CN115745788B - An ionizable lipid compound and its preparation method and application - Google Patents

Abstract

The present invention provides an ionizable cationic lipid compound and a preparation method and application _thereof . The compound has a structure as _shown in formula I; wherein n is an integer of _{1 to} 5 _{; R1} is _-CH3 , -CH2CH3, _-CH2CH2CH3 , _-CH2OH , _{-CH2CH2OH , -CH2CH2CH2OH , -CH2CH2CH2OH , -CH2CH2CH2CH2OH} or _{-CH2CH2NHCOCH3 ; R2} is _{-H ; R3} is X is an O, N or S heteroatom, n1 is selected from an integer of 1 to 8, m1 is selected from an integer of 1 to 8; R ₄ , R ₅ , and R ₆ are each independently a C10-20 alkyl, alkenyl or alkynyl group containing or not containing heteroatoms. The compounds provided by the present invention can be better applied to the delivery of bioactive molecules, especially nucleic acid molecules with negative charges, and provide a better choice for the development and application of bioactive molecule delivery and nucleic acid preventive and therapeutic agents.

Description

Ionizable lipid compound, and preparation method and application thereof

Technical Field

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

Background

Nucleic acids are important components of life bodies, including DNA (such as antisense oligonucleotides (ASO), plasmids) and RNA (small interfering RNA (siRNA), messenger RNA (mRNA), microRNA (miRNA), and play a key role in life activities, taking mRNA as an example, which is transcribed from DNA and carries corresponding genetic information to provide templates for the next translation into protein, nucleic acids have the application potential of preventing/treating vaccines, gene therapies, protein substitution therapies and other genetic disease therapies, since 2000, the design, modification and delivery methods of nucleic acid molecules have been greatly developed, siRNA drugs and mRNA vaccines have been brought into clinical use successively, in nucleic acid therapeutic applications, problems are faced, such as mRNA, which is very unstable in single-stranded structure and is degraded by nucleases soon after entry into the body, mRNA has a large molecular weight and has a large negative charge making it difficult to penetrate the cell membrane into target cells, so how to efficiently deliver nucleic acid to a target organ or target cell is a technical key to achieve its in vivo application, there is a need to develop more efficient delivery systems, particularly lipid compounds for non-liver targeted delivery, and related methods and compositions to facilitate extracellular or intracellular delivery of various therapeutic or prophylactic agents for therapeutic and/or prophylactic purposes.

Disclosure of Invention

In order to solve the technical problems, the invention provides an ionizable lipid compound, a preparation method and application thereof. The present invention provides novel ionizable lipid compounds that are useful for delivering biologically active molecules (e.g., DNA, mRNA, siRNA, miRNA, proteins, polypeptides, etc.), particularly useful for transporting nucleic acid molecules having a negative charge, such as DNA, mRNA, siRNA, etc. Providing more options for delivery of bioactive molecules and development and use of nucleic acid prophylactic and therapeutic agents.

The ionizable cationic lipid compound provided by the first aspect of the invention has a structure shown in a formula I:

Wherein n is an integer of 1 to 5, R ₁ is -CH₃,-CH₂CH₃,-CH₂CH₂CH₃,-CH₂OH, -CH₂CH₂OH,-CH₂CH₂CH₂OH,-CH₂CH₂CH₂CH₂OH or-CH ₂CH₂NHCOCH₃;R₂ is-H, R ₃ is X is O, N or S heteroatom, N1 is selected from integers of 1-8, m1 is selected from integers of 1-8, R ₄、R₅、R₆ is independently C10-20 alkyl, alkenyl or alkynyl containing or not containing heteroatom.

In a preferred embodiment of the invention, the R ₄、R₅、R₆ group is a linear or branched C10-20 alkyl, linear or branched C10-20 alkenyl, linear or branched C10-20 alkynyl, 1 or more C atoms of said alkyl, alkenyl or alkynyl being optionally replaced by heteroatoms independently selected from O, S and N, provided that R ₃,At least one of isWhen R ₃,At least two of them areWhen n1 and m1 in each of the groups are independent of each other, they may be the same or different.

In a preferred embodiment of the invention, n is 3, R ₁ is-CH ₃,R₂ is-H, preferably the compounds of the formula IWherein the R ₃ group isWherein X is O, N or S heteroatom, N1 is selected from integers of 1-8, m1 is selected from integers of 1-8, N1 and m1 are independent of each other and can be the same or different, R ₄、R₅、R₆ groups are independent of each other and are linear or branched C10-20 alkyl, linear or branched C10-20 alkenyl and linear or branched C10-20 alkynyl, and at least 1C atom of the alkyl, alkenyl or alkynyl is optionally replaced by a heteroatom independently selected from O, S or N.

As a preferred embodiment of the present invention, the compounds of formula I areN is 3, R ₁ is-CH ₃,R₂ is-H, wherein R ₃ isR ₄、R₅ isPreferably, X is an O or N heteroatom, more preferably, N1 is an integer selected from 4 to 8, and m1 is an integer selected from 4 to 8.

In a preferred embodiment of the invention, the ionizable cationic lipid compound is selected from the group consisting of a compound of formula a or a compound of formula B:

wherein n ₂ on each branched chain is independently selected from an integer of 1-8, preferably an integer of 4-8, m ₂ is independently selected from an integer of 1-8, preferably an integer of 4-8, preferably each n ₂ is selected from an integer of 4-8, and each m ₂ is selected from an integer of 4-8;

wherein n ₃ on each branched chain is independently selected from an integer of 1-8, preferably an integer of 4-8, m ₃ is independently selected from an integer of 1-8, preferably an integer of 4-8, preferably each n ₃ is selected from an integer of 4-8, and each m ₃ is selected from an integer of 4-8.

The novel ionizable lipid compounds provided by the invention, wherein R ₂ is-H, and the rest three hydrophobic tail chains are optimized, have higher cell transfection efficiency effect than the compounds with the existing four hydrophobic tail chain molecular structures, are possibly related to different configurations/conformations of the compounds, for example, in the slightly acidic lysosome microenvironment, the compounds containing the three hydrophobic tail chains tend to form a conical molecular structure, and can promote the hexagonal transformation of cell membranes and the escape of lysosomes. And on the basis, the lipid compound and the delivery system for non-liver targeting delivery with more excellent specific targeting effect can be provided by optimizing the X groups simultaneously, so that the nucleic acid can be delivered to a target organ more efficiently.

