Cu R1.2/1.3, 300mesh), and use Vitrobot Mark IV (ThermoFisher Scientific) to prepare cryo-electron microscopy samples.
  • Use Talos F200C equipped with a Ceta 4k ⁇ 4k camera for imaging The cryo-electron microscopy picture of the drug HG5-06 is shown in Figure 10.
  • the cryo-electron microscopy picture of the drug HG5-023 is shown in Figure 11.
  • this Preparation Example provides the preparation of drugs containing TM6 and TM7, in which the nucleic acid drug is mRNA encoding the SARS-CoV-2 virus spike glycoprotein (S protein).
  • S protein SARS-CoV-2 virus spike glycoprotein
  • the formulation information, particle size and encapsulation efficiency information of the prepared LNPs are shown in Table 5.
  • This example detects the expression level of the drug TP1 prepared in Preparation Example 2 in cells cultured in vitro.
  • the steps are as follows:
  • the 293T cell line a derivative of the human embryonic kidney cell line 293, was obtained by passage in the laboratory. It was cultured in RPMI-1640 medium containing 10% FBS and passaged 2 to 3 times a week. Take cells with a cell density of about 80% and plate them. After counting the cells, use culture medium to adjust the cell concentration to 2 ⁇ 10 5 /mL. In a 48-well plate, add 0.5mL of the above cell solution to each well, that is, the number of cells in each well is about is 1 ⁇ 10 5 . Each group was set with 3 duplicate holes, and the blank control group was also set with 3 duplicate holes.
  • Detect the total mRNA concentration based on the fluorescence of the mRNA add drugs containing 1 ⁇ g of mRNA to each well, and incubate for 18, 24, 48, and 72 hours respectively, and observe the protein expression at different incubation times.
  • the detection results of the protein expression of drug TP1 in 293T cells are shown in Figure 8.
  • the drug TP1 can be effectively transferred into 293T cells and expresses S protein.
  • the expression level continues to increase, and the protein expression level is also positively correlated with the dose.
  • LNP carriers can deliver drugs It can be transferred into cells and released, so that it can be used in the treatment of related diseases, which has practical application value.
  • mice Six female BALB/c mice aged 6 to 8 weeks were taken, and drug TP1 was injected intramuscularly (i.m.) on the 1st and 14th days respectively.
  • the anti-S protein antibody titers in mouse serum were detected by ELISA on days 10, 21, 28 and 35 after the first administration.
  • This example examines the efficacy of TM6 drugs and TM7 drugs with different prescriptions in Preparation Example 4 in cell and animal models.
  • Reporter gene concentration detection Dilute the above cell culture collection samples according to a certain ratio, and the dilution ratio is determined through preliminary experiments. The diluted samples were detected using Elisa kit.
  • Figure 14 compares the cell transfection effects of TM6 and TM7 drugs of various lipid formulations and controls them with drugs of similar lipid formulations containing TM3.
  • the transfection activity of drugs containing TM6 and TM7 is slightly higher. for medicines containing TM3.
  • the TM3 drug activity of HG-23 lipid prescription is also very high.
  • the particle size of TM series cationic LNPs has certain differences under different lipid ratios, the cell transfection activity is better.
