An ionizable lipid compound, its preparation method and application

Abstract

The present invention provides an ionizable cationic lipid compound, a preparation method and an application thereof. The compound has the structure shown in Formula I; wherein, n is an integer from 1 to 5; R1 is -CH3, -CH2CH3, -CH2CH2CH3, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH2CH2CH2CH2OH or -CH2CH2NHCOCH3; R2 is -H; R3 is X is a heteroatom O, N or S, n1 is selected from integers from 1 to 8, and m1 is selected from integers from 1 to 8; R4, R5, and R6 are each independently a C10-20 alkyl, alkenyl or alkynyl group containing or not containing a heteroatom. The compound provided by the present invention can be better applied to the delivery of bioactive molecules, especially negatively charged nucleic acid molecules, and provides a better choice for the development and application of bioactive molecule delivery and nucleic acid prophylactic and therapeutic agents.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal compounds, and particularly relates to an ionizable lipid compound, a preparation method thereof, and an application thereof. Background Art

[0002] Nucleic acids are important components of living organisms, including DNA types (such as antisense oligonucleotides (ASO), plasmids) and RNA types (small interfering RNA (siRNA), messenger RNA (mRNA), microRNA (miRNA)), and play a key role in life activities. Taking mRNA as an example, it is transcribed from DNA, carries corresponding genetic information, and provides a template for the next step of protein translation. Nucleic acids have the application potential in preventive / therapeutic vaccines, gene therapy, protein replacement therapy, and other genetic disease therapies. Since 2000, significant breakthroughs have been made in the research on the design, modification, and delivery methods of nucleic acid molecules, and siRNA drugs and mRNA vaccines have successively entered clinical applications. However, many problems are faced in nucleic acid therapy applications. For example, mRNA has a single-stranded structure and is very unstable, and will be quickly degraded by nucleases after entering the body. In addition, mRNA has a large molecular weight and carries a large number of negative charges, making it difficult to cross the cell membrane and enter target cells. Therefore, how to effectively deliver nucleic acids to target organs or target cells is the technical key to realizing their in vivo applications. Therefore, more efficient delivery systems need to be developed, especially lipid compounds for non-hepatic targeted delivery, as well as related methods and compositions, to promote the extracellular or intracellular delivery of various therapeutic or preventive agents for therapeutic and / or preventive purposes. Summary of the Invention

[0003] In order to solve the above technical problems, the present invention provides an ionizable lipid compound, a preparation method thereof, and an application thereof. The novel ionizable lipid compound provided by the present invention can be used to deliver bioactive molecules (such as DNA, mRNA, siRNA, miRNA, proteins, polypeptides, etc.), and is particularly suitable for transporting negatively charged nucleic acid molecules, such as DNA, mRNA, siRNA, etc. It provides more choices for the development and application of bioactive molecule delivery and nucleic acid preventive and therapeutic agents.

[0004] The ionizable cationic lipid compound provided in the first aspect of the present invention has the structure shown in Formula I:

[0005]

[0006] Among them, n is an integer from 1 to 5; R1 is -CH3, -CH2CH3, -CH2CH2CH3, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH2CH2CH2CH2OH or -CH2CH2NHCOCH3; R2 is -H; R3 is X is a heteroatom of O, N or S, n1 is selected from integers from 1 to 8, and m1 is selected from integers from 1 to 8; R4, R5, and R6 are each independently a C10-20 alkyl group, alkyl group, alkenyl group or alkynyl group that may or may not contain a heteroatom.

[0007] In a preferred embodiment of the present invention, the R4, R5, and R6 groups are linear or branched C10-20 alkyl groups, linear or branched C10-20 alkenyl groups, linear or branched C10-20 alkynyl groups, and one or more C atoms of the alkyl, alkenyl or alkynyl group are optionally independently replaced by heteroatoms selected from O, S and N, provided that at least one of R3, is When R3, at least two of them are at this time, n1 and m1 in each of the said groups are independent of each other and can be the same or different.

[0008] In a preferred embodiment of the present invention, n is 3, R1 is -CH3, R2 is -H; preferably, the compound of formula I is Among them, the R3 group is Among them, X is a heteroatom of O, N or S, n1 is selected from integers from 1 to 8, m1 is selected from integers from 1 to 8, n1 and m1 are independent of each other and can be the same or different; the R4, R5, and R6 groups are each independently a linear or branched C10-20 alkyl group, linear or branched C10-20 alkenyl group, linear or branched C10-20 alkynyl group, and at least 1 C atom of the alkyl, alkenyl or alkynyl group is optionally independently replaced by a heteroatom selected from O, S or N.

[0009] As a preferred embodiment of the present invention, the compound of formula I is n is 3, R1 is -CH3, R2 is -H; among them, R3 is R4 and R5 are Preferably, X is a heteroatom of O or N; more preferably, n1 is an integer selected from 4 to 8, and m1 is an integer selected from 4 to 8.

[0010] In a preferred embodiment of the present invention, the ionizable cationic lipid compound is selected from the compound of formula A or the compound of formula B:

[0011]

[0012] On each side chain, n2 are each independently selected from integers of 1 to 8, preferably integers of 4 to 8, and m2 are each independently selected from integers of 1 to 8, preferably integers of 4 to 8; preferably, each n2 is selected from integers of 4 to 8, and each m2 is selected from integers of 4 to 8;

[0013]

[0014] On each side chain, n3 are each independently selected from integers of 1 to 8, preferably integers of 4 to 8, and m3 are each independently selected from integers of 1 to 8, preferably integers of 4 to 8; preferably, each n3 is selected from integers of 4 to 8, and each m3 is selected from integers of 4 to 8.

[0015]

[0016] The novel ionizable lipid compound provided by the present invention has -H as R2 while optimizing the other three hydrophobic tail chains. This specific novel compound has a higher cell transfection rate effect than the compounds with the molecular structure of four hydrophobic tail chains existing in the prior art, which may also be related to the different configurations / conformations, etc. of this compound; for example, in the slightly acidic lysosomal microenvironment, the compound containing three hydrophobic tail chains tends to form a conical molecular structure, which can promote the hexagonal transformation of the cell membrane and lysosomal escape. And on this basis, optimizing the X group at the same time can provide a lipid compound and a delivery system for non-hepatic targeting delivery with more excellent specific targeting effect, and deliver nucleic acids to the target organ more efficiently.

[0017] The present invention also provides a synthesis method for these novel ionizable lipid compounds. The ionizable lipid compounds of the present invention can be synthesized by the methods existing in the art. For example, one equivalent or more than one equivalent of amine (the hydrophilic polar head containing an amino group) reacts with three equivalents or more than three equivalents of hydrophobic lipid tail chain compounds under suitable conditions to form. The synthesis of the ionizable lipid compound is carried out with or without a solvent, and the synthesis can be carried out at a relatively high temperature within the range of 25 - 120°C. The obtained ionizable lipid compound can be optionally purified. For example, a mixture of ionizable lipid compounds can be purified to obtain a specific ionizable lipid compound, such as a product containing three hydrophobic lipid tail chains. The hydrophobic lipid tail chain compounds can be commercially purchased or synthesized.

[0018] In some embodiments of the present invention, a preparation method for the ionizable cationic lipid compound is provided, including:

[0019] Compound synthesis route:

[0020]

[0021] (wherein each n4 on each side chain is independently selected from integers of 1 to 8, preferably integers of 4 to 8, and each m4 is independently selected from integers of 1 to 8, preferably integers of 4 to 8; preferably each n4 is selected from integers of 4 to 8, and each m4 is selected from integers of 4 to 8)

[0022] Specifically, it includes the following steps:

[0023] 1) Reduction: In the presence of a reducing agent, the carboxyl group of compound A1 is reduced to a hydroxyl group to obtain compound A2;

[0024] 2) Esterification: In the presence of acryloyl chloride, the hydroxyl group of compound A2 is esterified into an ester group to obtain compound A3;

[0025] 3) Michael addition: Compound A3 and an amine (for example, N,N-bis(3-aminopropyl)methylamine) undergo a Michael addition reaction to obtain the ionizable cationic lipid compound.