The invention also provides a synthesis method of the novel ionizable lipid compounds. The ionizable lipid compounds of the invention may be synthesized using methods known in the art, for example, by reacting one or more equivalents of an amine (hydrophilic polar head containing an amine group) with three or more equivalents of a hydrophobic lipid tail compound under suitable conditions. The synthesis of the ionizable lipid compounds is performed with or without a solvent, and the synthesis may be performed at a higher temperature in the range of 25-120 ℃. The resulting ionizable lipid compound may optionally be purified. For example, a mixture of ionizable lipid compounds may be purified to yield a particular ionizable lipid compound, such as a product containing three hydrophobic lipid tails. The hydrophobic lipid tail compounds may be commercially available or synthetically prepared.

In some embodiments of the invention there is provided a method of preparing the ionizable cationic lipid compound comprising:

Compound synthesis route:

(wherein n ₄ on each branch is independently selected from an integer of 1 to 8, preferably an integer of 4 to 8, m ₄ is independently selected from an integer of 1 to 8, preferably an integer of 4 to 8; preferably each n ₄ is selected from an integer of 4 to 8, and each m ₄ is selected from an integer of 4 to 8)

The method specifically comprises the following steps:

1) Reduction of the carboxyl group of the compound A1 to a hydroxyl group in the presence of a reducing agent to obtain a compound A2;

2) Esterifying the hydroxyl group of compound A2 to an ester group in the presence of acryloyl chloride to obtain compound A3;

3) Michael addition the ionizable cationic lipid compound is obtained by subjecting compound A3 to a Michael addition reaction with an amine (e.g., N, N-bis (3-aminopropyl) methylamine).

In some embodiments of the present invention, provided are methods of preparing the ionizable cationic lipid compounds comprising:

(wherein n ₄ on each branch is independently selected from an integer of 1 to 8, preferably an integer of 4 to 8, m ₄ is independently selected from an integer of 1 to 8, preferably an integer of 4 to 8; preferably each n ₄ is selected from an integer of 4 to 8, and each m ₄ is selected from an integer of 4 to 8)

1) Acyl chloride the carboxyl group of compound B1 is acyl-chlorinated in the presence of oxalyl chloride to obtain compound B2;

2) Substitution by converting the acid chloride group of compound B2 to an amide group in the presence of ammonium hydroxide to obtain compound B3;

3) Reduction of the amide of compound B3 to an amine in the presence of a reducing agent to obtain compound B4;

4) Amidation of the amine group of compound B4 to an amide group in the presence of acryloyl chloride to obtain compound B5;

3) Michael addition by reacting compound B5 with an amine to give the ionizable cationic lipid compound.

Examples of reducing agents according to the present invention include, but are not limited to, diisobutylaluminum hydride, lithium aluminum hydride, and the like. Examples of the solvent used in the reaction include, but are not limited to, halogenated hydrocarbons (such as chloroform, methylene chloride, dichloroethane, etc.), ethers (such as diethyl ether, tetrahydrofuran, etc.), hydrocarbons (such as n-pentane, benzene, toluene, etc.), and mixed solvents of 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, methylene chloride, and dichloroethane, etc.), hydrocarbons (such as n-pentane, benzene, and toluene, etc.), nitriles (such as acetonitrile, etc.), and mixed solvents of two or more of these solvents. The Michael addition reaction may optionally be carried out without a solvent, examples of which include, but are not limited to, isopropanol, t-butanol, tetrahydrofuran, and the like. The amine may be N, N-bis (3-aminopropyl) methylamine.

According to the invention, the starting material A1 in the preparation process is commercially available or can be synthesized by conventional methods.

The ionizable lipid provided by the invention contains two adjacent cis double bonds in the molecular structure, so that the ionizable lipid has higher encapsulation efficiency and better cell transfection efficiency when being subsequently applied to a delivery system for encapsulating active substances (such as mRNA), and in addition, the particle size of the obtained lipid nanoparticle can be more uniform due to the existence of the two adjacent cis double bonds in the tail chain when the lipid nanoparticle is prepared. The ionizable lipid compounds of the invention are particularly suitable for preparing nanoparticles of solid structure.

The invention also provides application of the ionizable cationic lipid compound in preparing a bioactive substance delivery system, and preferably, the delivery system is a microparticle, a nanoparticle, a liposome, a lipid nanoparticle or a microbubble.

In a preferred embodiment of the invention, when the ionizable cationic lipid compound is a compound of formula a, the use of said ionizable cationic lipid compound in the preparation of a specific spleen-targeted bioactive substance delivery system; when the ionizable cationic lipid compound is a compound of formula B, the use of the ionizable cationic lipid compound in the preparation of a specific lung-targeted bioactive substance delivery system;

wherein n ₂ on each branched chain is independently selected from an integer of 1-8, preferably an integer of 4-8, m ₂ is independently selected from an integer of 1-8, preferably an integer of 4-8, preferably each n ₂ is selected from an integer of 4-8, and each m ₂ is selected from an integer of 4-8;

wherein n ₃ on each branched chain is independently selected from an integer of 1-8, preferably an integer of 4-8, m ₃ is independently selected from an integer of 1-8, preferably an integer of 4-8, preferably each n ₃ is selected from an integer of 4-8, and each m ₃ is selected from an integer of 4-8.

In a preferred embodiment of the invention, the delivery system is a lipid nanoparticle.

In certain embodiments, all of the amino groups of the amine are fully reacted with the hydrophobic lipid tail compound to form the tertiary amine. In other embodiments, not all of the amino groups of the amine are fully reacted with the hydrophobic lipid tail compound, thereby producing a primary or secondary amine in the ionizable lipid compound. These primary or secondary amines are left intact or may be reacted with another electrophile such as a different hydrophobic lipid tail compound. It is known in the art that reacting an excess of amine with a hydrophobic lipid tail compound will produce a variety of different ionizable lipid compounds having a variety of tail numbers. For example, a diamine or polyamine may include one, two, three, or four tail compounds on various amino moieties of the molecule, thereby producing primary, secondary, and tertiary amines. In some embodiments, the same type of tail compound is used, or two types of tail compounds are used. In other embodiments, two or more different tail compounds are used.

The present invention also provides a bioactive substance delivery system comprising said ionizable cationic lipid compound, preferably said delivery system being a microparticle, nanoparticle, liposome, lipid nanoparticle or microbubble.

In one embodiment of the invention, the delivery system is a lipid nanoparticle. The lipid nanoparticle can be used for efficiently delivering bioactive substances (such as mRNA) into cells, tissues or organs, and realizing efficient regulation of the bioactive substances. In the present invention, the ionizable lipid compound is combined with a bioactive substance (e.g., mRNA) that is targeted for cellular or organ delivery or further comprises other substances (e.g., other anionic, cationic, or ionizable lipid compounds, synthetic or natural polymers, proteins, phospholipids, cholesterol, carbohydrates, surfactants, etc.) to form microbubbles, liposomes, lipid nanoparticles, or microparticles. 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 may then optionally be combined with pharmaceutical excipients to form a pharmaceutical composition.