  • blood was collected from the orbit after anesthesia. Collect about 50ul of whole blood in a 1.5mL EP tube. Place it at room temperature for 0.5h and then centrifuge at 7000g for 10min. After the collection is diluted according to a certain ratio, use (SARS-Cov2-2Spike RBD Antibody Titer Assay Kit(mouse)SinoBiological) to detect OD value. The results are shown in Figure 15.
  • TM6's HG-06 prescription and TM7's HG-23 prescription both performed well. It has clinical development value.
  • TMEA is a cationic lipid structure disclosed in CN 105085292B.
  • the hydrophilic and ionizable end groups of TM3 are the same, both are 3-((2-(dimethylamino)ethyl)(methyl)amino ) propionic acid, but the hydrophobic structure is different. So we compared the constructed mRNA-loaded LNPs. Table 6 compares three different lipid formulations. It can be seen that the in vivo protein expression activity of LNP obtained from TM3 is much better than that of TMEA LNP.
  • mice For in vivo protein expression experiments, Balb/c mice aged 6 to 8 weeks were selected. Each mouse was intravenously administered 10ug of nucleic acid LNP drug. Blood samples were taken 6 hours after administration, and the protein content in the serum was detected using an Elisa kit.

Abstract

本申请涉及药物载体的领域，具体公开了阳离子脂质化合物及其制备方法和应用，所述阳离子脂质化合物包括式I所示的化合物；式I中，n＝1、2、3或4；R1和R2各自独立地选自式II所示的二级长链烷基酯；式II中，n1＝2、4或6；n2＝5、7、9或11；n3＝5、7、9或11。将上述阳离子脂质化合物与磷脂、胆固醇和聚乙二醇脂质组合，可以制成LNP载体，进而包裹核酸分子制成相应的药物。本申请所述的阳离子脂质化合物的生产原料易得，合成步骤简单，构建的LNP载体具有良好的药物递送效果，相应的药物可高效进入靶细胞，或激活机体的免疫应答，具有实际应用的价值。

Description

阳离子脂质化合物及其制备方法和应用
相关申请的交叉引用
本申请要求于2022年6月14日提交的中国专利申请第2022106678978号的优先权。此申请的公开内容在此通过引用整体并入本文。
技术领域
本申请涉及药物载体的技术领域，更具体地说，涉及阳离子脂质化合物及其制备方法和应用。
背景技术
基因治疗(gene therapy)是将目的基因导入患者体内，以纠正或补偿缺陷和异常基因引起的疾病，从而使疾病得到治疗，是现代医学和分子生物学相结合而诞生的新技术。基因治疗作为疾病治疗的新手段，已有一些成功的应用，取得的科学性突破将继续推动基因治疗向主流医疗发展。常见的基因药物有质粒DNA(plasmid DNA，pDNA)、反义寡核苷酸(antisense ODN)、小干扰RNA(siRNA)、小发卡RNA(shRNA)和信使RNA(mRNA)。mRNA是一类包含特定遗传信息的单链核糖核酸，它能够将所携带的遗传信息传递到细胞内的核糖体，并在那里作为模板指导机体合成特定的目标蛋白。相比于难以跨过细胞膜的传统蛋白药物，mRNA的作用机制使其能够向机体递送跨膜或胞内蛋白，因此使得治疗或预防更多的疾病成为可能。mRNA的上述优势也使其成为未来药物开发的趋势。
基因治疗的关键在于将基因药物在体内输送到靶细胞，使其发挥作用。然而，直接将外源基因引入体内，会被体内的核酸酶降解，在未进入靶细胞之前，便被降解成小分子核苷酸，从而失去治疗作用。因此，实现基因治疗的关键在于构建高效、安全的基因递送系统。
基因载体在运送基因时要经历多个复杂的过程：通过血液循环到达靶细胞、细胞摄取、内涵体逃逸、胞内运动以及载体释放基因物质等，其主要障碍是复杂血液环境的细胞外障碍和溶酶体酶降解的细胞内障碍。因此如何寻找良好的基因载体，使得靶基因到达靶点发挥效用，是基因载体研究者亟待解决的问题。
目前，基因输送载体系统主要分为两大类：一是病毒载体系统，二是非病毒载体系统。病毒载体是一种天然的载体资源，病毒基因组结构简单、转染效率高、靶细胞特异性强，但其导向性差、携带能力低，且具有免疫原性和潜在致瘤性，使其难以达到临床应用的要求。因此，多样性、无免疫原性及易于控制生产的非病毒载体系统近年来备受 关注，并在很多治疗领域有所应用。
常用的非病毒载体系统主要是脂质载体。脂质载体通常含有阳离子脂质的正电荷，通过静电作用与带负电的基因药物结合，从而将基因物质浓缩包装成较小粒径的粒子，形成脂质纳米粒(LNP)。LNP在基因输送载体的制备和细胞转染方面显示出了其他类型脂质体无法比拟的优势。复合物较小的粒径降低了被体内巨嗜细胞识别、吞噬、清除的机会，提高了药物的体内生物利用度。同时，针对于肿瘤组织，复合物较小的粒径更容易通过渗透和滞留效应从血管内皮细胞间隙透过进入肿瘤实质，增加在肿瘤组织的药物聚集。在转染方面，由于细胞表面略微带有负电，带正电的脂质体更容易吸附到细胞表面，通过内吞等机制进入细胞，大大增加了脂质体的转染能力。
目前，LNP因其结构简单、操作简便、生物安全性高等特点成为了应用最为广泛的非病毒载体，但大部分LNP的制备过程复杂，不易进行放大生产。因此，如何提供一种制备方法简单、治疗效果好的非病毒载体系统，已成为亟待解决的问题。
发明内容
为了提高基因载体的药物递送效果并简化其合成步骤，本申请提供阳离子脂质化合物及其制备方法和应用。此类阳离子脂质化合物带有正电，更易进入细胞内，从而更好地递送药物，产生更好的治疗效果，且合成简单，容易制备，具有实际应用的价值。
第一方面，本申请提供阳离子脂质化合物，采用如下技术方案：
阳离子脂质化合物，所述阳离子脂质化合物包括式I所示的化合物；
式I中，n＝1、2、3或4；
R1和R2各自独立地选自式II所示的二级长链烷基酯；

式II中，n1＝2、4或6；n2＝5、7、9或11；n3＝5、7、9或11；波浪线代表式II与式I中其他部分的连接点。
本申请中，将3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸和二级长链烷基酯相结合获得的两亲性衍生物作为基因药物的载体系统中的阳离子脂质组分，其带有正电荷，因此能更好地递送基因药物，达到了更好的基因治疗效果。此类阳离子脂质化合物合成步骤简单，原材料简单易得，可以实现大规模生产。
本申请中，所述阳离子脂质化合物中共含有3个酯基，这3个酯基的空间位置直接影响阳离子脂质化合物功能的发挥，而烷基链上的碳原子数会影响酯基的空间结构，经试验验证，当n＝1、2、3或4，n1＝2、4或6，n2＝5、7、9或11，n3＝5、7、9或11时，阳离子化合物对基因药物的装载及递送效果最好，从而达到最佳的治疗效果。
作为优选技术方案，本申请所述阳离子脂质化合物包括式III、式IV、式V、式VI、式VII、式VIII或式IX所示的化合物。


第二方面，本申请提供一种阳离子脂质化合物的制备方法，采用如下技术方案：
一种阳离子脂质化合物的制备方法，所述阳离子脂质化合物的制备方法包括以下步骤：
以溴代酯类化合物为原料，依次进行烷基化反应、还原反应，
再与酰氯类化合物发生缩合反应，依次进行两步取代反应和一次水解反应，最后与一元仲醇类化合物进行酯化反应，制得所述阳离子脂质化合物。
作为优选技术方案，所述阳离子脂质化合物的制备方法包括以下步骤：
