Ionizable lipid compounds, compositions and applications

Abstract

The present invention discloses an ionizable lipid compound, a composition and an application, belonging to the field of biomedical technology. The ionizable lipid compound of the present invention has a high encapsulation rate for active agents, strong stability, high expression efficiency, is conducive to the release of active agents into cells, increases the expression amount, and can deliver nucleic acid molecules, small molecule compounds, polypeptides or proteins, etc. The carrier prepared using the ionizable lipid compound of the present invention has a high encapsulation efficiency for nucleic acid molecules, can successfully transport nucleic acid molecules into cells and express them, and the ionizable lipid compound of the present invention broadens the types and scope of ionizable lipid compounds, which is of great significance to the development and application of nucleic acid therapeutic drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an ionizable lipid compound, composition and application. Background Art

[0002] Gene therapy involves the introduction of exogenous, normal genes into target cells to correct or compensate for diseases caused by genetic defects and abnormalities, achieving therapeutic goals. Specifically, through gene transfer technology, exogenous genes are inserted into appropriate recipient cells in a patient, enabling the products produced by the exogenous genes to treat a specific disease. As a new approach to disease treatment that integrates modern medicine and molecular biology, gene therapy is gradually gaining a significant position in the medical field. Currently, treatments that express specific genes or proteins are primarily achieved through the introduction of plasmid DNA or mRNA, while treatments that inhibit specific genes are primarily achieved through siRNA or microRNA (miRNA) (i.e., RNAi technology).

[0003] Genes have great therapeutic potential in regulating protein expression in vivo, but the efficiency of naked genes internalized by cells is extremely low, and they are easily cleared by the kidneys or rapidly degraded by enzymes in the body during delivery in vivo, which greatly reduces their actual therapeutic effect. Since the first clinical study of therapeutic genes began in 2000, the design of gene molecules and research on their delivery methods have made significant progress. However, gene therapy still faces several challenges, including low cell permeability and high sensitivity to degradation of certain nucleic acid molecules (including RNA). Therefore, there is a need to develop more lipid compounds and compositions capable of delivering genes to promote the delivery of various nucleic acid molecules outside or inside cells for treatment and / or prevention purposes.

[0004] In view of this, this application is filed. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide an ionizable lipid compound, composition and application. The ionizable lipid compound of the present invention can successfully transport nucleic acid molecules into cells and express them. The ionizable lipid compound of the present invention broadens the types and scope of ionizable lipid compounds and is of great significance to the development and application of nucleic acid therapeutic drugs.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] An ionizable lipid compound, wherein the ionizable lipid compound is at least one of the compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof;

[0008]

[0009] In formula (I), G1 and G2 are each independently selected from a bond, a hydroxyl substituted or unsubstituted C 10 -C 18 Straight chain alkyl, C 10 -C 26 Branched alkyl, hydroxy substituted or unsubstituted C4-C 20 Straight chain alkenyl, hydroxy substituted or unsubstituted C4-C 20 Branched alkenyl, hydroxy substituted or unsubstituted C2-C 16 Straight chain alkylene, hydroxy substituted or unsubstituted C2-C 16 Straight chain alkylene, branched chain alkylene, hydroxy substituted or unsubstituted C2-C 16 Straight chain alkenylene, hydroxy substituted or unsubstituted C2-C 16 branched alkenylene;

[0010] Said X1, X2 and X3 are each independently selected from a bond, -O-, -NH-, -N(R6)-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R6)-, -N(R6)C(O)-, -SS-, -OC(O)O-, -ON=C(R6)-, -C(R6)=NO-, -OC(O)N(R6)-, -N(R6)C(O)N(R6)-, -N(R6)C(O)O-, -C(O)S-, -C(S)O- or -C(R6)=NOC(O)-;

[0011] Said R6 is selected from H, alkyl or nitrogen-containing heterocycle;

[0012] The R1 and R2 are each independently selected from C6-C 14 Straight chain alkyl, C6-C 14 Branched alkyl, C6-C 14 Straight chain alkenyl or C6-C 14 branched alkenyl;

[0013] The M is selected from a bond, an alkyl, an alkylene, an alkenyl, an alkenylene, an alkynyl, an imino, a cycloalkyl, an alkylamino, a dialkylamino, an aryl, a heterocycle or a substituted heterocyclic compound;

[0014] The aryl group is C6H4R7R8, and the R7 and R8 are each independently selected from H, halogen, -OH, alkyl, alkoxy, -NH2, R9, alkylamino or dialkylamino;

[0015] Said L is selected from alkylene, alkenylene or heterocycle;

[0016] Said R3 and R4 are each independently selected from a bond, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, R9, a heterocyclic ring or a substituted heterocyclic compound;

[0017] R9 is selected from the group consisting of PEG, poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N-vinyl pyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide], and poly(amino acid);

[0018] R5 is selected from amino, imino, alkylamino or does not exist, and when R5 does not exist, the dotted line does not exist;

[0019] The a is 0, 1, 2, 3, 4, 5 or 6.