The invention also provides a pharmaceutical composition comprising the bioactive substance delivery system.

In another aspect, the present invention also provides a lipid nanoparticle composition comprising lipid nanoparticles, the lipid nanoparticle comprises the ionizable cationic lipid compound.

According to the present invention, the lipid nanoparticle composition further comprises other lipid molecules. The additional 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, cholesterol, pegylated lipid molecules.

According to the present invention, the lipid nanoparticle composition, when used in a drug delivery system, can encapsulate a pharmaceutical agent, including nucleotides, small molecule compounds, proteins, polypeptides, metals, and the like. Such 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, and the like. The ionizable lipid compounds have several properties suitable for preparing drug delivery systems, 1) the ability to neutralize charge on negatively charged active substances, 2) the ability of lipids to complex and "protect" labile agents, 3) the ability to buffer pH values in vivo, 4) the ability to act as a "proton sponge" and cause dissolution in vivo.

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

According to some preferred embodiments of the invention, the mole percentage of the ionizable lipid molecules of formula I in the lipid of the lipid nanoparticle is 30-60 mole%, e.g. may be 30mol％, 31mol％,32mol％,33mol％,34mol％,35mol％,36mol％,37mol％, 38mol％,39mol％,40mol％,41mol％,42mol％,43mol％,44mol％, 45mol％,46mol％,47mol％,48mol％,49mol％,50mol％,51mol％, 52mol％,53mol％,54mol％,55mol％,56mol％,57mol％,58mol％, 59mol％,60mol％.

According to some preferred embodiments of the invention, the neutral lipid molecule is an uncharged lipid molecule or a zwitterionic lipid molecule, such as a phosphatidylcholine-like compound, or/and a phosphatidylethanolamine-like compound.

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

According to some preferred embodiments of the present invention, examples of neutral lipid molecules include, but are not limited to, dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), lysophosphatidylethanolamine, distearoyl phosphatidylcholine (DSPC), phosphorylcholine (DOPC), 5-heptadecylphenyl-1, 3-diol (resorcinol), dimyristoyl phosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DAPC), phosphatidylethanolamine (PE), lecithin phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (psc), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1, 2-di-arachidoyl-sn-3-phosphorylcholine (MPPC), stearoyl-glycero-3-phosphorylcholine (MPPC), and combinations thereof.

In one embodiment, the neutral lipid molecule may be selected from the group consisting of distearyl phosphatidylcholine (DSPC), distearyl 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).

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

According to the present invention, cholesterol lipid molecules include steroids, sterols, alkyl resorcinol, and the like, examples of which include, but are not limited to, cholesterol hemisuccinate, and 5-heptadecylresorcinol.

According to some preferred embodiments of the invention, the cholesterol lipid molecule is selected from one or more of cholesterol, cholesterol hemisuccinate and 5-heptadecylresorcinol.

In one embodiment, the cholesterol lipid molecule is Cholesterol (CHOL). In one embodiment, the cholesterol lipid molecule is cholesterol hemisuccinate.

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

According to some preferred embodiments of the present invention, the pegylated lipid molecule comprises a lipid moiety and a PEG-based polymer moiety, expressed as a number average molecular weight of the lipid moiety-PEG, the lipid moiety comprising one or more of diacylglycerol and/or diacylglycerol amides, preferably selected from dilauroylglycerol, dimyristoylglycerol, dipalmitoylglycerol, dimyristoylglycerol amides, dipalmitoylglycerol amides, dilauroylglycerol amides, 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-dimyristoyl-sn-glycerol-3-phosphoethanolamine, the PEG having a number average molecular weight of 130 to 50,000, preferably 150 to 10,000, further preferably 300 to 3,000, most preferably 1,500 to 2,500.

According to the 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 diacylglycerols or diacylglycerol amides (DIACYLGLYCAMIDE), including those comprising a dialkylglycerol or dialkylglyceramide 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 an amide or an ester. In some embodiments, the alkyl chain length comprises about C10 to C20. The dialkylglycerol or dialkylglyceramide group may further comprise one or more substituted alkyl groups. The chain length may be symmetrical or asymmetrical. As used herein, the term "PEG" means any polyethylene glycol or other polyalkylene ether polymer, unless otherwise indicated. In one embodiment, the PEG moiety is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In certain embodiments, the PEG moiety may be substituted with, for example, one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In one embodiment, the PEG moiety comprises a PEG copolymer such as PEG-polyurethane or PEG-polypropylene (see, e.g., J. Milton Harris, poly (ethylene glycol) chemistry: biotechnical and biomedical applications (1992)), or the PEG moiety does not comprise a PEG copolymer, e.g., it may be a PEG homopolymer. In one embodiment, the PEG has a molecular weight of about 130 to about 50,000, in a fruiting body embodiment, about 150 to about 30,000, in a fruiting body embodiment, about 150 to about 20,000, in a fruiting body embodiment, about 150 to about 15,000, in a fruiting body embodiment, about 150 to about 10,000, in a fruiting body embodiment, about 150 to about 6,000, in a fruiting body embodiment, about 150 to about 5,000, in a fruiting body embodiment, about 150 to about 4,000, in a fruiting body embodiment, about 150 to about 3,000, in a fruiting body embodiment, about 300 to about 3,000, in a fruiting body embodiment, about 1,000 to about 3,000, and in a fruiting body embodiment, about 1,500 to about 2,500. In certain embodiments, the PEG is "PEG 2000" having an average molecular weight of about 2,000 daltons. In some embodiments of the invention, PEG is represented by the formulaMeaning that for PEG-2000 where n is 45, meaning that the number average degree of polymerization comprises about 45 subunits, other PEG embodiments known in the art can also be used, including, for example, those wherein the number average degree of polymerization comprises about 23 subunits (n=23) and/or 68 subunits (n=68). In some embodiments, n may be in the range of about 30 to about 60. In some embodiments, n may be in the range of about 35 to about 55. In some embodiments, n may be in the range of about 40 to about 50. In some embodiments, n may be in the range of about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R may be unsubstituted C1-C30 alkyl, such as C1-C20 alkyl, C1-C10 alkyl, C1-C6 alkyl. In some embodiments, R may be H, methyl or ethyl.