(1)溴代酯类化合物与TosMIC(对甲苯磺酰基异腈)和NaH发生烷基化反应，生成中间产物2；
(2)中间产物2在酸性条件下发生反应，生成中间产物3；
(3)中间产物3与NaBH4发生还原反应，生成中间产物4；
(4)中间产物4与酰氯类化合物发生缩合反应，生成中间产物5；
(5)中间产物5与NaI发生取代反应，生成中间产物6；
(6)中间产物6与N,N,N'-三甲基乙二胺发生取代反应，生成中间产物7；
(7)中间产物7与LiOH发生水解反应，生成中间产物8；
(8)中间产物8与一元仲醇类化合物发生酯化反应，得到所述阳离子脂质化合物。
本申请中，步骤(1)中的原材料为溴代酯类化合物，在实际的生产过程中，可根据合成的具体的阳离子脂质化合物的结构进行具体地选择。
优选地，所述溴代酯类化合物包括8-溴辛酸乙酯、6-溴己酸乙酯或4-溴丁酸乙酯。
优选地，步骤(1)中，TBAI(四丁基碘化铵)作为反应的催化剂。
优选地，步骤(1)中，反应的溶剂包括DMSO(二甲基亚砜)溶液。
优选地，步骤(1)中，反应条件为在室温下进行。
优选地，步骤(2)中，反应的溶剂包括DCM(二氯甲烷)和浓硫酸的混合溶液，二者的体积比为DCM:浓硫酸＝(3.5～8):1。
本申请中，浓硫酸为步骤(2)中反应的进行提供酸性环境。
优选地，步骤(2)中，反应条件为在室温下进行。
在本申请中，中间产物3为对称的酮类化合物。
优选地，步骤(3)中，反应的溶剂包括THF(四氢呋喃)和乙醇的混合溶液，二者的体积比为THF:乙醇＝(2～4):1。
优选地，步骤(3)中，反应条件为在室温下进行。
在本申请中，中间产物4为醇类化合物。
在本申请中，步骤(4)中的酰氯类化合物为含有不同碳原子数的、末端未被卤素取代的一类化合物的统称，在实际的生产过程中，可根据合成的具体的阳离子脂质化合物的结构进行具体地选择。
优选地，所述酰氯类化合物包括3-氯丙酰氯、2-氯乙酰氯、4-氯丁酰氯或5-氯戊酰氯。
优选地，步骤(4)中，反应的溶剂包括含有吡啶的DCM溶液，其中，吡啶的浓度为73～134mM。
优选地，步骤(4)中，反应条件为在室温下进行。
优选地，步骤(5)中，反应的溶剂包括丙酮溶液。
优选地，步骤(5)中，反应条件为在68～72℃下进行，反应的温度例如可以使68℃、70℃、72℃、68～70℃、68～72℃或70～72℃等。
优选地，步骤(6)中，反应的溶剂包括DCM溶液。
优选地，步骤(6)中，反应条件为在室温下进行。
优选地，步骤(7)中，反应的溶剂包括乙醇和水的混合溶液，二者的体积比为乙醇:水＝1:(1.5～3)。
优选地，步骤(7)中，反应条件为在室温下进行。
优选地，步骤(8)中，DMAP(4-二甲氨基吡啶)和DCC(二环己基碳二亚胺)作为反应的催化剂。
优选地，步骤(8)中，反应的溶剂包括DCM溶液。
优选地，步骤(8)中，反应条件为在室温下进行。
在本申请中，在步骤(8)中，所述一元仲醇类化合物为含有不同碳原子数的一元醇，且羟基不在1号位上，在实际的生产过程中，可根据合成的具体的阳离子脂质化合物的结构进行具体地选择。
优选地，所述一元仲醇类化合物包括13-二十五醇、11-二十一醇、9-十七醇、7-十 五醇或9-二十一醇。
第三方面，本申请提供一种LNP载体，采用如下技术方案：
一种LNP载体，所述LNP载体含有第一方面所述的阳离子脂质化合物中的任意一种或至少两种的组合。
本申请中，所述的LNP载体中的阳离子脂质化合物可以为单一的阳离子脂质化合物，也可以为多种阳离子脂质化合物的组合。
优选地，以摩尔分数计，所述阳离子脂质化合物在所述LNP载体中的摩尔分数为15％～70％，例如可以是15％、30％、45％、60％、70％、15％～30％、15％～45％、15％～60％、15％～70％、30％～45％、30％～60％、30％～70％、45％～60％、45％～70％或60％～70％等。该摩尔分数是LNP载体中脂质材料的摩尔比例，不包括所含药物(如核酸药物)。
本申请中，当所述LNP载体中的阳离子脂质化合物为一种时，则其在LNP载体中的摩尔分数为15％～70％(例如，可以是15％、30％、45％、60％、70％、15％～30％、15％～45％、15％～60％、15％～70％、30％～45％、30％～60％、30％～70％、45％～60％、45％～70％或60％～70％等)；当所述LNP载体中的阳离子脂质化合物为多种的组合时，则多种阳离子脂质化合物的组合的总量在LNP载体中的摩尔分数为15％～70％(例如，可以是15％、30％、45％、60％、70％、15％～30％、15％～45％、15％～60％、15％～70％、30％～45％、30％～60％、30％～70％、45％～60％、45％～70％或60％～70％等)。在一些方面，上述的摩尔分数是LNP载体中脂质材料的摩尔比例，不包括所含药物(如核酸药物)。
在一些方面，以摩尔分数计，所述LNP载体还包括磷脂5％～30％(例如可以是5％～20％或8％～30％)和胆固醇15％～65％。该摩尔分数是LNP载体中脂质材料的摩尔比例，不包括所含药物(如核酸药物)。
在一些方面，以摩尔分数计，所述LNP载体还包括磷脂5％～30％(例如可以是5％～20％或8％～30％)、胆固醇15％～65％和聚乙二醇脂质1.5％～3％(例如可以是1％～3％或0.5％～2％)。该摩尔分数是LNP载体中脂质材料的摩尔比例，不包括所含药物(如核酸药物)。
优选地，所述磷脂在所述LNP载体中的摩尔分数为5％～40％，例如可以是8％、10％、15％、20％、25％、30％、5％～10％、8％～10％、5％～15％、8％～15％、5％～20％、8％～20％、5％～25％、8％～25％、5％～30％、8％～30％、10％～15％、10％～20％、10％～25％、10％～30％、15％～20％、15％～25％、15％～30％、20％～25％、20％～30％或25％～30％等。该摩尔分数是LNP载体中脂质材料的摩尔比例，不包括所含药物(如核酸药物)。
优选地，所述胆固醇在所述LNP载体中的摩尔分数为10％～65％，例如可以是10％，15％、30％、45％、65％、15％～30％、15％～45％、15％～65％、30％～45％、30％～65％或45％～65％等。该摩尔分数是LNP载体中脂质材料的摩尔比例，不包括所含药物(如核酸药物)。
在一些方面，以摩尔分数计，所述LNP载体还包括聚乙二醇脂质，其中所述聚乙二醇脂质在所述LNP载体中的摩尔分数为0.5％～3％，例如可以是1.5％、2％、2.5％、3％、0.5％～2％、1％～2％、1.5％～2％、0.5％～2.5％、1％～2％、1.5％～2.5％、0.5％～3％、1％～2％、1.5％～3％、2％～2.5％、1％～3％、2％～3％或2.5％～3％等。该摩尔分数是LNP中脂质材料的摩尔比例，不包括所含药物(如核酸药物)。
在本申请的一些方面，以摩尔分数计，所述LNP载体包括阳离子脂质化合物15％～70％(例如，可以是15％、30％、45％、60％、70％、30％～70％、15％～30％、15％～45％、15％～60％、15％～70％、30％～45％、30％～60％、30％～70％、45％～60％、45％～70％或60％～70％等)、磷脂5％～30％(例如可以是8％、10％、15％、20％、25％、30％、5％～10％、8％～10％、5％～15％、8％～15％、5％～20％、8％～20％、5％～25％、8％～25％、5％～30％、8％～30％、10％～15％、10％～20％、10％～25％、10％～30％、15％～20％、15％～25％、15％～30％、20％～25％、20％～30％或25％～30％等)和胆固醇10％～65％(例如可以是15％、30％、45％、65％、15％～30％、15％～45％、15％～65％、30％～45％、30％～65％或45％～65％等)。在一些方面，以摩尔分数计，所述LNP载体包括阳离子脂质化合物15％～70％(例如，可以是15％、30％、45％、60％、70％、30％～70％、15％～30％、15％～45％、15％～60％、15％～70％、30％～45％、30％～60％、30％～70％、45％～60％、45％～70％或60％～70％等)、磷脂5％～30％(例如可以是8％、10％、15％、20％、25％、30％、5％～10％、8％～10％、5％～15％、8％～15％、5％～20％、8％～20％、5％～25％、8％～25％、5％～30％、8％～30％、10％～15％、10％～20％、10％～25％、10％～30％、15％～20％、15％～25％、15％～30％、20％～25％、20％～30％或25％～30％等)和胆固醇10％～65％(例如可以是15％、30％、45％、65％、15％～30％、15％～45％、15％～65％、30％～45％、30％～65％或45％～65％等)和聚乙二醇脂质0.5％～2％(例如可以是1.5％、2％、2.5％、3％、0.5％～2％、1％～2％、1.5％～2％、0.5％～2.5％、1％～2％、1.5％～2.5％、0.5％～3％、1％～2％、1.5％～3％、2％～2.5％、2％～3％或2.5％～3％等)。该摩尔分数是LNP中脂质材料的摩尔比例，不包括所含药物(如核酸药物)。