[0026] In some embodiments of the present invention, the method for preparing the provided ionizable cationic lipid compound includes:

[0027]

[0028] (wherein each n4 on each side chain is independently selected from integers of 1 to 8, preferably integers of 4 to 8, and each m4 is independently selected from integers of 1 to 8, preferably integers of 4 to 8; preferably each n4 is selected from integers of 4 to 8, and each m4 is selected from integers of 4 to 8)

[0029] 1) Acyl chlorination: In the presence of oxalyl chloride, the carboxyl group of compound B1 is acyl chlorinated to obtain compound B2;

[0030] 2) Substitution: In the presence of ammonium hydroxide, the acyl chloride group of compound B2 is converted into an amide group to obtain compound B3;

[0031] 3) Reduction: In the presence of a reducing agent, the amide of compound B3 is reduced to an amine to obtain compound B4;

[0032] 4) Amidation: In the presence of acryloyl chloride, the amino group of compound B4 is amidated into an amide group to obtain compound B5;

[0033] 3) Michael addition: Compound B5 and an amine undergo a Michael addition reaction to obtain the ionizable cationic lipid compound.

[0034] According to the present invention, examples of the reducing agent include but are not limited to diisobutylaluminum hydride, lithium aluminum hydride, etc. Examples of the solvent used in the reaction include but are not limited to halogenated hydrocarbons (such as chloroform, dichloromethane, dichloroethane, etc.), ethers (such as diethyl ether, tetrahydrofuran, etc.), hydrocarbons (such as n-pentane, benzene, toluene, etc.), and mixed solvents formed by two or more of these solvents. Examples of the solvent used in the esterification reaction include but are not limited to halogenated hydrocarbons (such as chloroform, dichloromethane, dichloroethane, etc.), hydrocarbons (such as n-pentane, benzene, toluene, etc.), nitriles (such as acetonitrile, etc.), and mixed solvents formed by two or more of these solvents. The Michael addition reaction can be carried out with or without a solvent, and examples of the solvent used in the reaction include but are not limited to isopropyl alcohol, tert-butyl alcohol, tetrahydrofuran, etc. The amine can be N,N-bis(3-aminopropyl)methylamine.

[0035] According to the present invention, the raw material A1 in the preparation method can be commercially available or synthesized by a conventional method.

[0036] In the ionizable lipid molecular structure provided by the present invention, there are two adjacent cis double bonds, which enable it to have a higher encapsulation rate and better cell transfection rate when subsequently applied to encapsulate active substances (such as mRNA) in a delivery system; in addition, when preparing lipid nanoparticles, the presence of two adjacent cis double bonds in the tail chain can make the obtained lipid nanoparticles have a more uniform particle size. The ionizable lipid compound of the present invention is particularly suitable for preparing nanoparticles with a solid structure.

[0037] The present invention also provides the application of the described ionizable cationic lipid compound in the preparation of a delivery system for bioactive substances; preferably, the delivery system is a microparticle, nanoparticle, liposome, lipid nanoparticle or microbubble.

[0038] In a preferred embodiment of the present invention, when the ionizable cationic lipid compound is a compound of formula A, the application of the ionizable cationic lipid compound in the preparation of a delivery system for bioactive substances specifically targeting the spleen; when the ionizable cationic lipid compound is a compound of formula B, the application of the ionizable cationic lipid compound in the preparation of a delivery system for bioactive substances specifically targeting the lung;

[0039]

[0040] Wherein n2 on each side chain is independently selected from integers of 1 to 8, preferably integers of 4 to 8, and m2 is independently selected from integers of 1 to 8, preferably integers of 4 to 8; preferably, each n2 is selected from integers of 4 to 8, and each m2 is selected from integers of 4 to 8;

[0041]

[0042] wherein each n3 on each side chain is independently selected from integers of 1 to 8, preferably integers of 4 to 8, and each m3 is independently selected from integers of 1 to 8, preferably integers of 4 to 8; preferably, each n3 is selected from integers of 4 to 8, and each m3 is selected from integers of 4 to 8.

[0043] In a preferred embodiment of the present invention, the delivery system is a lipid nanoparticle.

[0044] In certain embodiments, all the amino groups of the amine react completely with the hydrophobic lipid tail chain compound to form a tertiary amine. In other embodiments, not all the amino groups of the amine react completely with the hydrophobic lipid tail chain compound, thereby generating a primary amine or a secondary amine in the ionizable lipid compound. These primary amines or secondary amines are left as they are or can react with another electrophilic reagent such as a different hydrophobic lipid tail chain compound. As is known to those skilled in the art, reacting an excess of amine with the hydrophobic lipid tail chain compound will produce a variety of different ionizable lipid compounds with various numbers of tails. For example, a diamine or a polyamine can include one, two, three, or four tail chain compounds on various amino moieties of the molecule, thereby generating primary amines, secondary amines, and tertiary amines. In certain embodiments, the same tail chain compound is used; or two tail chain compounds of the same type are used. In other embodiments, two or more different tail chain compounds are used.

[0045] The present invention also provides a bioactive substance delivery system containing the ionizable cationic lipid compound described above. Preferably, the delivery system is a microparticle, nanoparticle, liposome, lipid nanoparticle, or microbubble.

[0046] In one embodiment of the present invention, the delivery system is a lipid nanoparticle. Such lipid nanoparticles can efficiently deliver bioactive substances (such as mRNA) into cells, tissues, or organs, achieving efficient regulation of bioactive substances. In the present invention, the ionizable lipid compound is combined with a bioactive substance (e.g., mRNA) targeted to cells or organs for delivery or further contains other substances (e.g., other anions, cations, or ionizable lipid compounds, synthetic or natural polymers, proteins, phospholipids, cholesterol, carbohydrates, surfactants, etc.) to form microbubbles, liposomes, lipid nanoparticles, nanoparticles, or microparticles. And the bioactive substance can be in the form of a gas, liquid, or solid, and can be a protein, polypeptide, small molecule compound, or nucleotide. In the present invention, the delivery system can then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition.

[0047] The present invention also provides a pharmaceutical composition containing the bioactive substance delivery system described above.

[0048] On the other hand, the present invention also provides a lipid nanoparticle composition, which contains lipid nanoparticles, and the lipid nanoparticles contain the ionizable cationic lipid compound described above.

[0049] According to the present invention, the lipid nanoparticle composition further comprises other lipid molecules. The other lipid molecules may be lipid molecules known or conventionally used in the art for constructing lipid nanoparticles, including but not limited to neutral lipid molecules, lipidoid molecules, cholesterol, and PEGylated lipid molecules.

[0050] According to the present invention, when the lipid nanoparticle composition is used in a drug delivery system, it can encapsulate agents, including nucleotides, small molecule compounds, proteins, polypeptides, metals, etc. The nucleic acids include but are not limited to DNA, antisense nucleic acids (ASO), small interfering RNAs (siRNA), microRNAs (miRNA), small activating RNAs (saRNA), messenger RNAs (mRNA), aptamers, etc. The ionizable lipid compound has several properties suitable for preparing a drug delivery system: 1) the ability to neutralize the charge on negatively charged active substances; 2) the ability to complex lipids and "protect" unstable agents; 3) the ability to buffer the pH value in the body; 4) the ability to act as a "proton sponge" and cause dissolution in the body.