In some embodiments, the pegylated lipid molecule may be expressed as a "lipid fraction-PEG-number average molecular weight" or "PEG-number average molecular weight-lipid fraction" or "PEG-lipid fraction". The lipid moiety is a diacylglycerol or diacylglycerol amide selected from dilauryl glycerol, dimyristoyl glycerol amide, distearoyl glycerol, dilauryl glycerol amide, distearoyl glycerol amide, 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-dimyristoyl-sn-glycerol-3-phosphoethanolamine, and the PEG has a number average molecular weight of about 130 to about 50,000, for example, about 150 to about 30,000, about 150 to about 20,000, about 150 to about 15,000, about 150 to about 10,000, about 150 to about 6,000, about 150 to about 5,000, about 150 to about 4,000, about 150 to about 3,000, about 300 to about 3,000, about 1,000 to about 3,000, about 1,500 to about 2,500, for example, about 2000.

In some embodiments, the pegylated lipid molecule may be selected from the group consisting of PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dilauroylglycerol amide, PEG-dimyristoylglycerol amide, PEG-distearylglycerol (PEG-DSPE) and PEG-distearylglycerol amide, PEG-cholesterol (1- [8' - (cholest-5-en-3 [ beta ] -oxy) carboxamido-3 ',6' -dioxaoctyl ] carbamoyl- [ omega ] -methyl-poly (ethylene glycol), PEG-DMB (3, 4-ditetradecyloxybenzyl- [ omega ] -methyl-poly (ethylene glycol) ether), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (DMG-PEG 2000), 1, 2-distearoyl-sn-glycero-methoxypolyethylene glycol (DSG-PEG 2000), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000] (PEG-pe), poly (ethylene glycol) -2000-dimethacrylate (DMA-PEG 2000) and 1, 2-distearoyloxypropyl-3-amine-N- [ methoxy (polyethylene glycol) -2000] (DSA-PEG 2000). 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.

According to the invention, the mole percentage of pegylated lipid molecules in the lipid of the lipid nanoparticle is 0.5-5mol%, e.g. 0.5mol％,0.6mol％,0.7mol％, 0.8mol％,0.9mol％,1.0mol％,1.1mol％,1.2mol％,1.3mol％,1.4mol％, 1.5mol％,1.6mol％,1.7mol％,1.8mol％,1.9mol％,2.0mol％,2.1mol％, 2.2mol％,2.3mol％,2.4mol％,2.5mol％,2.6mol％,2.7mol％,2.8mol％, 2.9mol％,3.0mol％,3.1mol％,3.2mol％,3.3mol％,3.4mol％,3.5mol％, 3.6mol％,3.7mol％,3.8mol％,3.9mol％,4.0mol％,4.1mol％,4.2mol％, 4.3mol％,4.4mol％,4.5mol％,4.6mol％,4.7mol％,4.8mol％,4.9mol％, 5.0mol％ etc.

In some embodiments of the invention, the lipid nanoparticle comprises an ionizable cationic lipid molecule of formula a, a neutral lipid molecule, a cholesterol lipid molecule, a pegylated lipid molecule, wherein:

Formula A, wherein each n2 is independent of each other and can be the same or different, each n2 is selected from an integer of 1 to 8, each m2 is independent of each other and can be the same or different, each m2 is selected from an integer of 0 to 8, preferably, each n2 is selected from an integer of 4 to 8, each m2 is selected from an integer of 4 to 8, preferably, each n2 is the same as each other, each m2 is the same as each other, and the ionizable cationic lipid molecules represented by formula A account for 30 to 50mol%, preferably 35 to 48mol%, of the lipids in the lipid nanoparticle;

the neutral lipid molecules are selected from phosphatidylcholine compounds and phosphatidylethanolamine compounds, and the mole percentage of the neutral lipid molecules in the lipid nano-particles is 8-20mol%, preferably 8-18mol%, more preferably 9-16mol%;

The cholesterol lipid molecules are selected from cholesterol and cholesterol hemisuccinate, and the mole percentage of the cholesterol lipid molecules in the lipid nano particles is 30-50mol%, preferably 35-50mol%, more preferably 36-48mol%;

The pegylated lipid molecule is expressed as "lipid fraction-PEG-number average molecular weight", said lipid fraction being a diacylglycerol or diacylglycerol amide selected from dilauroylglycerol, dimyristoylglycerol, distearylglycerol, dilauryl glyceramide, dimyristoylglycerol amide, distearylglycerol amide, 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-dimyristoyl-sn-glycerol-3-phosphoethanolamine, the PEG having a number average molecular weight of 130 to 50,000, e.g. 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 2000, the pegylated lipid molecule comprising 0.5 to 5mol%, preferably 0.5 to 2.5mol%, more preferably 1.2 to 1.8mol% of the lipid in the lipid nanoparticle.

In some embodiments of the invention, the mole ratio of ionizable cationic lipid molecules of formula a, neutral lipid molecules, cholesterol, and pegylated lipid molecules is 35:15:48.5:1.5.

In some embodiments of the invention, the molar ratio of the ionizable cationic lipid molecule of formula a, the neutral lipid molecule, the cholesterol, and the pegylated lipid molecule is 45:15:38.5:1.5.

In some embodiments of the invention, the mole ratio of ionizable cationic lipid molecules of formula a, neutral lipid molecules, cholesterol, and pegylated lipid molecules is 40:10:48.5:1.5.

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

In one embodiment of the invention, the neutral lipid molecule is DSPC and the pegylated lipid molecule is DMG-PEG2000.

In one embodiment of the invention, the neutral lipid molecule is DOPE and the pegylated lipid molecule is DMG-PEG2000.

In one embodiment of the invention, the neutral lipid molecule is DSPC and the pegylated lipid molecule is DSPE-PEG2000.

In one embodiment of the invention, the neutral lipid molecule is DOPE and the pegylated lipid molecule is DSPE-PEG2000.

The three hydrophobic tail chain ionizable lipid compounds with adjacent cis double bond structures provided by the invention can provide higher active substance encapsulation efficiency and better cell or in vivo transfection efficiency, are especially suitable for preparing solid-structure nanoparticles, and in a lipid nanoparticle composition, the ionizable cationic lipid molecules, neutral lipid molecules, cholesterol lipid molecules and PEGylated lipid molecules of the formula I in the lipid nanoparticles are most preferably in a molar percentage of the total lipid molecules, and most importantly, the lipid nanoparticles have better specific spleen and/or lung targeting function.

The invention also provides a method for preparing the lipid nanoparticle composition, which comprises the steps of dissolving each lipid molecule by using an organic solvent according to a molar ratio to prepare a lipid-mixed solution, taking the lipid-mixed solution as an organic phase, taking an aqueous solution of a delivered object (such as mRNA) as an aqueous phase, and mixing the organic phase and the aqueous phase to prepare the lipid nanoparticle. Lipid nanoparticles may be prepared using other methods including, but not limited to, spray drying, solvent extraction, phase separation, nano-precipitation, single and double emulsion solvent evaporation, microfluidic, simple and complex coacervation, and others well known to those of ordinary skill in the art.