本申请中，通过对LNP载体的组分及其各自的添加量进行优化，产生了协同增益的技术效果，核酸药物的装载量更高，LNP的粒径分布更可控，对靶细胞的转染作用更强，从而提升药物的作用效果，降低毒副作用，治疗效果更好。
第四方面，本申请提供了一种药物，采用如下技术方案：
一种药物，所述药物包括第三方面所述的LNP载体。
优选地，所述药物还包括核酸药物。在一些方面，所述药物为基因药物。在一些方面，基因药物包括但不限于质粒DNA(plasmid DNA，pDNA)、反义寡核苷酸(antisense ODN)、小干扰RNA(siRNA)、小发卡RNA(shRNA)和信使RNA(mRNA)。在一些方面，所属药物为mRNA。在一些方面，所属药物为编码SARS-CoV-2病毒刺突糖蛋白(S蛋白)mRNA。
优选地，所述药物中，LNP载体中的阳离子脂质化合物的氮含量与核酸药物中的磷含量的摩尔比(即N/P摩尔比)为(2～15):1，例如可以是2:1、3:1、4:1、5:1、6:1、(2～3):1、(2～4):1、(2～5):1、(2～6):1、(3～4):1、(3～5):1、(3～6):1、(4～5):1、(4～6):1或(5～6):1等。
在一些方面，所述药物中，核酸药物(例如编码SARS-CoV-2病毒刺突糖蛋白的mRNA)占载药LNP的质量分数(载药量)为1％至10％，例如可以是1％～7％，1％～6％，2％～6％，2％～5％，或3％～5％等。
本申请中，阳离子脂质化合物与核酸药物的比例直接影响药物的包封率以及后续的释药效果，在本申请的另一些方面，当LNP载体中的阳离子脂质化合物的氮含量与核酸药物中的磷含量的摩尔比为(2～15):1时，载药结构具有更好的稳定性，在细胞内释放的效率最好。在本申请的一些方面，经试验验证，当LNP载体中的阳离子脂质化合物的氮含量与核酸药物中的磷含量的摩尔比为(2～6):1时，药物的包封率最高，治疗效果最好，并且可以提高原料的利用率，节约生产成本。在本申请的一些方面，当核酸药物在载药LNP中的质量分数(载药量)为3％至10％时，药物的包封率较好，并且可以提高原料的利用率，节约生产成本。
本申请中，所述药物的粒径与粒径分布影响载药能力以及后续的实际应用。在本申请的一些方面，所述药物的平均粒径为30nm～300nm，例如可以是30nm～270nm，30nm～250nm，30nm～200nm，50nm～200nm，50nm～150nm，或60nm～120nm等。在本申请的一些方面，所述药物的polydispersity(PDI)为小于0.15，例如可以是小于0.13，小于0.12，小于0.11，小于0.1，或小于0.09等。在本申请的一些方面，所述药物的polydispersity(PDI)为0.02～0.25，例如可以是0.02～0.20，0.05～0.20，0.05～0.25，0.05～0.1，或0.05～0.25等。在本申请的一些方面，所属药物的PDI小于0.1。
在本申请的一些方面，所述药物包含LNP载体与mRNA(例如编码SARS-CoV-2病毒刺突糖蛋白的mRNA),其中mRNA载药量为2-6％(wt.),其中的LNP载体包括 60mol％～70mol％的TM3脂质化合物，7mol％～12mol％的磷脂(例如HSPC)，20mol％～25mol％的胆固醇，和1mol％～2mol％的DSPE-PEG2000。在一些方面，上述药物的平均粒径在150nm至200nm之间。在一些方面，上述药物的粒径分布PDI小于0.1。在一些方面，上述药物的包封率大于85％。在一些方面，上述药物的N/P值为5.5至6.5。
在本申请的一些方面，所述药物包含LNP载体与核酸药物，其中核酸药物与LNP载体的质量比为5％至7％，其中的LNP载体包括60mol％～70mol％的TM3脂质化合物，10mol％～15mol％的磷脂(例如HSPC)，15mol％～25mol％的胆固醇，和1mol％～2mol％的DSPE-PEG2000。在一些方面，上述药物的平均粒径在50nm至100nm之间。在一些方面，上述药物的PDI小于0.1或小于0.05。在一些方面，上述药物的包封率大于80％或大于90％。
在本申请的一些方面，所述药物包含LNP载体与核酸药物，其中核酸药物与LNP载体的质量比为4％至5％，其中的LNP载体包括60mol％～70mol％的TM3脂质化合物，5mol％～8mol％的磷脂(例如HSPC)，25mol％～35mol％的胆固醇，和1mol％～2mol％的DSPE-PEG2000。在一些方面，上述药物的平均粒径在100nm至150nm之间。在一些方面，上述药物的PDI小于0.1。在一些方面，上述药物的包封率大于80％或大于90％。
在本申请的一些方面，所述药物包含LNP载体与核酸药物，其中核酸药物与LNP载体的质量比为4％至5％，其中的LNP载体包括60mol％～70mol％的TM3脂质化合物，10mol％～15mol％的磷脂(例如HSPC)，15mol％～25mol％的胆固醇，和2mol％～3mol％的DSPE-PEG2000。在一些方面，上述药物的平均粒径在100nm至150nm之间。在一些方面，上述药物的平均粒径在100nm至150nm之间。在一些方面，上述药物的PDI小于0.1。在一些方面，上述药物的包封率大于80％或大于90％。
在本申请的一些方面，所述药物包含LNP载体与核酸药物，其中核酸药物与LNP载体的质量比为2％至3％，其中的LNP载体包括25mol％～35mol％的TM3脂质化合物，5mol％～12mol％的磷脂(例如HSPC)，50mol％～60mol％的胆固醇，和1mol％～2mol％的DSPE-PEG2000。在一些方面，上述药物的平均粒径在50nm至100nm之间。在一些方面，上述药物的PDI小于0.1。在一些方面，上述药物的包封率大于80％或大于90％。
在本申请的一些方面，所述药物包含LNP载体与核酸药物，其中核酸药物与LNP 载体的质量比为5％至6％，其中的LNP载体包含35mol％～45mol％的TM3脂质化合物，12mol％～18mol％的磷脂(例如HSPC)，40mol％～50mol％的胆固醇，和0.5mol％～1mol％的DSPE-PEG2000。在一些方面，上述药物的平均粒径在100nm至150nm之间。在一些方面，上述药物的PDI小于0.1。在一些方面，上述药物的包封率大于80％或大于90％。
在本申请的一些方面，所述药物包含LNP载体与核酸药物(例如编码SARS-CoV-2病毒刺突糖蛋白的mRNA),其中的LNP载体包含60mol％～70mol％的TM6脂质化合物，10mol％～15mol％的磷脂(例如HSPC)，15mol％～25mol％的胆固醇，和2mol％～3mol％的DSPE-PEG2000。在一些方面，上述药物的平均粒径在100nm至150nm之间。在一些方面，上述药物的粒径分布PDI小于0.1。在一些方面，上述药物的包封率大于90％。在一些方面，上述药物的其中N/P值为5.5至6.2。
在本申请的一些方面，所述药物包含LNP载体与核酸药物(例如编码SARS-CoV-2病毒刺突糖蛋白的mRNA),其中N/P值为5.5至6.2，其中的LNP载体包含25mol％～35mol％的TM6脂质化合物，5mol％～15mol％的磷脂(例如HSPC)，55mol％～65mol％的胆固醇，和1mol％～2mol％的DSPE-PEG2000。在一些方面，上述药物的平均粒径在150nm至200nm之间。在一些方面，上述药物的粒径分布PDI小于0.1。在一些方面，上述药物的包封率大于85％。