[0051] According to some preferred embodiments of the present invention, in the lipid nanoparticle composition, the lipid nanoparticles contain: 30 - 60 mol% of the ionizable cationic lipid compound of formula I, 5 - 20 mol% of neutral lipid molecules, 30 - 50 mol% of cholesterol lipid molecules, and 0.5 - 5 mol% of PEGylated lipid molecules; preferably contain 30 - 50 mol% of ionizable cationic lipid molecules, 8 - 18 mol% of neutral lipid molecules, 35 - 50 mol% of cholesterol lipid molecules, and 0.5 - 2.5 mol% of PEGylated lipid molecules; more preferably contain 35 - 48 mol% of the ionizable cationic lipid molecules of formula I, 9 - 16 mol% of neutral lipid molecules, 36 - 48 mol% of cholesterol lipid molecules, and 1.2 - 1.8 mol% of PEGylated lipid molecules.

[0052] According to some preferred embodiments of the present invention, the molar percentage of the ionizable lipid molecule of formula I in the lipids of the lipid nanoparticle is 30-60 mol%, for example, it can be 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, 60 mol%.

[0053] According to some preferred embodiments of the present invention, the neutral lipid molecule is an uncharged lipid molecule or a zwitterionic lipid molecule, such as phosphatidylcholine compounds, or / and phosphatidylethanolamine compounds.

[0054] According to some preferred embodiments of the present invention, the neutral lipid molecule is selected from phosphatidylcholine compounds and / or phosphatidylethanolamine compounds.

[0055] According to some preferred embodiments of the present invention, examples of the neutral lipid molecule include but are not limited to dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), lysophosphatidylethanolamine, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), 5-heptadecylbenzene-1,3-diol (resorcinol), dimyristoyl phosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), 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-eicosadienoyl-sn-glycero-3-phosphocholine (DEPC), lysophosphatidylcholine, and combinations thereof.

[0056] In one embodiment, the neutral lipid molecule may be selected from the group consisting of distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylethanolamine (DSPE), and dioleoyl phosphatidylethanolamine (DOPE). In another embodiment, the neutral lipid molecule may be dimyristoyl phosphatidylethanolamine (DMPE). In another embodiment, the neutral lipid molecule may be dimyristoyl phosphatidylcholine (DMPC).

[0057] According to the present invention, the molar percentage of the neutral lipid molecule in the lipids of the lipid nanoparticle is 5-20 mol%, for example, it may be 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%.

[0058] According to the present invention, the cholesterol lipid molecules include steroids, sterols, alkylresorcinols, etc., and examples include but are not limited to cholesterol, cholesterol hemisuccinate, and 5-heptadecylresorcinol.

[0059] According to some preferred embodiments of the present invention, the cholesterol lipid molecules are selected from one or more of cholesterol, cholesterol hemisuccinate, and 5-heptadecylresorcinol.

[0060] In one embodiment, the cholesterol lipid molecule is cholesterol (CHOL). In one embodiment, the cholesterol lipid molecule is cholesterol hemisuccinate.

[0061] According to the present invention, the molar percentage of the cholesterol lipid molecule in the lipids of the lipid nanoparticle is 30-50 mol%, for example, it may be 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%.

[0062] According to some preferred embodiments of the present invention, the PEGylated lipid molecule comprises a lipid moiety and a PEG-based polymer moiety, represented by the number average molecular weight of lipid moiety-PEG. The lipid moiety includes diacylglycerol and / or diacylglycerol amide, preferably selected from one or more of glycerol dilaurate, glycerol dimyristate, glycerol dipalmitate, glycerol dimyristamide, glycerol dipalmitamide, glycerol dilauramide, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; the number average molecular weight of the PEG is 130 to 50,000, preferably 150 to 10,000, more preferably 300 to 3,000, and most preferably 1,500 to 2,500.

[0063] According to the present invention, the PEGylated lipid molecule comprises a lipid moiety and a PEG-based polymer moiety. In some embodiments, the lipid moiety may be derived from diacylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having an alkyl chain length independently comprising from about C4 to about C30 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups such as amide or ester. In some embodiments, the alkyl chain length comprises from about C10 to C20. The dialkylglycerol or dialkylglycamide group may further comprise one or more substituted alkyls. The chain length may be symmetric or asymmetric. Unless otherwise specified, as used in the present invention, the term "PEG" means any polyethylene glycol or other polyalkylene ether polymer. In one embodiment, the PEG moiety is an optionally substituted straight-chain or branched-chain polymer of ethylene glycol or ethylene oxide. In certain embodiments, the PEG moiety may be substituted by, for example, one or more alkyls, alkoxys, acyls, hydroxyls or aryls. In one embodiment, the PEG moiety comprises a PEG copolymer, such as PEG-polyurethane or PEG-polypropylene (see, for example, J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); alternatively, the PEG moiety does not comprise a PEG copolymer, for example, it may be a PEG homopolymer. In one embodiment, the molecular weight of PEG is from about 130 to about 50,000, in a sub-embodiment, from about 150 to about 30,000, in a sub-embodiment, from about 150 to about 20,000, in a sub-embodiment, from about 150 to about 15,000, in a sub-embodiment, from about 150 to about 10,000, in a sub-embodiment, from about 150 to about 6,000, in a sub-embodiment, from about 150 to about 5,000, in a sub-embodiment, from about 150 to about 4,000, in a sub-embodiment, from about 150 to about 3,000, in a sub-embodiment, from about 300 to about 3,000, in a sub-embodiment, from about 1,000 to about 3,000, and in a sub-embodiment, from about 1,500 to about 2,500. In certain embodiments, the PEG is "PEG2000" having an average molecular weight of about 2,000 daltons. In some embodiments of the present invention, PEG in the present invention is represented by the following formula Indicates that for PEG-2000 where n is 45, the number average degree of polymerization includes approximately 45 subunits; other PEG embodiments known in the art can also be used, including those where the number average degree of polymerization includes approximately 23 subunits (n = 23) and / or 68 subunits (n = 68), for example. In some embodiments, n can range from about 30 to about 60. In some embodiments, n can range from about 35 to about 55. In some embodiments, n can range from about 40 to about 50. In some embodiments, n can range from about 42 to about 48. In some embodiments, n can be 45. In some embodiments, R can be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R can be an unsubstituted C1-C30 alkyl, such as a C1-C20 alkyl, a C1-C10 alkyl, or a C1-C6 alkyl. In some embodiments, R can be H, methyl, or ethyl.

[0064] In some embodiments, the PEGylated lipid molecule can be represented as "lipid moiety - PEG - number average molecular weight" or "PEG - number average molecular weight - lipid moiety" or "PEG - lipid moiety". The lipid moiety is a diacylglycerol or a diacylglycerol amide, selected from dilauroyl glycerol, dimyristoyl glycerol, dimyristyl glycerol amide, distearoyl glycerol, dilauryl glycerol amide, distearoyl glycerol amide, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; the number average molecular weight of PEG is from about 130 to about 50,000, for example, from about 150 to about 30,000, from about 150 to about 20,000, from about 150 to about 15,000, from about 150 to about 10,000, from about 150 to about 6,000, from about 150 to about 5,000, from about 150 to about 4,000, from about 150 to about 3,000, from about 300 to about 3,000, from about 1,000 to about 3,000, from about 1,500 to about 2,500, for example, about 2000.