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

In some embodiments, the volume ratio of the organic phase to the aqueous phase is (2-4): 1, e.g., 3:1.

In some embodiments, the nanoparticle is prepared using a microfluidic platform.

According to the present invention, the preparation method further comprises the step of separating and purifying the lipid nanoparticle.

According to the present invention, the preparation method further comprises a step of lyophilizing the lipid nanoparticle.

The particle size of the lipid nanoparticle in the present invention ranges from 1nm to 1000 nm.

The delivery system formed from the ionizable lipid compounds of the invention may also be modified with targeting molecules that render them targeted to specific cells, tissues or organs. The targeting molecule may be included in the entire delivery system or may be located only on its surface. The targeting molecule may be a protein, small molecule, nucleic acid, polypeptide, glycoprotein, lipid, etc., examples of which include, but are not limited to, antibodies, antibody fragments, low Density Lipoproteins (LDL), sialic acid, aptamers, transferrin (transferrin), asialoglycoprotein (asialycoprotein), receptor ligands, etc.

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

The delivery system formed from the ionizable lipid compounds of the invention may 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 nontoxic, 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 (Tween 80), colorants, sweeteners, flavoring and aromatics, preservatives and antioxidants, buffers such as phosphate buffer solutions, citrate buffers, etc.

The pharmaceutical compositions of the present invention may be administered orally, rectally, intravenously, intramuscularly, intranasally, intraperitoneally, intravaginally, bucally, or in the form of oral or nasal spray, etc., to humans and/or animals.

The nucleic acid drug delivery system provided by the invention can be used for efficiently and specifically delivering nucleic acid drug molecules into spleen and/or lung and effectively translating the nucleic acid drug molecules into target molecules, and simultaneously reducing the accumulated side effects of liposome in liver, thereby having important significance for targeted drug administration, development and application of nucleic acid drugs.

The term "alkyl" in the present invention means a saturated hydrocarbon group obtained by removing a single hydrogen atom from a hydrocarbon moiety containing 1 to 30 carbon atoms. Examples of alkyl groups 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, and the like. The term "alkenyl" refers to a monovalent group derived from a hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl and the like. The term "alkynyl" refers to a monovalent group derived from a hydrocarbon having at least one carbon-carbon triple bond by removal of a single hydrogen atom. Representative alkynyl groups include ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like. And/or is to be taken as a specific disclosure of each of two specified features or components with or without the other. Thus, the term "and/or" as used in phrases 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 for the inclusion of additional elements or steps but not required. When the terms "comprising" or "including" are used herein, the term "consisting of" and/or "consisting essentially of" is therefore also included and disclosed. The term "about" used in conjunction with a numerical value means the range of accuracy familiar to and acceptable to those skilled in the art. Typically, this accuracy is in the interval of + -10%.

Drawings

In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a hydrogen spectrum of compound (9Z, 12Z) -9, 12-octadecadien-1-ol (linolic alcohol, a 2) in the examples of the present invention;

FIG. 2 is a hydrogen spectrum of the compound (9Z, 12Z) -9, 12-dienearyl acrylate (a 3) in the example of the present invention;

FIG. 3 is a hydrogen spectrum of the compound N34-O18-2 (3T) in the examples of the present invention;

FIG. 4 is a mass spectrum of the compound N34-O18-2 (3T) in the example of the invention;

FIG. 5 is a hydrogen spectrum of compound N34-O18-2 (4T) in the examples of the present invention;

FIG. 6 is a mass spectrum of the compound N34-O18-2 (4T) in the example of the invention;

FIG. 7 is a hydrogen spectrum of compound N34-N18-2 (3T) in the examples of the present invention;

FIG. 8 is a mass spectrum of compound N34-N18-2 (3T) in the examples of the present invention;

FIG. 9 is a hydrogen spectrum of compound N34-N18-2 (4T) in the examples of the present invention;

FIG. 10 is a mass spectrum of compound N34-N18-2 (4T) in the examples of the present invention;

FIG. 11 is a graph showing dissociation constants (pKa) of the compound N34-O18-2 (3T) (A), N34-O18-2 (4T) (B) in examples of the present invention;

FIG. 12 is a graph showing the expression level of Luciferase protein 24 hours after transfection of 293T cells with LNP envelope LuciferasemRNA (LucRNA) prepared from N34-O18-2 (3T) and N34-O18-2 (4T) in the examples of the present invention;

FIG. 13 is a graph showing the expression level of Lucifease protein after transfection of 293T cells with LNP envelope LuciferaseDNA (pDNA) prepared as described in example N34-O18-2 (3T) for 24 h;

FIG. 14 is a graph showing the expression level of the Luciferase protein after transfection of 293T cells with LNP encapsulated Luciferase siRNA (siRNA) prepared by N34-O18-2 (3T) in the examples of the present invention for 24 hours;

FIG. 15 is a graph showing the expression level of Luciferase protein after transfection of 293T cells with N34-O18-2 (3T), LNP envelope LuciferasemRNA (LucRNA) prepared by ALC-0315 for 24 hours in examples of the present invention;

FIG. 16 is a graph of cytotoxicity of N34-O18-2 (3T) -LNP and ALC-0315-LNP against 293T in examples of the present invention;

FIG. 17 shows fluorescence expression of various organs of mice according to the example of the present invention after intravenous injection of N34-O18-2 (3T) -LucRNA h;

FIG. 18 shows the fluorescence expression of various organs of mice according to the examples of the present invention after intravenous injection of N34-N18-2 (3T) -LucRNA h.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.

Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.

The examples are not intended to identify the particular technology or conditions, and are either conventional or are carried out according to the technology or conditions described in the literature in this field or are carried out according to the product specifications. The reagents and instruments used, etc. are not identified to the manufacturer and are conventional products available for purchase by regular vendors.

EXAMPLE 1 Synthesis of the ionizable lipid N34-O18-2 (3T), N34-O18-2 (4T)

Synthesis of (9Z, 12Z) -9, 12-octadecadien-1-ol (linoleyl alcohol, a 2) LiAlH ₄ (7.0 g), linoleic acid (50 g, a 1) was added to 950mL of tetrahydrofuran at 0℃and the mixture was stirred at 25℃for 2h. After the completion of the reaction, which was shown by Thin Layer Chromatography (TLC), the reaction mixture was quenched by adding water (8.0 mL), naOH aqueous solution (8.0 mL, mass fraction 15%) and water (25 mL) in this order, and after stirring for 15 minutes with an appropriate amount of Na ₂SO₄, the mixture was filtered through a buchner funnel and the cake was washed with ethyl acetate, and the filtrate was collected and concentrated by evaporation to give the target product linoleyl (a 2) 51g in 100% yield, and the hydrogen spectrum of the compound a2 was shown in fig. 1.