在本申请的一些方面，所述药物包含LNP载体与核酸药物(例如编码SARS-CoV-2病毒刺突糖蛋白的mRNA),其中N/P值为3.8至4.2，其中的LNP载体包含45mol％～55mol％的TM6脂质化合物，5mol％～15mol％的磷脂(例如HSPC)，35mol％～45mol％的胆固醇，和1mol％～2mol％的DSPE-PEG2000。在一些方面，上述药物的平均粒径在50nm至100nm之间。在一些方面，上述药物的粒径分布PDI小于0.1。在一些方面，上述药物的包封率大于85％。
在本申请的一些方面，所述药物包含LNP载体与核酸药物(例如编码SARS-CoV-2病毒刺突糖蛋白的mRNA),其中N/P值为5.5至6.2，其中的LNP载体包含60mol％～70mol％的TM7脂质化合物，10mol％～15mol％的磷脂(例如HSPC)，15mol％～25mol％的胆固醇，和2mol％～3mol％的DSPE-PEG2000。在一些方面，上述药物的平均粒径在100nm至150nm之间。在一些方面，上述药物的粒径分布PDI小于0.15。在一些方面，上述药物的包封率大于70％。
在本申请的一些方面，所述药物包含LNP载体与核酸药物(例如编码SARS-CoV- 2病毒刺突糖蛋白的mRNA),其中N/P值为5.5至6.2，其中的LNP载体包含25mol％～35mol％的TM7脂质化合物，5mol％～15mol％的磷脂(例如HSPC)，55mol％～65mol％的胆固醇，和1mol％～2mol％的DSPE-PEG2000。在一些方面，上述药物的平均粒径在50nm至1100nm之间。在一些方面，上述药物的粒径分布PDI小于0.1。在一些方面，上述药物的包封率大于95％。
在本申请的一些方面，所述药物包含LNP载体与核酸药物(例如编码SARS-CoV-2病毒刺突糖蛋白的mRNA),其中N/P值为3.8至4.2，其中的LNP载体包含45mol％～55mol％的TM7脂质化合物，5mol％～15mol％的磷脂(例如HSPC)，35mol％～45mol％的胆固醇，和1mol％～2mol％的DSPE-PEG2000。在一些方面，上述药物的平均粒径在100nm至150nm之间。在一些方面，上述药物的粒径分布PDI小于0.1。在一些方面，上述药物的包封率大于90％。
在一些方面，本申请中的药物可以用于治疗或预防某些疾病，比如COVID感染，病毒和病菌感染，肿瘤，代谢疾病，自身免疫疾病，心血管疾病和遗传性疾病。
第五方面，本申请提供上述药物的制备方法，采用如下技术方案：
一种药物的制备方法，所述药物的制备方法包括以下步骤：
称取除阳离子脂质化合物外的其它组分，溶解，制成储备液；
向所述储备液中加入阳离子脂质化合物，混合，得到脂质醇相；
稀释核酸药物，制成核酸水相；
将所述脂质醇相和核酸水相混合，制成药物中间品，透析，得到所述药物。
优选地，磷脂、胆固醇和聚乙二醇脂质溶解时，使用的溶剂包括乙醇(例如无水乙醇)。
优选地，稀释核酸药物时，使用酸性缓冲盐溶液进行稀释，例如使用pH＝4.0的酸性缓冲盐溶液进行稀释。
本申请中，使用透析装置进行透析，以除去溶剂。在一些方面，在透析步骤后，本申请的制备方法还包括将LNP分散在生物相容的注射缓冲液中。。
第六方面，本申请提供一种疫苗，采用如下技术方案：
一种疫苗，所述疫苗包括第三方面所述的LNP载体。
第七方面，本申请提供第一方面所述的阳离子脂质化合物在制备LNP载体、药物或疫苗中的应用，或第三方面所述的LNP载体在制备药物和/或疫苗中的应用。
综上所述，本申请具有以下有益效果：
1.本申请将3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸和二级长链烷基酯相结合获得的两亲性衍生物作为基因药物的载体系统中的阳离子脂质组分，其带有正电荷，从而更容易吸附到细胞表面并进入细胞中，传递药物的效率更高，在一些方面使得使基因转染的效率更高，治疗效果更好；生产原材料易得，制备方法简单，合成效率高，为大规模生产创造了条件；
2.使用上述阳离子脂质化合物制备得到的LNP载体具有良好的载药及传递效率，在一些方面，包封率可达88％以上，与核酸分子制成药物后，可以高效地在体内表达相关的蛋白，从而作为治疗药物发挥治疗疾病的作用，或作为疫苗产生激活免疫的效果，具有广阔的应用前景。在一些方面，使用上述阳离子脂质化合物制备得到的LNP载体可以准确高效地进入细胞中并表达相关的蛋白，发挥治疗的效果。
附图说明
图1是本申请中阳离子脂质化合物TM3的合成路线示意图。
图2是本申请制备例1中中间产物2的氢谱检测结果图片。
图3是本申请制备例1中中间产物3的氢谱检测结果图片。
图4是本申请制备例1中中间产物4的氢谱检测结果图片。
图5是本申请制备例1中中间产物5的氢谱检测结果图片。
图6是本申请制备例1中中间产物7的氢谱检测结果图片。
图7是本申请制备例1中阳离子脂质化合物TM3的氢谱检测结果图片。
图8是本申请实施例1中药物TP1在293T细胞内蛋白表达量的检测结果图片。
图9是本申请实施例2中小鼠血清中抗S蛋白抗体滴度的检测结果图片。
图10是本申请实施例3中的HG-06LNP的冷冻电镜结构图。
图11是本申请实施例3中的HG-23LNP的冷冻电镜结构图。
图12是本申请实施例3中的HG-23LNP在荧光标记后进入细胞在内吞体中聚集的图像。
图13是本申请实施例中阳离子脂质化合物TM6的氢谱检测结果图片。
图14是多个处方的TM6和TM7LNP的细胞转染的结果比较。
图15是多个处方的TM6和TM7LNP对小鼠进行免疫后诱导的抗体水平比较。
具体实施方式
本申请提供了阳离子脂质化合物，所述阳离子脂质化合物包括式I所示的化合物；
式I中，n＝1、2、3或4；
R1和R2各自独立地选自式II所示的二级长链烷基酯；
式II中，n1＝2、4或6；n2＝5、7、9或11；n3＝5、7、9或11；波浪线代表式II与式I中其他部分的连接点。
在本申请的一些方面，n＝1、2或3。
在本申请的一些方面，n＝2。
在本申请的一些方面，R1中的n1与R2中的n1值相同。
具体地，所述阳离子脂质化合物包括式III所示的TM1、式IV所示的TM2、式V所示的TM3、式VI所示的TM4或式VII所示的TM5。
具体地，本申请以TM3为例，说明所述阳离子脂质化合物的制备方法，其合成路线示意图如图1所示，具体流程如下：
(1)8-溴辛酸乙酯在DMSO溶液中，以TBAI作为催化剂，与TosMIC和NaH在室温条件下发生烷基化反应，生成中间产物2：
18～22mmol 8-溴辛酸乙酯溶解于55～65mL无水DMSO中，8～15℃下搅拌5～10min，加入8～12mmol TosMIC，搅拌5～10min，分多次加入23～28mmol NaH，最后再加入1.8～2.2mmol TBAI，缓慢升高至室温，搅拌1～3h。
通过TLC监测反应进度，待反应完全时，将体系在冰水浴中冷却并加入145～155mL冰水淬灭，再用DCM分3次萃取，每次90～110mL。收集所有有机相，使用90～110mL水清洗，再用饱和碳酸氢钠分2次清洗，每次145～155mL，干燥浓缩后过硅胶柱纯 化得到中间产物2。
(2)中间产物2在DCM和浓硫酸的酸性混合溶液(体积比为(3.5～8):1)中，在室温条件反应，生成中间产物3：
称取3.9～4.2g中间产物2，溶解于45～55mL的DCM溶液中，搅拌3～7min。加入7～12mL浓硫酸，室温条件下搅拌2～5h。
通过TLC监测反应进度，待反应完全后，向体系中加入45～55mL水，混匀后静置分层，使用45～55mL DCM萃取水层，合并所有有机相，用45～55mL饱和碳酸氢钠洗脱有机相，干燥浓缩后过硅胶柱纯化，得到中间产物3。
(3)中间产物3在THF和乙醇的混合溶液(体积比为(2～4):1)中，与NaBH4在室温条件下发生还原反应，生成中间产物4：
称取3～5mmol中间产物3溶解于45～55mL THF和乙醇的混合溶液(体积比为(2～4):1)中，0～4℃搅拌3～7min。分多次缓慢加入3～5mmol NaBH4，室温下反应3～5h。
TLC检测反应进度，待反应完全后，向体系中加入90～110mL冰水淬灭，使用DCM分3次萃取，每次90～110mL。合并所有有机相，使用45～55mL饱和碳酸氢钠洗脱有机相，干燥浓缩后过硅胶柱纯化，得到中间产物4。
(4)中间产物4在含有吡啶的DCM溶液(吡啶的浓度为73～134mM)中，与3-氯丙酰氯在室温条件下发生缩合反应，生成中间产物5：
称取4～6mmol中间产物4溶解于45～55mL DCM溶液中，再加入4～6mmol吡啶，在0～4℃下搅拌3～7min，缓慢加入7～9mmol 3-氯丙酰氯，室温下反应0.5～2h。