[0065] In some embodiments, the PEGylated lipid molecules may be selected from PEG-dilauroyl glycerol, PEG-dimyristoyl glycerol (PEG-DMG), PEG-dilauryl glycerol amide, PEG-dimyristyl glycerol amide, PEG-distearoyl glycerol (PEG-DSPE) and PEG-distearoyl glycerol amide, PEG-cholesterol (1-[8'-(cholest-5-en-3[β]-yloxy)carbamoyl-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] (DMG-PEG2000), 1,2-distearoyl-sn-glycero-methoxypolyethylene glycol (DSG-PEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), poly(ethylene glycol)-2000-dimethacrylate (DMA-PEG2000) and 1,2-distearoyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (DSA-PEG2000). In one embodiment, the PEGylated lipid molecule may be DMG-PEG2000. In one embodiment, the PEGylated lipid molecule may be C-DMA-PEG2000. In one embodiment, the PEGylated lipid molecule may be DSA-PEG2000. In one embodiment, the PEGylated lipid molecule may be PEG2000-C11. In some embodiments, the PEGylated lipid molecule may be DSG-PEG2000. In one embodiment, the PEGylated lipid molecule may be DSPE-PEG2000. In one embodiment, the PEGylated lipid molecule may be DMA-PEG2000. In some embodiments, the PEGylated lipid molecule may be PEG2000-C14. In some embodiments, the PEGylated lipid molecule may be PEG2000-C16. In some embodiments, the PEGylated lipid molecule may be PEG2000-C18.

[0066] According to the present invention, the molar percentage of the PEGylated lipid molecule in the lipids of the lipid nanoparticle is 0.5-5 mol%, for example, it can be 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.8 mol%, 0.9 mol%, 1.0 mol%, 1.1 mol%, 1.2 mol%, 1.3 mol%, 1.4 mol%, 1.5 mol%, 1.6 mol%, 1.7 mol%, 1.8 mol%, 1.9 mol%, 2.0 mol%, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%, 2.5 mol%, 2.6 mol%, 2.7 mol%, 2.8 mol%, 2.9 mol%, 3.0 mol%, 3.1 mol%, 3.2 mol%, 3.3 mol%, 3.4 mol%, 3.5 mol%, 3.6 mol%, 3.7 mol%, 3.8 mol%, 3.9 mol%, 4.0 mol%, 4.1 mol%, 4.2 mol%, 4.3 mol%, 4.4 mol%, 4.5 mol%, 4.6 mol%, 4.7 mol%, 4.8 mol%, 4.9 mol%, 5.0 mol% and the like.

[0067] In some embodiments of the present invention, the lipid nanoparticle contains an ionizable cationic lipid molecule, a neutral lipid molecule, a cholesterol lipid molecule, and a PEGylated lipid molecule shown in Formula A, wherein:

[0068]

[0069] Formula A, wherein each n2 is independent of each other, can be the same or different, each n2 is selected from an integer of 1-8, each m2 is independent of each other, can be the same or different, each m2 is selected from an integer of 0-8; preferably, each n2 is selected from an integer of 4-8, each m2 is selected from an integer of 4-8; preferably, each n2 is the same as each other, each m2 is the same as each other; the molar percentage of the ionizable cationic lipid molecule shown in Formula A in the lipids of the lipid nanoparticle is 30-50 mol%, preferably 35-48 mol%;

[0070] The neutral lipid molecule is selected from phosphatidylcholine compounds and phosphatidylethanolamine compounds; the molar percentage of the neutral lipid molecule in the lipids of the lipid nanoparticle is 8-20 mol%, preferably 8-18 mol%, more preferably 9-16 mol%;

[0071] The cholesterol lipid molecule is selected from cholesterol and cholesterol hemisuccinate; the molar percentage of the cholesterol lipid molecule in the lipids of the lipid nanoparticle is 30-50 mol%, preferably 35-50 mol%, more preferably 36-48 mol%;

[0072] The PEGylated lipid molecule is represented as "lipid moiety - PEG - number average molecular weight", where the lipid moiety is diacylglycerol or diacylglycerol amide, selected from dilauroyl glycerol, dimyristoyl glycerol, distearoyl glycerol, dilauryl glycerol amide, dimyristyl glycerol amide, distearyl glycerol amide, 1,2 - distearoyl - sn - glycero - 3 - phosphoethanolamine, 1,2 - dimyristoyl - sn - glycero - 3 - phosphoethanolamine; the number average molecular weight of PEG is 130 - 50,000, such as 150 - 30,000, 150 - 20,000, 150 - 15,000, 150 - 10,000, 150 - 6,000, 150 - 5,000, 150 - 4,000, 150 - 3,000, 300 - 3,000, 1,000 - 3,000, 1,500 - 2,500, about 2,000; the molar percentage of the PEGylated lipid molecule in the lipids of the lipid nanoparticles is 0.5 - 5 mol%, preferably 0.5 - 2.5 mol%, more preferably 1.2 - 1.8 mol%.

[0073] In some embodiments of the present invention, the molar ratio of the ionizable cationic lipid molecule, neutral lipid molecule, cholesterol and PEGylated lipid molecule shown in formula A is 35:15:48.5:1.5.

[0074] In some embodiments of the present invention, the molar ratio of the ionizable cationic lipid molecule, neutral lipid molecule, cholesterol and PEGylated lipid molecule shown in formula A is 45:15:38.5:1.5.

[0075] In some embodiments of the present invention, the molar ratio of the ionizable cationic lipid molecule, neutral lipid molecule, cholesterol and PEGylated lipid molecule shown in formula A is 40:10:48.5:1.5.

[0076] In one embodiment of the present invention, the ionizable cationic lipid molecule of formula A is compound N34 - O18 - 2(3T).

[0077] In one embodiment of the present invention, the neutral lipid molecule is DSPC, and the PEGylated lipid molecule is DMG - PEG2000.

[0078] In one embodiment of the present invention, the neutral lipid molecule is DOPE, and the PEGylated lipid molecule is DMG - PEG2000.

[0079] In one embodiment of the present invention, the neutral lipid molecule is DSPC, and the PEGylated lipid molecule is DSPE - PEG2000.

[0080] In one embodiment of the present invention, the neutral lipid molecule is DOPE, and the PEGylated lipid molecule is DSPE-PEG2000.

[0081] The ionizable lipid compound with three hydrophobic tail chains having adjacent cis double bond structures provided by the present invention can provide a higher encapsulation rate of active substances, a better transfection rate in cells or in vivo, is particularly suitable for preparing solid-structured nanoparticles, and in the lipid nanoparticle composition, the molar percentage of the ionizable cationic lipid molecule, neutral lipid molecule, cholesterol lipid molecule, and PEGylated lipid molecule in the lipid nanoparticles of Formula I is most preferably selected. Most importantly, the above lipid nanoparticles have a better function of specifically targeting the spleen and / or lungs.

[0082] The present invention also provides a method for preparing a lipid nanoparticle composition, including: dissolving the above lipid molecules in an organic solvent according to a molar ratio to form a solution of mixed lipids, using the solution of mixed lipids as the organic phase, using an aqueous solution of the substance to be delivered (for example, mRNA) as the aqueous phase, mixing the organic phase and the aqueous phase to prepare lipid nanoparticles. Methods including but not limited to spray drying, solvent extraction, phase separation, nanoprecipitation, single and double emulsion solvent evaporation, microfluidics, simple and complex coacervation, and other methods well-known to those of ordinary skill in the art can be used to prepare lipid nanoparticles.

[0083] In some embodiments, the organic solvent is an alcohol, such as ethanol.

[0084] In some embodiments, the volume ratio of the organic phase to the aqueous phase is (2 - 4):1, such as 3:1.

[0085] In some embodiments, a microfluidic platform is used to prepare the nanoparticles.