¹H NMR(400MHz,Chloroform-d)δ5.47-5.26(m,4H),3.64(t,J＝ 6.6Hz,2H),2.77(t,J＝6.5Hz,2H),2.08-2.01(m,4H),1.57(p,J＝6.6Hz,2H),1.39-1.25(m,16H),0.89(t,J＝6.7Hz,3H).

Synthesis of (9Z, 12Z) -9, 12-dienearyl acrylate (a 3) to 30.0mL of methylene chloride were added (9Z, 12Z) -9, 12-octadecadien-1-ol (3.2 g), triethylamine (3.64 g) at 0℃and then a methylene chloride (10.0 mL) solution containing acryloyl chloride (1.65 g) was added dropwise to the reaction system, and the reaction solution was stirred at 20℃for 2 hours. The triethylamine salt precipitated in the reaction solution was filtered through a buchner funnel, and the filtrate was collected, washed with water, 5% by mass of hydrochloric acid, water and then dried over magnesium sulfate. Evaporation to dryness followed by purification of the residue by flash column eluting with EtOAc/petroleum ether (0% -60%) gave the desired product (9Z, 12Z) -9, 12-dienearyl acrylate (2.7 g), 70% yield, hydrogen profile of compound a3 as shown in FIG. 2.

¹H NMR(400MHz,Chloroform-d)δ6.40(dd,J＝17.3,1.5Hz, 1H),6.12(dd,J＝17.4,10.4Hz,1H),5.81(dd,J＝10.4,1.5Hz,1H),5.32-5.40(m,4H),4.15(t,J＝6.7Hz,2H),2.77(t,J＝6.5Hz,2H),2.05 (q,J＝6.9Hz,4H),1.67(p,J＝6.8Hz,2H),1.38-1.27(m,16H),0.89(t, J＝6.7Hz,3H).

Synthesis of N34-O18-2 (3T), N34-O18-2 (4T) to 320mg of N, N-bis (3-aminopropyl) methylamine solution was added (9Z, 12Z) -9, 12-dienecacrylate (2.5 g) at room temperature, after which the mixture was heated to 120℃and stirred continuously for 48h. When the thin layer chromatography plate detection reaction was completed, 2.65g of a crude product was obtained, and then the target product was purified by eluting with methylene chloride/methanol by a rapid column chromatography to obtain 45mg of N34-O18-2 (3T), 66mg of N34-O18-2 (4T), the hydrogen spectrum of the compound N34-O18-2 (3T) was shown in FIG. 3, the mass spectrum was shown in FIG. 4, the hydrogen spectrum of the compound N34-O18-2 (4T) was shown in FIG. 5, and the mass spectrum was shown in FIG. 6.

N34-O18-2(3T):

¹H NMR(400MHz,Chloroform-d)δ5.45-5.26(m,12H),4.12-3.98 (m,6H),2.89(t,J＝6.5Hz,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＝6.8Hz,12H), 1.74-1.54(m,10H),1.40-1.25(m,48H),0.89(t,J＝6.7Hz,9H).

MALDI-TOFMS:m/z 1107.030[M+H]⁺.

N34-O18-2(4T):

¹H NMR(400MHz,Chloroform-d)δ5.42-5.29(m,16H),4.04(t,J ＝6.8Hz,8H),2.76(q,J＝7.5,7.0Hz,16H),2.44-2.41(m,J＝7.1Hz,12H),2.29–2.16(m,5H),2.05(q,J＝6.8Hz,16H),1.63-1.60(m,12H), 1.38-1.26(m,66H),0.89(t,J＝6.7Hz,12H).

MALDI-TOFMS:m/z 1427.310[M+H]⁺.

EXAMPLE 2 Synthesis of ionizable lipid N34-N18-2 (3T), N34-N18-2 (4T)

Synthesis of N34-N18-2 (3T), N34-N18-2 (4T) to 400mg of N, N-bis (3-aminopropyl) methylamine solution was added (9Z, 12Z) -9, 12-dienecarboxamide (3.08 g) at room temperature, after which the mixture was heated to 120℃and stirred continuously for 48h. When the thin layer chromatography plate detection reaction was completed, 3.10g of a crude product was obtained, and then the target product was purified by eluting with methylene chloride/methanol by a rapid column chromatography to obtain 100mg of N34-N18-2 (3T), 120mg of N34-N18-2 (4T), the hydrogen spectrum of the compound N34-N18-2 (3T) was shown in FIG. 7, the mass spectrum was shown in FIG. 8, the hydrogen spectrum of the compound N34-N18-2 (4T) was shown in FIG. 9, and the mass spectrum was shown in FIG. 10.

N34-N18-2(3T):

¹H NMR (400MHz,Chloroform-d)δ7.11(br,3H),5.50-5.23(m, 12H),3.28-3.14(m,6H),2.99(s,3H),2.77(t,J＝6.5Hz,6H),2.70(t,J ＝6.3Hz,4H),2.50(s,5H),2.37(d,J＝6.4Hz,6H),2.24(s,3H),2.05(q,J＝7.4,6.7Hz,12H),1.79(s,2H),1.63-1.40(m,10H),1.36-1.24(m, 48H),0.92-0.86(m,9H).

MALDI-TOFMS:m/z 1104.177[M+H]⁺.

N34-N18-2(4T):

¹H NMR(400MHz,Chloroform-d)δ7.14(br,3H),5.40-5.29(m, 16H),3.18(q,J＝6.8Hz,8H),2.77(t,J＝6.5Hz,8H),2.68(t,J＝6.1 Hz,8H),2.47(t,J＝6.5Hz,4H),2.35(t,J＝6.0Hz,8H),2.05(q,J＝7.0Hz,16H),1.70(s,3H),1.49(q,J＝7.2Hz,8H),1.37-1.25(m,72H),0.89 (t,J＝6.7Hz,12H).

MALDI-TOFMS:m/z 1423.553[M+H]⁺.