通过TLC检测反应进度，待反应完全后，向体系中加入90～110mL冰水淬灭，使用90～110mL水清洗，再用饱和碳酸氢钠溶液分2次清洗，每次140～160mL，干燥浓缩后过硅胶柱纯化得到中间产物5。
(5)中间产物5在丙酮溶液中，与NaI在68～72℃条件下发生取代反应，生成中间产物6：
称取3～5mmol中间产物5溶解于45～55mL丙酮溶液中，缓慢加入3～6mmol NaI，升高温度至68～72℃，反应12～18h。
通过TLC检测反应进度，待反应完全后，用45～55mL饱和硫代硫酸钠洗脱有机相，干燥浓缩后过硅胶柱纯化，得到中间产物6。
(6)中间产物6在DCM溶液中，与N,N,N'-三甲基乙二胺在室温条件下发生 取代反应，生成中间产物7：
称取3～5mmol中间产物6溶解于45～55mL DCM溶液中，加入5～7mmol N,N,N'-三甲基乙二胺，室温下搅拌反应1～3h。
通过TLC检测反应进度，待反应完全后，用45～55mL饱和碳酸氢钠洗脱有机相，干燥浓缩后过硅胶柱纯化，得到中间产物7。
(7)中间产物7在乙醇和水的混合溶液(体积比为1:(1.5～3))中，与LiOH在室温条件下发生水解反应，生成中间产物8：
称取4～6mmol中间产物7和18～22mmoL LiOH溶解于90～110mL的乙醇和水的混合溶液(体积比为1:(1.5～3))中，室温条件下搅拌4～7h。
通过TLC检测反应进度，待反应完全后，旋干乙醇后用1.5～2.5M盐酸酸化至pH约为2，使用DCM分3次萃取，每次90～110mL。合并所有有机相，用45～55mL饱和碳酸氢钠洗脱有机相，干燥浓缩后过硅胶柱纯化，得到中间产物8。
(8)中间产物8在DCM溶液中，在DMAP及DCC的作用下，与9-十七醇在室温条件下发生酯化反应，生成所述阳离子脂质化合物TM3：
称取4～6mmol中间产物8、14～16mmol DMAP和16～20mmol 9-十七醇溶解于55～65mL的DCM中，搅拌3～7min，加入13～17mmol DCC，室温条件下搅拌过夜。
通过TLC检测反应进度，待反应完全后，分3次水洗，每次55～65mL。再用饱和碳酸氢钠分2次清洗，每次45～55mL。干燥浓缩后过硅胶柱纯化，得到阳离子脂质化合物TM3。
本申请还提供了一种LNP载体，以摩尔分数计，所述LNP载体包括阳离子脂质化合物15％～70％、磷脂8％～30％、胆固醇15％～65％和聚乙二醇脂质1.5％～3％。
本申请还提供了一种LNP载体，以摩尔分数计，所述LNP载体包括阳离子脂质化合物15％～70％、磷脂8％～40％、和胆固醇10％～65％。
本申请还提供了一种药物，所述药物包括LNP载体和核酸药物，其中，所述药物中，核酸药物的载药量在1.0-10.0％之间。在一些方面，LNP载体中的阳离子脂质化合物的氮含量与核酸药物中的磷含量的摩尔比为2:1到15:1、2:1到10:1、2:1到6:1、或4:1到6:1。
在一些方面，所述药物可以通过如下方法进行制备：
根据处方准确称取脂质，加入乙醇，在磁力搅拌器上，搅拌溶解。mRNA原液加入计算量的pH＝4的柠檬酸盐缓冲液，混合均匀。
用注射器吸取上述脂质乙醇溶液和mRNA-柠檬酸盐缓冲液水溶液，使用LNP混合泵，启动后收集稳定混合阶段的溶液。将上述混合后mRNA-LNP中间品加入透析袋，加入4℃预冷的透析缓冲液，放置于2-8℃冰箱，200rpm搅拌透析过夜。
使用0.22um无菌针式过滤器(Millipore)过滤除菌，放置于无菌无酶离心管。使用Ribogreen检测试剂(thermo)检测过滤后mRNA-LNP的mRNA载量和包封率。使用PCS检测仪检测过滤后mRNA-LNP的zeta平均粒径。
在一些方面，所述药物可以通过如下方法进行制备：
称取1.5～1.8mg磷脂、1.7～1.9mg胆固醇和0.8～1mg聚乙二醇脂质，在55～65℃条件下溶解于0.8～1.5mL无水乙醇中，制成储备液；
在室温条件下向所述储备液中加入13～15mg阳离子脂质化合物，混合，得到脂质醇相；
使用酸性缓冲盐溶液稀释核酸药物至终浓度为0.5～0.7mg/mL，混合均匀，制成核酸水相；
将所述脂质醇相和核酸水相按体积比为1:(2～5)在室温下通过混合器混合，制成药物中间品，通过透析装置进行透析，去除溶剂，并置换为含0.2％～0.5％氯化钠、4％～6％蔗糖的浓度为18～22mM的Tris溶液，得到所述药物。
在一些方面，所述药物可以通过如下方法进行制备：
称取1.5～1.8mg磷脂、1.7～1.9mg胆固醇和0.8～1mg聚乙二醇脂质，在55～65℃条件下溶解于0.8～1.5mL无水乙醇中，制成储备液；
在室温条件下向所述储备液中加入13～15mg阳离子脂质化合物，混合，得到脂质醇相；
使用酸性缓冲盐溶液稀释核酸药物至终浓度为0.05～0.3mg/mL，混合均匀，制成核酸水相；
将所述脂质醇相和核酸水相按体积比为1:(2～5)在室温下通过混合器混合，制成药物中间品，通过透析装置进行透析，去除溶剂，并置换为含0.2％～0.5％氯化钠、4％～6％蔗糖的浓度为18～22mM的Tris溶液，得到所述药物。
列举的实施例
以下列举的实施例代表本发明的一些方面
实施例1.阳离子脂质化合物，其特征在于，所述阳离子脂质化合物包括式I所示的化合物；
式I中，n＝1、2、3或4；
R1和R2各自独立地选自式II所示的二级长链烷基酯；
式II中，n1＝2、4或6；n2＝5、7、9或11；n3＝5、7、9或11。
实施例2.根据实施例1所示的阳离子脂质化合物，其特征在于，所述阳离子脂质化合物包括式III、式IV、式V、式VI或式VII所示的化合物；

实施例3.如实施例1或2所述的阳离子脂质化合物的制备方法，其特征在于，所述阳离子脂质化合物的制备方法包括以下步骤：
以溴代酯类化合物为原料，依次进行烷基化反应、还原反应，
再与酰氯类化合物发生缩合反应，依次进行两步取代反应和一次水解反应，最后与一元仲醇类化合物进行酯化反应，制得所述阳离子脂质化合物。
实施例4.一种LNP载体，其特征在于，所述LNP载体含有实施例1或2所述的阳离子脂质化合物中的任意一种或至少两种的组合。
实施例5.根据实施例4所述的LNP载体，其特征在于，以摩尔分数计，所述LNP 载体包括阳离子脂质化合物15％～70％；
优选的，以摩尔分数计，所述LNP载体还包括磷脂8％～30％、胆固醇5％～65％和聚乙二醇脂质1.5％～3％。
实施例6.一种药物，其特征在于，所述药物包括实施例4或5所述的LNP载体。
实施例7.根据实施例6所述的药物，其特征在于，所述药物还包括核酸药物；
优选地，所述药物中，LNP载体中的阳离子脂质化合物的氮含量与核酸药物中的磷含量的摩尔比为(2～6):1。
实施例8.如实施例6或7所述的药物的制备方法，其特征在于，所述药物的制备方法包括以下步骤：
称取除阳离子脂质化合物外的其它组分，溶解，制成储备液；
向所述储备液中加入阳离子脂质化合物，混合，得到脂质醇相；
稀释核酸药物，制成核酸水相；
将所述脂质醇相和核酸水相混合，制成药物中间品，透析，得到所述药物。
实施例9.一种疫苗，其特征在于，所述疫苗包括实施例4或5所述的LNP载体。
实施例10.如实施例1或2所述的阳离子脂质化合物在制备LNP载体、药物或疫苗中的应用，或实施例4或5所述的LNP载体在制备药物和/或疫苗中的应用。
以下结合附图1～9、制备例1～2和实施例1～2对本申请作进一步详细说明。
制备例
制备例1
本制备例提供一种阳离子脂质化合物TM3，所述阳离子脂质化合物TM3的结构式如式V所示。
所述阳离子脂质化合物TM3通过如下方法进行制备，合成路线图如图1所示：
(1)8-溴辛酸乙酯在DMSO溶液中，以TBAI为催化剂，与TosMIC和NaH在室温条件下发生烷基化反应，生成中间产物2：
在500mL的单口瓶中，称取5g 8-溴辛酸乙酯(20mmol)溶解于60mL无水DMSO中，10℃下搅拌7min，加入1.9g TosMIC(10mmol)，搅拌7min，分多次加入1g NaH(25mmol)，最后再加入0.7g TBAI(2mmol)，由10℃缓慢升高至室温，搅拌2h。
通过TLC监测反应进度，待反应完全时，将体系在冰水浴中冷却并加入150mL冰水淬灭，再用DCM分3次萃取，每次100mL。收集所有有机相，使用100mL水清洗，再用饱和碳酸氢钠分2次清洗，每次150mL，干燥浓缩后过硅胶柱纯化，得到中间产物2。中间产物2的氢谱检测结果如图2所示。