[0020] As an embodiment of the present application, the term "pharmaceutically acceptable salt" includes acid addition salts or base addition salts.

[0021] "Acid addition salts" refers to salts that retain the biological effectiveness and properties of the free acids, are not biologically or otherwise undesirable, and are formed with acids including, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, decanoic acid, hexanoic acid, octanoic acid, carbonic acid, cinnamic acid, citric acid, cycloamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid , galactonic acid, gentisic acid, glucoheptanoic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-dicarboxylic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0022] "Base addition salts" refer to salts that retain the biological effectiveness and properties of the free base and are not biologically or otherwise undesirable. These salts are prepared by the addition of inorganic or organic bases to free acids. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts; and organic bases include, but are not limited to, ammonia, isopropylamine, trimethylamine, diethylamine, tripropylamine, diethanolamine, ethanolamine, dealcoholization, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, caffeine, procaine, hydrazine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, purines, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Preferably, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0023] The base addition salt refers to a salt prepared by adding an inorganic base or an organic base to a free base compound. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, and aluminum salts; the organic base includes, but is not limited to, ammonia, isopropylamine, trimethylamine, diethylamine, tripropylamine, diethanolamine, ethanolamine, dealcoholization, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, caffeine, procaine, hydrazine, choline, betaine, benephrine (benethamine), benzathine penicillin (benzathine), ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Preferably, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0024] It should be further understood that the compound of formula (I) or its pharmaceutically acceptable salt can be isolated in the form of a solvate, and therefore any such solvate is included within the scope of the present invention. For example, the compound of formula (I) or its pharmaceutically acceptable salt can exist in an unsolvated form as well as in a solvated form with a pharmaceutically acceptable solvent (such as water, ethanol, etc.).

[0025] The term "stereoisomer" includes geometric isomers, diastereomers and enantiomers. Therefore, the compounds claimed in the present disclosure also include racemic mixtures, single stereoisomers and optically active mixtures. It should be understood by those skilled in the art that one stereoisomer may have better efficacy and / or lower side effects than other stereoisomers. Single stereoisomers and optically active mixtures can be obtained by chiral source synthesis, chiral catalysis, chiral resolution and other methods. Racemates can be chirally resolved by chromatographic resolution or chemical resolution. For example, chiral acid resolution reagents such as chiral tartaric acid and chiral malic acid can be added to the compounds of the present disclosure to form salts, and the physical and chemical properties of the products, such as different solubility, can be used for separation.

[0026] The term "solvate" means a physical association of a compound of the invention with one or more solvent molecules. This physical association involves varying degrees of ionic bonding, including hydrogen bonding. In certain instances, the solvate will be capable of separation, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" encompasses both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methoxides, and the like.

[0027] The term "delivery" refers to the act or manner of delivering a compound, substance, entity, moiety, cargo, or payload.

[0028] The term "feature" refers to a characteristic, property, or distinctive element.

[0029] As used herein, the term "fragment" refers to a portion. For example, a protein fragment may include a polypeptide obtained by digesting a full-length protein isolated from cultured cells.

[0030] As used herein, the term "treatment" refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of the disease to cure, alleviate, relieve, improve or affect the disease or the symptoms of the disease. In the context of this application, unless specifically stated to the contrary, the term "treatment" may also include prevention.

[0031] As an embodiment of the present application, at least one of the following (a) to (d) is satisfied:

[0032] (a) When X1 and X2 are a bond, R1 and R2 are alkyl or alkenyl groups;

[0033] (b) X1 and X2 are each independently a bond, -S-, -C(O)O-, -OC(O)-, -C(O)N(R6)- or -OC(O)O-;

[0034] (c) X3 is selected from a bond, -C(O)O- or -OC(O)-;

[0035] (d) R7 and R8 are each independently selected from H or alkoxy.

[0036] As an embodiment of the present application, said R3 and R4 form a heterocyclic ring with the nitrogen atom to which they are attached; or

[0037] One of R3 and R4 is selected from an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a heterocyclic ring or a substituted heterocyclic compound, and the other forms a heterocyclic ring or a heteroaryl group with the nitrogen atom to which they are attached and R5.