Example 3 dissociation constant (pKa) of ionizable lipid N34-O18-2 (3T), N34-O18-2 (4T)

Ionizable lipids have two main roles, binding nucleic acids and allowing the release of nucleic acid molecules in cells. The pKa of lipids is an important factor because lipids need to be positively charged at low pH to bind nucleic acids, but not charged at neutral pH, so LNP does not cause toxicity. As shown in FIG. 11, the ionizable lipid N34-O18-2 (3T) had a pKa of 6.72 (A) and N34-O18-2 (4T) had a pKa of 5.92 (B) as determined by the TNS dye binding assay. It can be seen that both molecules are positively charged under acidic conditions and that RNA is carried, and that they are uncharged at neutral pH (pH=7.4)

EXAMPLE 4 preparation of lipid nanoparticles by encapsulation of mRNA with N34-O18-2 (3T), N34-O18-2 (4T)

The ionizable lipids N34-O18-2 (3T) or N34-O18-2 (4T), DSPC, cholesterol and DMG-PEG2000 were each prepared in a molar ratio of 45% to 15% to 38.5% to 1.5% with an ethanol solution as the organic phase and LuciferasemRNA (LucRNA) dissolved in an aqueous solution at pH=4 as the aqueous phase. According to the volume ratio of the water phase to the organic phase of 3:1, preparing the nanoparticle suspension by using a microfluidic technology on a nano-drug manufacturing instrument (Micana). And after the preparation, performing ultrafiltration and concentration to obtain the final LucRNA-LNP lipid nanoparticle, and storing at 2-8 ℃ for later use.

Characterization of LucRNA-LNP particle size and Zeta potential was performed using ZetasizerPro nm particle size potentiometers (malverpanaceae). The encapsulation efficiency of LucRNA-LNP was measured by the method of F-280 fluorescence spectrophotometer (Tianjin Gangdong) Ribogreen. The test results of example 2 are shown in Table 1.

TABLE 1 detection results

From the results of example 4, it can be seen that the lipid nanoparticle LucRNA-LNP particle size prepared from the novel lipid compound N34-O18-2 (3T) was around 120nm, the LucRNA-LNP particle size distribution was narrow (PDI was small), and the encapsulation efficiency was as high as 98%.

In addition, transfection efficiency of the prepared LucRNA-LNP cells was examined by the multifunctional enzyme-labeled instrument (BioTek, model SLXFATS) fluorescein reporter method. The method of in vitro transcription LucRNA is that 293T cells are plated at a cell density of 1X 10 ⁴ cells/well and transfected at a cell fusion of 30% -50%. 1.0 mu gLucRNA was transfected with the transfection reagent Lipofectamine2000 (ThermoFisher Scientific) and the transfection procedure was performed according to the transfection reagent product instructions. And (5) detecting the protein expression quantity by using a multifunctional enzyme-labeled instrument after 24 hours of transfection. The negative control was cell culture medium without LucRNA-LNP added. In vitro cell transfection efficiency as shown in FIG. 12, LNP-coated mRNA prepared from the ionizable lipid N34-O18-2 (3T) was shown to have extremely high cell transfection efficiency, which was significantly lower than that of the conventional ionizable lipid N34-O18-2 (4T) containing four hydrophobic tails, which was an order of magnitude lower than that of the conventional ionizable lipid N34-O18-2 (4T). It can be seen that the ionizable lipid N34-O18-2 (3T) containing three hydrophobic tails has higher cell transfection efficiency than the conventional ionizable lipid N34-O18-2 (4T) containing four hydrophobic tails, and the advantage is very obvious.

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 better physicochemical characteristics and cell transfection efficiency in vitro is about 3-5 times higher than that of commercial Lipofectamine 2000.

EXAMPLE 5 preparation of lipid nanoparticles by encapsulation of DNA with N34-O18-2 (3T)

The ionizable lipids N34-O18-2 (3T), DSPC, cholesterol and DMG-PEG2000 were prepared as an organic phase with 45% to 15% to 38.5% to 1.5% ethanol solution, respectively, and LuciferaseDNA (pDNA) was dissolved in an aqueous solution at ph=4 as an aqueous phase. The nanoparticle suspension was prepared by microfluidic techniques on a nanopharmaceutical manufacturing apparatus (PNI company, canada, model Ignite) with a volume ratio of aqueous phase to organic phase of 3:1. And after the preparation, performing ultrafiltration concentration to obtain the final pDNA-LNP lipid nanoparticle, and storing at 2-8 ℃ for later use.

Characterization of pDNA-LNP particle size and Zeta potential was performed using ZetasizerPro nm particle size potentiometers (Markov panaceae). The results of the test in example 5 are shown in Table 2, and the particle size of the lipid nanoparticle pDNA-LNP prepared by the compatibility of the novel lipid compound is about 303nm, and the particle size distribution of the pDNA-LNP is narrower (PDI is smaller).

TABLE 2 detection results

Transfection efficiency of the prepared pDNA-LNP 293T cells was measured by a multifunctional enzyme-labeled instrument (BioTek, model SLXFATS) fluorescein reporter method. The in vitro transcription method is as follows, 293T cells are plated at a cell density of 1X 10 ⁴ cells/well and transfected at a cell fusion of 30% -50%. Transfection procedure was performed using the transfection reagent Lipofectamine2000 (ThermoFisher Scientific) to transfect 2. Mu. gpDNA according to the transfection reagent product instructions. And (5) detecting the protein expression quantity by using a multifunctional enzyme-labeled instrument after 24 hours of transfection. The negative control was cell culture medium without pDNA-LNP added. In vitro cell transfection efficiency as shown in FIG. 13, LNP coated DNA prepared from the ionizable lipid N34-O18-2 (3T) was shown to have high cell transfection efficiency.

From the results of example 5, it can be seen that the lipid nanoparticle pDNA-LNP prepared from the novel lipid compound has good physicochemical characteristics and high in vitro cell transfection efficiency.

EXAMPLE 6 preparation of lipid nanoparticles by N34-O18-2 (3T) -coated siRNA

The ionizable lipids N34-O18-2 (3T), DSPC, cholesterol and DMG-PEG2000 were prepared as an organic phase by preparing an ethanol solution at a molar ratio of 45% to 15% to 38.5% to 1.5%, respectively, and the Luciferase siRNA (siRNA) was dissolved in an aqueous solution at pH=4 as an aqueous phase. The nanoparticle suspension was prepared by microfluidic techniques on a nanopharmaceutical manufacturing apparatus (PNI company, canada, model Ignite) with a volume ratio of aqueous phase to organic phase of 3:1. And after the preparation, performing ultrafiltration concentration to obtain the final siRNA-LNP lipid nanoparticle, and storing at 2-8 ℃ for later use.

Characterization of siRNA-LNP particle size and Zeta potential was performed using ZetasizerPro nm particle size potentiometers (malverpa). The test results of example 6 are shown in Table 3. The particle size of the lipid nanoparticle siRNA-LNP prepared by the compatibility of the novel lipid compound is about 225 nm.