(2)中间产物2在DCM和浓硫酸的酸性混合溶液(体积比为5:1)中，在室温条件反应，生成中间产物3：
在250mL的单口瓶中，称取4.1g中间产物2，溶解于50mL的DCM溶液中，搅拌5min。加入10mL浓硫酸，室温条件下搅拌3h。
通过TLC监测反应进度，待反应完全后，向体系中加入50mL水，混匀后静置分层，使用50mL DCM萃取水层，合并所有有机相，用50mL饱和碳酸氢钠洗脱有机相，干燥浓缩后过硅胶柱纯化，得到中间产物3。中间产物3的氢谱检测结果如图3所示。
(3)中间产物3在THF和乙醇的混合溶液(体积比为3:1)中，与NaBH4在室温条件下发生还原反应，生成中间产物4：
在250mL的单口瓶中，称取1.5g中间产物3(4mmol)溶解于50mL THF和乙醇的混合溶液(体积比为3:1)中，0℃搅拌5min。分多次缓慢加入0.15g NaBH4(4mmol)，室温下反应4h。
TLC检测反应进度，待反应完全后，向体系中加入100mL冰水淬灭，使用DCM分3次萃取，每次100mL。合并所有有机相，使用50mL饱和碳酸氢钠洗脱有机相，干燥浓缩后过硅胶柱纯化，得到中间产物4。中间产物4的氢谱检测结果如图4所示。
(4)中间产物4在含有吡啶的DCM溶液中(吡啶的浓度为100mM)，与3-氯丙酰氯在室温条件下发生缩合反应，生成中间产物5：
在250mL的单口瓶中，称取1.8g中间产物4(5mmol)溶解于50mL DCM溶液中，再加入0.4g吡啶(5mmol)，在0℃下搅拌5min，缓慢加入1g 3-氯丙酰氯(8mmol)，室温下反应1h。
通过TLC检测反应进度，待反应完全后，向体系中加入100mL冰水淬灭，使用100mL水清洗，再用饱和碳酸氢钠溶液分2次清洗，每次150mL，干燥浓缩后过硅胶柱纯化得到中间产物5。中间产物5的氢谱检测结果如图5所示。
(5)中间产物5在丙酮溶液中，与NaI在70℃条件下发生取代反应，生成中间产物6：
在250mL的单口瓶中，称取1.8g中间产物5(4mmol)溶解于50mL丙酮溶液中，缓慢加入0.8g NaI(5mmol)，升高温度至70℃，反应16h。
通过TLC检测反应进度，待反应完全后，用50mL饱和硫代硫酸钠洗脱有机相，干燥浓缩后过硅胶柱纯化，得到中间产物6。
(6)中间产物6在DCM溶液中，与N,N,N'-三甲基乙二胺在室温条件下发生取代反应，生成中间产物7：
在250mL的单口瓶中，称取2.2g中间产物6(4mmol)溶解于50mL DCM溶液中，加入0.6g N,N,N'-三甲基乙二胺(6mmol)，室温下搅拌反应2h。
通过TLC检测反应进度，待反应完全后，用50mL饱和碳酸氢钠洗脱有机相，干燥浓缩后过硅胶柱纯化，得到中间产物7。中间产物7的氢谱检测结果如图6所示。
(7)中间产物7在乙醇和水的混合溶液(体积比为1:2)中，与LiOH在室温条件下发生水解反应，生成中间产物8：
在250mL的单口瓶中，称取3.8g中间产物7(5mmol)和0.5g LiOH(20mmoL)溶解于100mL的乙醇和水的混合溶液(体积比为1:2)中，室温条件下搅拌5h。
通过TLC检测反应进度，待反应完全后，旋干乙醇后用2M盐酸酸化至pH约为2，使用DCM分3次萃取，每次100mL。合并所有有机相，用50mL饱和碳酸氢钠洗脱有机相，干燥浓缩后过硅胶柱纯化，得到中间产物8。
(8)中间产物8在DCM溶液中，在DMAP及DCC的作用下，与9-十七醇在室温条件下发生酯化反应，生成所述阳离子脂质化合物TM3：
在250mL的单口瓶中，称取3.6g中间产物8(5mmol)、1.8g DMAP(15mmol)和4.6g 9-十七醇(18mmol)溶解于60mL的DCM中，搅拌5min，加入3.1g DCC(15mmol)，室温条件下搅拌过夜。
通过TLC检测反应进度，待反应完全后，分3次水洗，每次60mL。再用饱和碳酸氢钠分2次清洗，每次50mL。干燥浓缩后过硅胶柱纯化，得到所述阳离子脂质化合物TM3。TM3的氢谱检测结果如图7所示。
上述反应中，各个步骤获得的产物的质量及得率的统计结果如表1所示。
表1各个步骤获得的产物的质量及得率的统计结果
制备例2
本制备例提供一种药物TP1，其中为核酸药物为编码SARS-CoV-2病毒刺突糖蛋白(S蛋白)mRNA，通过如下方法进行制备：
称取1.64mg HSPC、1.83mg胆固醇和0.89mg DSPE-PEG2000，在60℃条件下溶解于1mL无水乙醇中，制备为储备液。
在室温条件下向储备液中加入13.41mg TM3，混合，得到脂质醇相。
将1.6mL 1mg/mL的mRNA溶解于1.4mL pH 4.0的D-柠檬酸溶液中，混合均匀，制备为核酸水相。
将1mL脂质醇相与3mL核酸水相在室温下通过混合器混合，制备为药物中间品。
将该中间品通过透析装置去除乙醇和柠檬酸，并置换为含0.4％氯化钠、5％蔗糖的浓度为20mM的Tris溶液，制备为药物TP1。
性能测试
对制备例2制备的药物TP1进行性能测试，具体包括粒径检测、分散程度检测和包封率检测。
粒径及分散程度检测
使用激光散射粒度分析仪(PCS，丹东百特BeNano 180 Zeta pro激光粒度仪)测定纳米粒子粒径大小及分散程度(PDI)，使用671nm固体激光器为入射光，动力学光散射试验在25℃进行，散射光路采用173°背散射检测。连续检测3次的平均值作为检测数据。
包封率检测
包封率检测采用荧光定量的检测方法，使用Quant-itTMRiboGreen RNA Assay Kit作为检测试剂盒。加入Triton破乳后释放制剂中核酸，加入特异性核酸染料ribogreen，通过ALLSHENG Feyond-A300酶标仪，设置激发光波长为470nm，发射光波长为525nm，检测样品吸光度值，通过标准曲线计算总核酸含量；进一步，通过不加Triton处理的LNP样品，加入特异性核酸染料ribogreen，通过ALLSHENG Feyond-A300酶标仪，设置激发光波长为470nm，发射光波长为525nm，检测样品吸光度值，通过标准曲线计算游离核酸含量，公式如下：
包封率＝(总核酸含量-游离核酸含量)/总核酸含量×100％。
粒径、分散以及包封率的检测结果如表2所示。
表2 TP1的粒径、分散以及包封率的检测结果
由表2可以看出，按照制备例2的配方和方法制备得到的药物TP1的平均粒径为169nm，PDI为0.07.具有适宜的粒径大小和均一的粒径分布，同时也具有较高的包封率，表明其具有良好的载药能力，为后续的实际应用创造了条件。
制备例3
本制备例提供了七种药物的制备，其中核酸药物为编码荧光素酶的mRNA。具体的脂质处方信息如表3所示。
表3.七种含TM3的LNP药物的脂质处方信息
制备方法：根据以上处方，准确称取脂质，加入无水乙醇，在磁力搅拌器上，搅拌溶解。mRNA原液加入计算量的pH＝4的柠檬酸盐缓冲液，混合均匀。用注射器吸取上述脂质 乙醇溶液和mRNA-柠檬酸盐缓冲液水溶液，使用LNP混合泵，启动后收集稳定混合阶段的溶液。将上述混合后mRNA-LNP中间品加入透析袋，加入4℃预冷的透析缓冲液，放置于2-8℃冰箱，200rpm搅拌透析过夜。使用0.22um无菌针式过滤器(Millipore)过滤除菌，放置于无菌无酶离心管。
使用Ribogreen检测试剂(thermo)检测过滤后mRNA-LNP的mRNA载量和包封率。使用PCS检测仪检测过滤后mRNA-LNP的zeta平均粒径。检测结果如表4所示。
表4七种含TM3的药物的粒径以及包封率的检测结果
冷冻电镜：(取2.5μL LNP溶液滴上grid(Quantifoil Cu R1.2/1.3,300mesh),，用Vitrobot Mark IV(ThermoFisher Scientific)制备冷冻电镜样品。用Talos F200C equipped with a Ceta 4k×4k camera成像。药物HG5-06的冷冻电镜图片如图10所示。药物HG5-023的冷冻电镜图片如图11所示。
制备例4
采用与制备例3相似的方法，本制备例提供了含有TM6和TM7的药物的制备，其中核酸药物为编码SARS-CoV-2病毒刺突糖蛋白(S蛋白)mRNA。TM7的核磁共振图谱如图12所示。
制备所得的LNP的处方信息和粒径和包封率信息如表5所示。
表5含TM6和TM7的药物及粒径等检测结果

实施例1
本实施例对制备例2制备的药物TP1在体外培养细胞内的表达量进行检测，步骤如下：