[0086] According to the present invention, the preparation method further includes the step of separating and purifying to obtain the lipid nanoparticles.

[0087] According to the present invention, the preparation method further includes the step of lyophilizing the lipid nanoparticles.

[0088] The particle size range of the lipid nanoparticles in the present invention is in the range of 1 nm to 1000 nm.

[0089] The delivery system formed by the ionizable lipid compound of the present invention can also be modified with targeting molecules to make it a targeting agent capable of targeting specific cells, tissues or organs. The targeting molecules can be included throughout the delivery system or can be located only on its surface. The targeting molecules can be proteins, small molecules, nucleic acids, polypeptides, glycoproteins, lipids, etc. Examples thereof include (but are not limited to) antibodies, antibody fragments, low-density lipoproteins (LDLs), sialic acids, aptamers, transferrins, asialycoproteins, receptor ligands, etc.

[0090] The active substance delivered by the delivery system formed by the ionizable lipid compound of the present invention can be a therapeutic agent, a diagnostic agent or a prophylactic agent. The nature of the active substance can be nucleic acids, proteins, polypeptides, small molecule compounds, metals, isotopically labeled compounds, vaccines, etc.

[0091] The delivery system formed by the ionizable lipid compound of the present invention can be combined with one or more pharmaceutical excipients to form a pharmaceutical composition suitable for administration to animals (including humans). The term "pharmaceutical excipient" means any type of non-toxic, inert solid, semi-solid or liquid filler, diluent, etc., including but not limited to cellulose and its derivatives, such as sodium carboxymethyl cellulose and cellulose acetate; sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; gelatin; talc; glycols, such as propylene glycol; esters, such as ethyl oleate and ethyl laurate; oils, such as peanut oil, cottonseed oil, corn oil and soybean oil; surfactants, such as Tween 80; coloring agents, sweeteners, flavoring agents and fragrances, preservatives and antioxidants; buffers, such as phosphate buffer solution, citrate buffer, etc.

[0092] The pharmaceutical composition of the present invention can be administered to humans and / or animals orally, rectally, intravenously, intramuscularly, intranasally, intraperitoneally, vaginally, buccally or in the form of an oral or nasal spray, etc.

[0093] The nucleic acid drug delivery system provided by the present invention can efficiently and specifically deliver nucleic acid drug molecules to the spleen and / or lungs and effectively translate them into target molecules, while reducing the side effects of liposome accumulation in the liver, which is of great significance for the targeted administration, development and application of nucleic acid drugs.

[0094] Term Explanation in the Present Invention: The term "alkyl" refers to a saturated hydrocarbon group obtained by removing a single hydrogen atom from a hydrocarbon moiety containing 1 to 30 carbon atoms. Examples of alkyl include (but are not limited to) methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, n-dodecyl, etc. The term "alkenyl" denotes a monovalent group obtained by removing a single hydrogen atom from a hydrocarbon moiety having at least one carbon-carbon double bond. Alkenyl includes, for example, vinyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, etc. The term "alkynyl" refers to a monovalent group obtained by removing a single hydrogen atom from a hydrocarbon having at least one carbon-carbon triple bond. Representative alkynyls include ethynyl, 2-propynyl (propargyl), 1-propynyl, etc. "And / or" will be regarded as a specific disclosure of each of two designated features or components with or without the other. Thus, the term "and / or" used in a phrase such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). "Comprising" and "including" have the same meaning and are intended to be open and allow, but not require, the inclusion of additional elements or steps. When the term "comprising" or "including" is used herein, the terms "consisting of" and / or "consisting essentially of" are thus also included and disclosed. "About": The term "about" used in conjunction with a numerical value represents an accuracy range familiar and acceptable to those skilled in the art. Generally, such an accuracy range is ±10%. Brief Description of the Drawings

[0095] To more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0096] Figure 1 1H NMR spectrum of compound (9Z,12Z)-9,12-octadecadien-1-ol (linoleyl alcohol, a2) in the embodiment of the present invention;

[0097] Figure 2 1H NMR spectrum of compound (9Z,12Z)-9,12-dienyl octadecane acrylate (a3) in the embodiment of the present invention;

[0098] Figure 3 1H NMR spectrum of compound N34-O18-2(3T) in the embodiment of the present invention;

[0099] Figure 4 Mass spectrum of compound N34-O18-2(3T) in the embodiment of the present invention;

[0100] Figure 5 This is the hydrogen spectrum of compound N34-O18-2(4T) in the embodiments of the present invention;

[0101] Figure 6 This is the mass spectrum of compound N34-O18-2(4T) in the embodiments of the present invention;

[0102] Figure 7 This is the hydrogen spectrum of compound N34-N18-2(3T) in the embodiments of the present invention;

[0103] Figure 8 This is the mass spectrum of compound N34-N18-2(3T) in the embodiments of the present invention;

[0104] Figure 9 This is the hydrogen spectrum of compound N34-N18-2(4T) in the embodiments of the present invention;

[0105] Figure 10 This is the mass spectrum of compound N34-N18-2(4T) in the embodiments of the present invention;

[0106] Figure 11 This is the dissociation constant (pKa) graph of compounds N34-O18-2(3T)(A) and N34-O18-2(4T)(B) in the embodiments of the present invention;

[0107] Figure 12 This is the graph of the expression level of Luciferase protein after 24 hours of transfection of 293T cells with LNP encapsulating Luciferase mRNA (LucRNA) prepared from N34-O18-2(3T) and N34-O18-2(4T) in the embodiments of the present invention;

[0108] Figure 13 This is the graph of the expression level of Luciferase protein after 24 hours of transfection of 293T cells with LNP encapsulating Luciferase DNA (pDNA) prepared from N34-O18-2(3T) in the embodiments of the present invention;

[0109] Figure 14 This is the graph of the expression level of Luciferase protein after 24 hours of transfection of 293T cells with LNP encapsulating Luciferase siRNA (siRNA) prepared from N34-O18-2(3T) in the embodiments of the present invention;

[0110] Figure 15 This is the graph of the expression level of Luciferase protein after 24 hours of transfection of 293T cells with LNP encapsulating Luciferase mRNA (LucRNA) prepared from N34-O18-2(3T) and ALC-0315 in the embodiments of the present invention;

[0111] Figure 16 This is the cytotoxicity graph of N34-O18-2(3T)-LNP and ALC-0315-LNP against 293T cells in the embodiments of the present invention;

[0112] Figure 17 This is the fluorescence expression of each organ in mice after intravenous injection of N34-O18-2(3T)-LucRNA for 6 h in the embodiments of the present invention;

[0113] Figure 18 This is the fluorescence expression of each organ in mice after intravenous injection of N34-N18-2(3T)-LucRNA for 6 h in the embodiments of the present invention. Detailed implementation manners

[0114] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0115] It should be understood that the following embodiments are only used to illustrate and explain the present invention by way of example, 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.

[0116] Unless otherwise specified, the raw materials and reagents used in the following embodiments are all commercially available products, or can be prepared by known methods.

[0117] For those not specified in the embodiments regarding specific technologies or conditions, they are all conventional methods or carried out according to the technologies or conditions described in the literature in this field, or according to the product instructions. For those reagents and instruments not specified regarding the manufacturer, they are all conventional products that can be obtained through regular channels.