TABLE 3 detection results

Transfection efficiency of the prepared siRNA-LNP 293T cells was measured by a multifunctional enzyme-labeled instrument (BioTek, model SLXFATS) fluorescein reporter method. The in vitro transcription method is as follows, the 293T cells reported by stable transfer of Luciferase are plated at a cell density of 1X 10 ⁴ cells/well and transfected at a cell fusion of 30% -50%. siRNA was transfected with transfection reagent Lipofectamine2000 (ThermoFisher Scientific) and transfection procedures were performed according to the transfection reagent product instructions. And (5) detecting the protein expression quantity by using a multifunctional enzyme-labeled instrument after 24 hours of transfection. The negative control was cell culture medium without siRNA-LNP added. In vitro cell transfection efficiency as shown in FIG. 14, LNP-coated siRNA prepared from the ionizable lipid N34-O18-2 (3T) was shown to have extremely high protein knockdown efficiency.

From the results of example 6, it can be seen that the lipid nanoparticle siRNA-LNP particle size prepared from the novel lipid compound was around 225 nm. In vitro cell transfection and knockdown efficiency was higher than commercial Lipofectamine 2000.

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

ALC-0315 has the formula of ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate).

The structural formula of ALC-0315 is:

Lipid nanoparticles were prepared according to the method described in example 4, using N34-O18-2 (3T) and ALC-0315, respectively, in a specific molar ratio of ：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.

The physicochemical quality control data of the prepared lipid nanoparticle are shown in the following table (table 4):

TABLE 4 physical and chemical control data for lipid nanoparticles

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

The same transfection method as in example 4 is adopted to transfect the prepared lipid nanoparticle into cells, the protein expression condition is known, and the result is shown in fig. 15, wherein after the lipid nanoparticle prepared by N34-O18-2 (3T) carries mRNA to transfect the cells, the protein expression quantity in the cells is higher than that of Lipofectamine2000, and the protein expression quantity in the cells transfected by the corresponding mRNA concentration is also higher than ALC-0315, which indicates that the cell transfection efficiency of the lipid nanoparticle prepared by N34-O18-2 (3T) is very high.

In addition, the MTT method is adopted to measure the cytotoxicity of N34-O18-2 (3T) -LNP and ALC-0315-LNP, and the influence of factors such as vector dosage, acting time and the like on the proliferation of normal cells (such as 293T) cells is examined. As shown in FIG. 16, the lipid nanoparticle prepared from N34-O18-2 (3T) remained relatively active at a relatively high dose (2. Mu.g/mL) after 48 hours of transfection of cells with mRNA, indicating that the cytotoxicity of the lipid nanoparticle prepared from N34-O18-2 (3T) was very low.

From the results of example 7, it can be seen that lipid nanoparticles prepared from the novel lipid compounds have low cytotoxicity and high mRNA transfection efficiency.

Example 8 transfection experiments of N34-O18-2 (3T) -LNP, N34-N18-2 (3T) -LNP lipid nanoparticles in animals

Lipid nanoparticles were prepared using N34-O18-2 (3T) or N34-N18-2 (3T) in the molar ratios N34-O18-2 (3T) DSPC: cholestol DMG-PEG 2000=45:15:38.5:1.5, N/P ratio 10:1, N34-N18-2 (3T) DSPC: cholestol DMG-PEG 2000=45:15:38.5:1.5, and N/P ratio 10:1 according to the procedure described in example 3. Wherein mRNA is mRNA for expressing Luciferase fluorescent protein, the dosage of the mRNA is 10 mug, the total amount of N34-O18-2 (3T) or N34-N18-2 (3T) and DSPC, cholesterol, DMG-PEG2000 is 100 mug, a neutral PBS buffer solution with 200 mug is adopted to quickly change the liposome environment, and the liposome is quickly injected into 6-8 week female C57 mice through tail vein, and intravenous injection is controlled to 10 mug gmRNA.

Mice were injected with N34-O18-2 (3T) -LNP, PBS (blank) via the tail vein and fluorescent expression of each organ after 6h is shown in FIG. 17. The results showed that after intravenous Injection of (IV) lipid nanoparticles of N34-O18-2 (3T) -LNP, the fluorescent expression levels in various organs of mice were mainly distributed in about 100% of spleen, 0% of heart, 0% of liver, 0% of lung, and 0% of kidney, which was found to target spleen specifically. Mice were injected with N34-N18-2 (3T) -LNP, PBS (blank) via the tail vein and fluorescent expression of each organ after 6h is shown in FIG. 18. The results showed that after intravenous Injection of (IV) lipid nanoparticles of N34-N18-2 (3T) -LNP, the fluorescent expression levels in various organs of mice were mainly distributed at about 0% in spleen, 0% in heart, 0% in liver, 100% in lung, and 0% in kidney, which was seen to specifically target the lung. Therefore, the invention forms an isomerism compound based on three lipid hydrophobic tail chains, and further realizes the specific targeting of different organs by adjusting the X hetero atoms in the lipid hydrophobic tail chains to be O or N.

It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the technical solution described in the above-mentioned embodiments may be modified or some technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solution of the embodiments of the present invention.

Claims (10)

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 a structure shown in Formula I: ; wherein n is an integer of 1 to 5; R ₁ is CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , CH ₂ OH, CH ₂ CH ₂ OH, CH ₂ CH ₂ CH ₂ OH, CH ₂ CH ₂ CH ₂ CH ₂ OH or CH ₂ CH ₂ NHCOCH ₃ ; R ₂ is H; X is O, R ₃ Both , n1 is selected from an integer of 1 to 8, and m1 is selected from an integer of 1 to 8. 2. Use of an ionizable cationic lipid compound in the preparation of a bioactive substance delivery system targeted to the lungs, wherein the ionizable cationic lipid compound has a structure shown in Formula I: ; wherein n is an integer of 1 to 5; R ₁ is -CH ₃ , -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₃ , -CH ₂ OH, -CH ₂ CH ₂ OH, -CH ₂ CH ₂ CH ₂ OH, -CH ₂ CH ₂ CH ₂ CH ₂ OH or -CH ₂ CH ₂ NHCOCH ₃ ; R ₂ is -H; X is N, R ₃ Both , n1 is selected from an integer of 1 to 8, and m1 is selected from an integer of 1 to 8. 3. The use according to claim 1 or 2, characterized in that n is 3, R1 is CH3, and R2 is H. 4. The use 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 use according to claim 1 or 2, characterized in that the delivery system is microparticles. 6. The use according to claim 1 or 2, characterized in that the delivery system is lipid nanoparticles. 7. The use according to claim 1 or 2, characterized in that the delivery system is nanoparticles. 8. The use according to claim 1 or 2, characterized in that the delivery system is microbubbles. 9. The use according to claim 1 or 2, characterized in that the delivery system is a liposome. 10. The use according to claim 1 or 2, characterized in that the bioactive substance is selected from DNA, mRNA, siRNA, and miRNA.