(1)细胞的培养和收集：
人胚肾细胞株293衍生株293T细胞系由实验室传代获得，采用含10％FBS的RPMI-1640培养基培养，每周传代2～3次。取细胞密度约为80％的细胞进行铺板，细胞计数后使用培养基调整细胞浓度为2×105/mL，在48孔板中，每孔加入0.5mL上述细胞液，即每孔细胞数量约为1×105。每组设置3个复孔，空白对照组也设置3个复孔。
(2)细胞与药物共孵育：
根据mRNA的荧光检测总mRNA浓度，每孔加入含1μg mRNA的药物，分别孵育18、24、48和72h，观察不同孵育时间内蛋白的表达量。
(3)蛋白表达检测：
将细胞培养板在800g下离心5min，取上清，分别用ddH2O稀释10倍、50倍和100倍，制备为待测样品。根据表达的蛋白，以SARS-CoV-2病毒刺突糖蛋白(S蛋白)为标准品绘制标准曲线，采用ELISA方法进行检测。
(4)结果分析：
用待测样品的OD值减去空白对照的OD值，以校准标准曲线的吸光度值。以标准浓度为x轴绘制标准曲线，根据标准曲线计算样本OD值所对应的S蛋白的浓度。
药物TP1在293T细胞内蛋白表达量的检测结果如图8所示。由图8可以看出，药物TP1能够有效地转入293T细胞中并表达S蛋白，并且随着孵育时间的增加，表达量也持续增加，且蛋白的表达量也与剂量呈正相关。上述结果表明LNP载体可以将药物 转入细胞中并释放，从而用于相关疾病的治疗中，具有实际应用的价值。
实施例2
本实施例对制备例2制备的药物TP1在体内的免疫原性进行检测，步骤如下：
取6只6～8周的雌性BALB/c小鼠，分别在第1天和第14天通过肌肉(i.m.)注射药物TP1。在首次给药后第10、21、28和35天用ELISA方法检测小鼠血清中的抗S蛋白抗体滴度。
小鼠血清中抗S蛋白抗体滴度的检测结果如图9所示。由图9可以看出，在首次给药后第10天已有抗体产生，在第14天给予加强针后，第21天抗体表达量有显著增加，并且随着时间的推移持续表达，第35天时仍有表达。上述结果表明，所述药物可以有效激活动物的免疫应答，给药后动物体内的抗体滴度有显著提升，因而可作为疫苗，刺激机体产生相应的免疫应答，效果显著。
实施例3
本实施例对制备例4中的不同处方的TM6药物和TM7药物在细胞和动物模型中的药效进行了考察。
制剂体外细胞转染活性评价：于37℃融化冻存的细胞，并移到5mL培养液中，1000rpm离心5min后弃去上清并用培养液重悬细胞，取10μL用台泮蓝染色后计数/活率并将余下细胞悬液移到细胞培养瓶中，放置于37℃培养箱静置培养。待细胞增长至约90％丰度时，消化离心收集细胞，重悬后计数，调整细胞浓度为2*10^5/ml,48孔板(corning)每孔加入0.5ml细胞悬液。根据具体实验进行设组，每组设置3-4个复孔。每孔加入1ug mRNA对应的mRNA-LNP,给药后吹摇细胞培养板，放回细胞培养相继续培养。细胞培养24小时候，将细胞培养上清液移取到相应EP管中，1300rpm离心5min后用于检测。
报告基因浓度检测：将上述细胞培养收集样品按照一定比例稀释，稀释比例通过预实验确定。稀释后样品采用Elisa kit检测。
附图14对多种脂质处方的TM6和TM7药物的细胞转染效果进行了比较，并与含有TM3的类似脂质处方的药物进行了对照，含有TM6和TM7的药物的转染活性略高于含有TM3的药物。但是HG-23号脂质处方的TM3药物活性也很高。总的来说，TM系列的阳离子构成的LNP，虽然在不同的脂质配比条件下粒径有一定的差别，但是细胞转染活性均表现较好。
我们进一步地对这些药物的小鼠体内免疫效果进行了评价。动物模型为6-8周雌 性Balb/c小鼠，饲养于SPF级动物饲养中心。根据试验方案，i.m.给药，每组n＝6，每只给药量为10ug。免疫二周后，通过麻醉后眼眶采血，收集50ul左右全血于1.5mL EP管中，室温放置0.5h后7000g离心10min,收集按照一定比例稀释后，采用(SARS-Cov2-2Spike RBD Antibody Titer Assay Kit(mouse)SinoBiological)检测OD值。结果见附图15。
根据小鼠实验的结果分析，三种不同结构的脂质及其处方诱导抗COVID S蛋白的抗体的滴度相当，其中TM6的HG-06号处方和TM7的HG-23号处方均表现优异，具有临床开发价值。
实施例4：TMEA为CN 105085292B中公开的阳离子脂质结构，与TM3的亲水和可电离端基相同，都是3-((2-(二甲氨基)乙烷基)(甲基)氨基)丙酸，但疏水结构不同。所以我们对其构建的载mRNA LNP进行了对比。表6比较了三种不同的脂质处方，可以看到，TM3得到的LNP的体内蛋白表达活性都远好于TMEA LNP。
体内蛋白表达实验选用6-8周Balb/c小鼠，每只小鼠静注给药10ug核酸的LNP药物，于给药后6小时取血样，用Elisa试剂盒检测血清中的蛋白含量。
表6.TMEA和TM3脂质的比较研究
本具体实施例仅仅是对本申请的解释，其并不是对本申请的限制，本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改，但只要在本申请的权利要求范围内都受到专利法的保护。

Claims (15)

  1. 阳离子脂质化合物，其特征在于，所述阳离子脂质化合物包括式I所示的化合物；
    式I中，n＝1、2、3或4；
    R1和R2各自独立地选自式II所示的二级长链烷基酯；
    式II中，n1＝2、4或6；n2＝5、7、9或11；n3＝5、7、9或11；波浪线代表式II与式I中其他部分的连接点。
  2. 根据权利要求1中任一项所示的阳离子脂质化合物，其中n＝1、2或3。
  3. 根据权利要求1或2中任一项所示的阳离子脂质化合物，其中n＝2。
  4. 根据权利要求1到3中任一项所示的阳离子脂质化合物，其中R1中的n1与R2中的n1值相同。
  5. 根据权利要求1所示的阳离子脂质化合物，其特征在于，所述阳离子脂质化合物包括式III、式IV、式V、式VI、式VII、式VIII或式IX所示的化合物；


  6. 如权利要求1到5中任一项所述的阳离子脂质化合物的制备方法，其特征在于，所述阳离子脂质化合物的制备方法包括以下步骤：
    以溴代酯类化合物为原料，依次进行烷基化反应、还原反应，
    再与酰氯类化合物发生缩合反应，依次进行两步取代反应和一次水解反应，最后与一元仲醇类化合物进行酯化反应，制得所述阳离子脂质化合物。
  7. 一种LNP载体，其特征在于，所述LNP载体含有权利要求1到5中任一项所述的阳离子脂质化合物中的任意一种或至少两种的组合。
  8. 根据权利要求7所述的LNP载体，其特征在于，以摩尔分数计，所述LNP载体包括阳离子脂质化合物15％～70％；
    优选的，以摩尔分数计，所述LNP载体还包括磷脂5％～40％和胆固醇10％～65％。
  9. 一种药物，其特征在于，所述药物包括权利要求7或8所述的LNP载体。
  10. 根据权利要求9所述的药物，其特征在于，所述药物还包括核酸药物；
    优选地，所述药物中，LNP载体中的阳离子脂质化合物的氮含量与核酸药物中的磷含量的摩尔比为2:1到15:1。
  11. 根据权利要求10所述的药物，其中LNP载体中的阳离子脂质化合物的氮含量与核酸药物中的磷含量的摩尔比为4:1到6:1。
  12. 根据权利要求9所述的药物，特征在于，所述药物还包括核酸药物；
    优选地，所述药物中，核酸药物在含药LNP中的质量分数(载药量)在1.0-15.0％之间。特别优选的，核酸药物的载药量在3.0-8.0wt.％之间。
  13. 如权利要求9到12中任一项所述的药物的制备方法，其特征在于，所述药物 的制备方法包括以下步骤：
    称取除阳离子脂质化合物外的其它组分，溶解，制成储备液；
    向所述储备液中加入阳离子脂质化合物，混合，得到脂质醇相；
    稀释核酸药物，制成核酸水相；
    将所述脂质醇相和核酸水相混合，制成药物中间品，透析，得到所述药物。
  14. 一种疫苗，其特征在于，所述疫苗包括权利要求7或8所述的LNP载体。
  15. 如权利要求1到5中任一项所述的阳离子脂质化合物在制备LNP载体、药物或疫苗中的应用，或权利要求7或8所述的LNP载体在制备药物和/或疫苗中的应用。
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