[0118] Example 1 Synthesis of ionizable lipids N34-O18-2(3T) and N34-O18-2(4T)

[0119]

[0120] Synthesis of (9Z,12Z)-9,12-octadecadien-1-ol (linoleyl alcohol, a2): At 0 °C, LiAlH4 (7.0 g) and linoleic acid (50 g, a1) were added to 950 mL of tetrahydrofuran. Then the mixture was stirred at 25 °C for 2 h. After the reaction was completed as shown by thin layer chromatography (TLC), water (8.0 mL), aqueous NaOH solution (8.0 mL, 15% by mass), and water (25 mL) were successively added to the reaction solution to quench it. An appropriate amount of Na2SO4 was added and stirred for 15 minutes, then filtered through a Buchner funnel and the filter cake was washed with ethyl acetate. The filtrate was collected and evaporated to concentrate to obtain 51 g of the target product linoleyl alcohol (a2) with a yield of 100%. The 1H NMR spectrum of compound a2 is shown in Figure 1 .

[0121] 1 1H NMR (400 MHz, Chloroform-d) δ 5.47 - 5.26 (m, 4H), 3.64 (t, J = 6.6 Hz, 2H), 2.77 (t, J = 6.5 Hz, 2H), 2.08 - 2.01 (m, 4H), 1.57 (p, J = 6.6 Hz, 2H), 1.39 - 1.25 (m, 16H), 0.89 (t, J = 6.7 Hz, 3H).

[0122] Synthesis of (9Z,12Z)-9,12-dienyl octadecyl acrylate (a3): At 0 °C, (9Z,12Z)-9,12-octadecadien-1-ol (3.2 g) and triethylamine (3.64 g) were added to 30.0 mL of dichloromethane. Subsequently, a solution of acryloyl chloride (1.65 g) in dichloromethane (10.0 mL) was added dropwise to the reaction system, and the reaction solution was stirred at 20 °C for 2 h. The triethylamine salt precipitated in the reaction solution was filtered through a Buchner funnel, and the filtrate was collected. The filtrate was successively washed with water, 5% hydrochloric acid by mass, and water, and the organic phase was dried over magnesium sulfate. Evaporated to dryness, and then the residue was purified by flash column chromatography eluting with EtOAc / petroleum ether (0% - 60%) to obtain 2.7 g of the target product (9Z,12Z)-9,12-dienyl octadecyl acrylate with a yield of 70%. The 1H NMR spectrum of compound a3 is shown in Figure 2 .

[0123] 11H NMR (400 MHz, Chloroform-d) δ 6.40 (dd, J = 17.3, 1.5 Hz, 1H), 6.12 (dd, J = 17.4, 10.4 Hz, 1H), 5.81 (dd, J = 10.4, 1.5 Hz, 1H), 5.32 - 5.40 (m, 4H), 4.15 (t, J = 6.7 Hz, 2H), 2.77 (t, J = 6.5 Hz, 2H), 2.05 (q, J = 6.9 Hz, 4H), 1.67 (p, J = 6.8 Hz, 2H), 1.38 - 1.27 (m, 16H), 0.89 (t, J = 6.7 Hz, 3H).

[0124] Synthesis of N34 - O18 - 2(3T) and N34 - O18 - 2(4T): At room temperature, (9Z,12Z)-9,12-dienoctadecyl acrylate (2.5 g) was added to a solution of 320 mg of N,N-bis(3-aminopropyl)methylamine. Then the mixture was heated to 120 °C and stirred continuously for 48 h. When thin-layer chromatography detected the end of the reaction, 2.65 g of the crude product was obtained. Then the target products were purified by flash column chromatography eluting with dichloromethane / methanol to obtain 45 mg of N34 - O18 - 2(3T) and 66 mg of N34 - O18 - 2(4T). The 1H NMR spectrum of compound N34 - O18 - 2(3T) is shown in Figure 3 , and the mass spectrum is shown in Figure 4 , and the 1H NMR spectrum of compound N34 - O18 - 2(4T) is shown in Figure 5 , and the mass spectrum is shown in Figure 6 .

[0125] N34 - O18 - 2(3T):

[0126] 1 1H NMR (400 MHz, Chloroform-d) δ 5.45 - 5.26 (m, 12H), 4.12 - 3.98 (m, 6H), 2.89 (t, J = 6.5 Hz, 2H), 2.79 - 2.74 (m, 10H), 2.68 (s, 2H), 2.54 (s, 2H), 2.47 - 2.28 (m, 10H), 2.21 (s, 3H), 2.05 (q, J = 6.8 Hz, 12H), 1.74 - 1.54 (m, 10H), 1.40 - 1.25 (m, 48H), 0.89 (t, J = 6.7 Hz, 9H).

[0127] MALDI-TOF MS: m / z 1107.030 [M + H] + .

[0128] N34 - O18 - 2(4T):

[0129] 1 1H NMR (400 MHz, Chloroform-d) δ 5.42 - 5.29 (m, 16H), 4.04 (t, J = 6.8 Hz, 8H), 2.76 (q, J = 7.5, 7.0 Hz, 16H), 2.44 - 2.41 (m, J = 7.1 Hz, 12H), 2.29–2.16 (m, 5H), 2.05 (q, J = 6.8 Hz, 16H), 1.63 - 1.60 (m, 12H), 1.38 - 1.26 (m, 66H), 0.89 (t, J = 6.7 Hz, 12H).

[0130] MALDI - TOFMS: m / z 1427.310 [M + H] + .

[0131] Synthesis of Ionizable Lipids N34 - N18 - 2(3T) and N34 - N18 - 2(4T) in Example 2

[0132]

[0133] Synthesis of N34 - N18 - 2(3T) and N34 - N18 - 2(4T): At room temperature, (9Z,12Z) - 9,12 - dioctadeceneacrylamide (3.08 g) was added to a solution of 400 mg of N,N - bis(3 - aminopropyl)methylamine. Then the mixture was heated to 120 °C and stirred for 48 h. When thin - layer chromatography detected the end of the reaction, 3.10 g of the crude product was obtained. Then the target products were purified by flash column chromatography eluting with dichloromethane / methanol to obtain 100 mg of N34 - N18 - 2(3T) and 120 mg of N34 - N18 - 2(4T). The 1H NMR spectrum of compound N34 - N18 - 2(3T) is shown in Figure 7 , and the mass spectrum is shown in Figure 8 ; the 1H NMR spectrum of compound N34 - N18 - 2(4T) is shown in Figure 9 , and the mass spectrum is shown in Figure 10 .

[0134] N34 - N18 - 2(3T):

[0135] 11H NMR (400 MHz, Chloroform-d) δ 7.11 (br, 3H), 5.50 - 5.23 (m, 12H), 3.28 - 3.14 (m, 6H), 2.99 (s, 3H), 2.77 (t, J = 6.5 Hz, 6H), 2.70 (t, J = 6.3 Hz, 4H), 2.50 (s, 5H), 2.37 (d, J = 6.4 Hz, 6H), 2.24 (s, 3H), 2.05 (q, J = 7.4, 6.7 Hz, 12H), 1.79 (s, 2H), 1.63 - 1.40 (m, 10H), 1.36 - 1.24 (m, 48H), 0.92 - 0.86 (m, 9H).

[0136] MALDI - TOFMS: m / z 1104.177 [M + H] + .

[0137] N34 - N18 - 2(4T):

[0138] 1 1H NMR (400 MHz, Chloroform-d) δ 7.14 (br, 3H), 5.40 - 5.29 (m, 16H), 3.18 (q, J = 6.8 Hz, 8H), 2.77 (t, J = 6.5 Hz, 8H), 2.68 (t, J = 6.1 Hz, 8H), 2.47 (t, J = 6.5 Hz, 4H), 2.35 (t, J = 6.0 Hz, 8H), 2.05 (q, J = 7.0 Hz, 16H), 1.70 (s, 3H), 1.49 (q, J = 7.2 Hz, 8H), 1.37 - 1.25 (m, 72H), 0.89 (t, J = 6.7 Hz, 12H).