[0038] As an embodiment of the present application, the group represented by formula (II) is a group containing a protonatable tertiary amine;

[0039]

[0040] The group containing a protonatable tertiary amine may be selected from one of the following groups:

[0041]

[0042]

[0043] As an embodiment of the present application, G1 and G2 can each independently be selected from one of the following groups:

[0044]

[0045]

[0046] As an embodiment of the present application, R1 and R2 can be independently selected from one of the following groups:

[0047]

[0048] As an embodiment of the present application, the ionizable lipid compound is at least one of the compounds described in Formula (Ii), Formula (I-ii), Formula (I-iii), or pharmaceutically acceptable salts, stereoisomers, and solvates thereof;

[0049]

[0050]

[0051] In formula (Ii), formula (I-ii), and formula (I-iii);

[0052] The z, p, q, t, and u are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0053] The R 11、R 12 、R 100、 R 200 Each independently selected from C5-C 15 Straight chain alkyl, C5-C 15 Branched alkyl, C5-C 15 Straight chain alkenyl, C5-C 15 Branched alkenyl, C5-C 15 Straight chain alkynyl or C5-C 15 branched alkynyl;

[0054] The R 300 Selected from C 12 -C 24 Straight chain alkyl, C 12 -C 24 Branched alkyl, C 12 -C 24 Straight chain alkenyl, C 12 -C 24 Branched alkenyl, C 12 -C 24 Straight chain alkynyl or C 12 -C 24 branched alkynyl;

[0055] The W is a group containing a protonatable tertiary amine.

[0056] As an embodiment of the present application, the W is selected from one of the following groups:

[0057]

[0058]

[0059] As an embodiment of the present application, at least one of the following (1) to (4) is satisfied:

[0060] (1) p and t in formula (Ii) are each independently 4, 5 or 6;

[0061] (2) q and u in formula (Ii) are each independently 0, 1, 2, 3 or 4;

[0062] (3) in formula (I-ii), z and t are each independently 0, 1, 2, 3, 4, 5 or 6;

[0063] (4) Formula (I-iii) and t is 0, 1, 2, 3, 4, 5 or 6.

[0064] As an embodiment of the present application, the ionizable lipid compound is selected from at least one of the compounds shown below:

[0065]

[0066]

[0067]

[0068]

[0069]

[0070] The present invention also provides an application of an ionizable lipid compound in preparing a bioactive substance delivery system, wherein the delivery system is a microparticle, a nanoparticle, a liposome, a lipid nanoparticle or a microbubble.

[0071] The present invention also provides an application of an ionizable lipid compound in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

[0072] The present invention also provides an ionizable lipid composition, comprising a carrier and an active agent, wherein the carrier comprises an ionizable lipid compound, and the ionizable lipid compound is the above-mentioned ionizable lipid compound.

[0073] More specifically, the active agent is encapsulated within or associated with a carrier.

[0074] As an embodiment of the present application, the molar percentage of the ionizable lipid compound in the carrier is 20-60%, for example, it can be 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or a range consisting of any two values ​​therein.

[0075] As an embodiment of the present application, the carrier further includes neutral lipids, PEG lipids, and sterols.

[0076] As an embodiment of the present application, the molar percentage of the neutral lipid in the carrier is 5-25%, for example, it can be 5%, 10%, 15%, 20%, 25% or a range consisting of any two values ​​therein.

[0077] As an embodiment of the present application, the molar percentage of the PEG lipid in the carrier is 0.5-15%, for example, it can be 0.5%, 1%, 2%, 4%, 5%, 6%, 8%, 10%, 12%, 14%, 15% or a range consisting of any two values ​​therein.

[0078] As an embodiment of the present application, the molar percentage of the sterol in the carrier is 25-55%, for example, it can be 25%, 30%, 35%, 40%, 45%, 50%, 55% or a range consisting of any two values ​​therein.

[0079] As an embodiment of the present application, the neutral lipids include at least one of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC), and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE).

[0080] As an embodiment of the present application, the PEG lipid includes at least one of 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), and PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).

[0081] As an embodiment of the present application, the sterol includes at least one of cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and corticosteroids.

[0082] As an embodiment of the present application, the active agent comprises at least one mRNA encoding a protein or a fragment or epitope thereof.

[0083] As an embodiment of the present application, the active agent includes at least one of a nucleic acid molecule, a small molecule compound, a polypeptide, and a protein.