[0139] MALDI - TOFMS: m / z 1423.553 [M + H] + .

[0140] Dissociation constants (pKa) of ionizable lipids N34 - O18 - 2(3T), N34 - O18 - 2(4T)

[0141] Ionizable lipids have two main functions: binding nucleic acids and allowing nucleic acid molecules to be released in cells. The pKa of the lipid is an important factor because the lipid needs to be positively charged at low pH to bind to nucleic acids, but uncharged at neutral pH so that the LNP does not cause toxicity. As Figure 11, the pKa of ionizable lipid N34-O18-2(3T) was determined to be 6.72 (A) by TNS dye binding assay, and the pKa of N34-O18-2(4T) was 5.92 (B). It can be seen that both molecules are capable of being positively charged under acidic conditions and carrying RNA, and uncharged at neutral pH (pH = 7.4).

[0142] Example 4 Preparation of lipid nanoparticles by encapsulating mRNA with N34-O18-2(3T) and N34-O18-2(4T)

[0143] Ionizable lipids N34-O18-2(3T) or N34-O18-2(4T), DSPC, Cholesterol, and DMG-PEG2000 were respectively configured into an ethanol solution as the organic phase according to a molar ratio of 45%:15%:38.5%:1.5%. Luciferase mRNA (LucRNA) was dissolved in an aqueous solution with pH = 4 as the aqueous phase. According to a volume ratio of the aqueous phase to the organic phase of 3:1, a nanoparticle suspension was prepared using microfluidic technology on a nano-drug manufacturing instrument (Mai'anna). After preparation, it was ultrafiltered and concentrated to obtain the final LucRNA-LNP lipid nanoparticles, which were stored at 2-8 °C for later use.

[0144] The particle size and Zeta potential of LucRNA-LNP were characterized using a Zetasizer Pro nanoparticle size and potential analyzer (Malvern Panalytical). The encapsulation efficiency of LucRNA-LNP was detected by the Ribogreen method using an F-280 fluorescence spectrophotometer (Tianjin Gangdong). The detection results of Example 2 are shown in Table 1.

[0145] Table 1 Detection results

[0146]

[0147] From the results of Example 4, it can be seen that the particle size of the lipid nanoparticles LucRNA-LNP prepared from the novel lipid compound N34-O18-2(3T) is about 120 nm, the particle size distribution of LucRNA-LNP is narrow (small PDI), and the encapsulation efficiency is as high as 98%.

[0148] In addition, a multifunctional microplate reader (BioTek, model SLXFATS) was used to detect the transfection efficiency of the prepared LucRNA-LNP cells by the luciferase reporter gene method. The method for in vitro transcription of LucRNA is as follows: 293T cells were plated at a cell density of 1×10 4Cells / well, and transfection was performed when the cell confluence was 30%-50%. 1.0 μg of LucRNA was transfected using the transfection reagent Lipofectamine 2000 (ThermoFisher Scientific), and the transfection operation was carried out according to the product instruction manual of the transfection reagent. After 24 h of transfection, a multifunctional microplate reader was used to detect the protein expression level. The negative control was the cell culture medium without LucRNA-LNP. The in vitro cell transfection efficiency was as Figure 12 shown, indicating that the LNP encapsulating mRNA prepared from the ionizable lipid N34-O18-2(3T) has extremely high cell transfection efficiency. Compared with the traditional ionizable lipid N34-O18-2(4T) with four hydrophobic tail chains, the cell transfection efficiency of the LNP encapsulating mRNA prepared by it is much lower, and there is about an order of magnitude decrease in transfection efficiency. It can be seen that compared with the traditional ionizable lipid N34-O18-2(4T) with four hydrophobic tail chains, the ionizable lipid N34-O18-2(3T) with three hydrophobic tail chains has higher cell transfection efficiency, and the advantage is very obvious.

[0149] From the results of Example 4, it can also be seen that the lipid nanoparticle LucRNA-LNP prepared from the novel lipid compound N34-O18-2(3T) has good physicochemical characteristics, and the in vitro cell transfection efficiency is about 3-5 times higher than that of the commercial Lipofectamine 2000.

[0150] Example 5 Preparation of lipid nanoparticles by encapsulating DNA with N34-O18-2(3T)

[0151] The ionizable lipid N34-O18-2(3T), DSPC, Cholesterol, and DMG-PEG2000 were respectively configured into an ethanol solution as the organic phase according to the molar ratio of 45%:15%:38.5%:1.5%. Luciferase DNA (pDNA) was dissolved in an aqueous solution with pH = 4 as the aqueous phase. According to the volume ratio of the aqueous phase to the organic phase of 3:1, a nanoparticle suspension was prepared by microfluidic technology on a nano drug manufacturing instrument (PNI, Canada, Ignite model). After preparation, it was ultrafiltered and concentrated to obtain the final pDNA-LNP lipid nanoparticles, which were stored at 2-8 °C for later use.

[0152] The particle size and Zeta potential of pDNA-LNP were characterized using a Zetasizer Pro nanoparticle size and zeta potential analyzer (Malvern Panalytical). The detection results of Example 5 are shown in Table 2. The particle size of the lipid nanoparticle pDNA-LNP prepared by the novel lipid compound formulation is about 303 nm, and the particle size distribution of pDNA-LNP is narrow (small PDI).

[0153] Table 2 Detection Results

[0154]

[0155] The transfection efficiency of the prepared pDNA-LNP 293T cells was detected by the luciferase reporter gene method using a multifunctional microplate reader (BioTek, model SLXFATS). The method of in vitro transcription is as follows: 293T cells were plated at a cell density of 1×10 4 cells / well, and transfection was performed when the cell confluence was 30%-50%. 2 μg of pDNA was transfected using the transfection reagent Lipofectamine 2000 (ThermoFisher Scientific), and the transfection operation was carried out according to the product instruction manual of the transfection reagent. The protein expression level was detected using a multifunctional microplate reader 24 h after transfection. The negative control was the cell culture medium without pDNA-LNP. The in vitro cell transfection efficiency is as Figure 13 shown, indicating that the LNP encapsulating DNA prepared from the ionizable lipid N34-O18-2(3T) has a high cell transfection efficiency.

[0156] It can be seen from the results of Example 5 that the lipid nanoparticles pDNA-LNP prepared from the novel lipid compound have good physicochemical characteristics and a very high in vitro cell transfection efficiency.

[0157] Example 6 Preparation of Lipid Nanoparticles by Encapsulating siRNA with N34-O18-2(3T)

[0158] An ethanol solution was prepared as the organic phase with the ionizable lipid N34-O18-2(3T), DSPC, Cholesterol, and DMG-PEG2000 at a molar ratio of 45%:15%:38.5%:1.5% respectively, and Luciferase siRNA (siRNA) was dissolved in an aqueous solution with pH = 4 as the aqueous phase. According to the volume ratio of the aqueous phase to the organic phase of 3:1, a nanoparticle suspension was prepared by microfluidic technology on a nano drug manufacturing instrument (PNI, Canada, Ignite model). After preparation, it was ultrafiltered and concentrated to obtain the final siRNA-LNP lipid nanoparticles, which were stored at 2-8 °C for later use.

[0159] The particle size and Zeta potential of siRNA-LNP were characterized using a ZetasizerPro nanoparticle size and zeta potential analyzer (Malvern Panalytical). The detection results of Example 6 are shown in Table 3. The particle size of the lipid nanoparticles siRNA-LNP prepared by the combination of the novel lipid compound is about 225 nm.

[0160] Table 3 Detection Results

[0161]

[0162] The transfection efficiency of the prepared siRNA-LNP in 293T cells was detected by the fluorescence reporter gene method using a multifunctional microplate reader (BioTek, model SLXFATS). The in vitro transcription method is as follows: 293T cells stably transfected with Luciferase reporter were plated at a cell density of 1×10 4 cells / well, and transfection was performed when the cell confluence reached 30%-50%. siRNA was transfected using the transfection reagent Lipofectamine 2000 (ThermoFisher Scientific), and the transfection operation was carried out according to the product instruction manual of the transfection reagent. The protein expression level was detected using a multifunctional microplate reader 24 hours after transfection. The negative control was the cell culture medium without siRNA-LNP. The in vitro cell transfection efficiency is as Figure 14 shown, indicating that the LNP encapsulating siRNA prepared from the ionizable lipid N34-O18-2(3T) has extremely high protein knockdown efficiency.

[0163] As can be seen from the results of Example 6, the particle size of the lipid nanoparticle siRNA-LNP prepared from the novel lipid compound is about 225 nm. The in vitro cell transfection and knockdown efficiency are higher than those of the commercial Lipofectamine 2000.

[0164] Example 7 Comparison of the effects of N34-O18-2(3T) and the commercial ionizable cationic lipid molecule ALC-0315

[0165] The molecular formula of ALC-0315 is: ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate).

[0166] The structural formula of ALC-0315 is:

[0167]

[0168] According to the method described in Example 4, lipid nanoparticles were prepared using N34-O18-2(3T) and ALC-0315 respectively, and the specific molar ratios were: N34-O18-2(3T):DSPC:Cholesterol:DMG-PEG2000 = 45:15:38.5:1.5; ALC-0315:DSPC:Cholesterol:DMG-PEG2000 = 45:15:38.5:1.5.

[0169] The physicochemical quality control data of the prepared lipid nanoparticles are shown in the following table (Table 4):

[0170] Table 4 Physicochemical quality control data of lipid nanoparticles

[0171]

[0172] As can be seen from the above table, the encapsulation efficiency of the lipid nanoparticles prepared by N34-O18-2(3T) is as high as 98.7%, which is higher than that of the lipid nanoparticles of ALC-0315.

[0173] Using the same transfection method as in Example 4, the prepared lipid nanoparticles were transfected into cells to understand the protein expression. The results are as Figure 15 shown. After the lipid nanoparticles prepared by N34-O18-2(3T) carried mRNA and transfected the cells, the protein expression level in the cells was higher than that of Lipofectamine2000, and the protein expression level in the cells transfected with the corresponding mRNA concentration was also higher than that of ALC-0315, indicating that the cell transfection efficiency of the lipid nanoparticles prepared by N34-O18-2(3T) is very high.

[0174] In addition, the MTT method was used to determine the cytotoxicity of N34-O18-2(3T)-LNP and ALC-0315-LNP, and the effects of factors such as vector dose and action time on the proliferation of normal cells (such as 293T) were investigated. The results are as Figure 16 shown. After the lipid nanoparticles prepared by N34-O18-2(3T) carried mRNA and transfected the cells for 48 hours, good cell viability was still maintained at a relatively high dose (2 μg / mL), indicating that the cytotoxicity of the lipid nanoparticles prepared by N34-O18-2(3T) is very low.

[0175] It can be seen from the results of Example 7 that the lipid nanoparticles prepared from the novel lipid compound have low cytotoxicity and very high mRNA transfection efficiency.

[0176] Example 8 Transfection experiments of N34-O18-2(3T)-LNP and N34-N18-2(3T)-LNP lipid nanoparticles in animals

[0177] By means of tail vein injection of nano-lipid particles in mice, lipid nanoparticles were prepared using N34-O18-2(3T) or N34-N18-2(3T) according to the method described in Example 3. The specific molar ratio was: N34-O18-2(3T):DSPC:Cholesterol:DMG-PEG2000 = 45:15:38.5:1.5; the N / P ratio was 10:1, N34-N18-2(3T):DSPC:Cholesterol:DMG-PEG2000 = 45:15:38.5:1.5; the N / P ratio was 10:1. Among them, the mRNA was the mRNA expressing Luciferase fluorescent protein, the dosage of mRNA was 10 μg, and the total amount of N34-O18-2(3T) or N34-N18-2(3T), DSPC, Cholesterol, and DMG-PEG2000 was 100 μg. A 200 μL neutral PBS buffer solution was used to rapidly convert the liposome environment and was quickly injected into 6-8 week-old female C57 mice through the tail vein, controlling the intravenous injection of 10 μg mRNA.

[0178] The fluorescence expression in each organ of mice after tail vein injection of N34-O18-2(3T)-LNP, PBS (blank control) for 6 h is shown in Figure 17 . The results showed that after intravenous injection (IV) of the lipid nanoparticles of N34-O18-2(3T)-LNP, the fluorescence expression levels in each organ of the mice were mainly distributed in the spleen at about 100%, the heart at 0%, the liver at 0%, the lungs at 0%, and the kidneys at 0%. It can be seen that it can specifically target the spleen. The fluorescence expression in each organ of mice after tail vein injection of N34-N18-2(3T)-LNP, PBS (blank control) for 6 h is shown in Figure 18 . The results showed that after intravenous injection (IV) of the lipid nanoparticles of N34-N18-2(3T)-LNP, the fluorescence expression levels in each organ of the mice were mainly distributed in the spleen at about 0%, the heart at 0%, the liver at 0%, the lungs at 100%, and the kidneys at 0%. It can be seen that it can specifically target the lungs. It can be seen that the present invention is based on a compound formed by three lipid hydrophobic tail chains, and further by adjusting the X heteroatom in the lipid hydrophobic tail chain to "O" or "N", the specific targeting of different organs is better achieved.

[0179] 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 foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of an ionizable cationic lipid compound in the preparation of a bioactive substance delivery system targeting the spleen, wherein the ionizable cationic lipid compound has the structure shown in Formula I: ； Among them, n is an integer from 1 to 5; R1 is CH3, CH2CH3, CH2CH2CH3, CH2OH, CH2CH2OH, CH2CH2CH2OH, CH2CH2CH2CH2OH or CH2CH2NHCOCH3; R2 is H; X is O, R3 both are , n1 is selected from integers from 1 to 8, and m1 is selected from integers from 1 to 8.

2. Use of an ionizable cationic lipid compound in the preparation of a bioactive substance delivery system targeting the lung, wherein the ionizable cationic lipid compound has the structure shown in Formula I: ； Among them, n is an integer from 1 to 5; R1 is -CH3, -CH2CH3, -CH2CH2CH3, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH2CH2CH2CH2OH or -CH2CH2NHCOCH3; R2 is -H; X is N, R3 both are , n1 is selected from integers from 1 to 8, and m1 is selected from integers from 1 to 8.

3. The application according to claim 1 or 2, characterized in that, n is 3, R1 is CH3, and R2 is H.

4. The application according to claim 1 or 2, characterized in that, n1 is an integer selected from 4 to 8, and m1 is an integer selected from 4 to 8.

5. The application according to claim 1 or 2, characterized in that, The delivery system is a microparticle.

6. The application according to claim 1 or 2, characterized in that, The delivery system is a lipid nanoparticle.

7. The application according to claim 1 or 2, characterized in that, The delivery system is a nanoparticle.

8. The application according to claim 1 or 2, characterized in that, The delivery system is a microbubble.

9. The application according to claim 1 or 2, characterized in that The delivery system is a liposome.

10. The application according to claim 1 or 2, characterized in that, The bioactive substance is selected from DNA, mRNA, siRNA, miRNA.

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