[0084] Specifically, the small molecule compounds include but are not limited to active ingredients of therapeutic and / or preventive agents, which are currently known drugs, such as anti-tumor drugs, anti-infective drugs, local anesthetics, antidepressants, anticonvulsants, antibiotics / antibacterial agents, antifungals, antiparasitic drugs, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-glaucoma agents, anesthetics, or imaging agents.

[0085] As an embodiment of the present application, the nucleic acid includes at least one of single-stranded DNA, double-stranded DNA, short isomers, agomir, antagomir, antisense molecules, small interfering RNA, asymmetric interfering RNA (aiRNA), microRNA, Dicer-substrateRNA, small hairpin RNA, transfer RNA (tRNA), messenger RNA, locked nucleic acid, peptide nucleic acid, morpholino ring oligonucleotide, aptamer, and ribozyme.

[0086] As an embodiment of the present application, the mass ratio of the carrier to the active agent is (5-50):1, for example, it can be 5:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1 or a range consisting of any two values ​​therein.

[0087] As an embodiment of the present application, the carrier of the present invention may further include one or more other ionizable lipid compounds.

[0088] More specifically, the other ionizable lipid compounds include but are not limited to:

[0089] 3-(Docosylamino)-N1,N1,4-triadecyl-1-piperazineethylamine (KL10), N1-[2-(Docosylamino)ethyl]-N1,N4,N4-triadecyl-1,4-piperazinedienamide (KL22), 14,25-tricosyl-15,18,21,24-tetraazaoctaconane (KL25), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLinDMA), in-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N ,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2R)), (2S)-2-({ 8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethylethyl-3-[(9Z-,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)), (12Z,15Z)-N,Ndimethyl-2-nonyldocosyl-12,15-den-1-amine, N,N-dimethyl-1-{(1S,2R)-2-octylcyclopropyl}heptadecan-8-amine;

[0090] Or, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEP C); 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1); N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[bis(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-bis[oleyloxy]-benzamide (MVL5); dioctadecylamino-glycyl spermine (DOGS); 3b-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol ... Dimethylammonium bromide (DDAB); SAINT-2, N-methyl-4-(dioleyl) methylpyridine; 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE); 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORIE); 1,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride (DORI); di-alkylated amino acids (DILA2); dioleyldimethylammonium chloride (DODAC); 1-palmitoyl-2-oleoyl -sn-glycero-3-ethylphosphocholine (POEPC); 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC); (R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride (DODAPen-Cl); (R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride (DOPen-G); and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-ammonium chloride (DOTAPen);

[0091] Or cationic lipids having a head group charged at physiological pH, such as a primary amine (e.g., DODAG N', N'-dioctadecyl-N-4,8-diaza-10-aminodecanoylglycine amide) and a guanidinium head group (e.g., bis-guanidinium-spermidine-cholesterol (BGSC), bis-guanidinium trisaminoethylamine-cholesterol (BGTC), PONA, and (R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride (DOPen-G)). Another suitable cationic lipid is (R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride (DODAPen-Cl). In certain embodiments, the cationic lipid is a specific enantiomer or racemic form, and includes various salt forms (e.g., chlorides or sulfates) of the above cationic lipids;

[0092] or N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP-Cl), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium sulfate (DOTAP-sulfate);

[0093] Or dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptathriacontadecan-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), morpholino cholesterol (Mo-CHOL).

[0094] The present invention also provides an application of an ionizable lipid composition in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

[0095] The beneficial effects of the present invention are as follows: the ionizable lipid compound of the present invention has a high encapsulation rate for the active agent, strong stability, and high expression efficiency, which is conducive to the release of the active agent into the cell and increases the expression amount. It can deliver nucleic acid molecules, small molecule compounds, polypeptides or proteins, etc., and can successfully transport nucleic acid molecules into cells and express them. The ionizable lipid compound of the present invention broadens the types and scope of ionizable lipid compounds and is of great significance to the development and application of nucleic acid therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 Transmission electron microscopy images of lipid nanoparticles.

[0097] Figure 2 This is a stability test of lipid nanoparticles within 20 hours.

[0098] Figure 3 Figure 3 is the toxicity of different lipid nanoparticles to ATP7B knockout hepatocytes at different concentrations.

[0099] Figure 4 The effects of different lipid nanoparticles at different concentrations on the proliferation of ATP7B knockout hepatocytes. DETAILED DESCRIPTION

[0100] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of this application, not all of them. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0101] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0102] In this application, when referring to numerical ranges, unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0103] In the present application, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0104] Unless otherwise specified, the components, raw materials or instruments used in the embodiments and comparative examples of the present invention are all commercially available raw materials or instruments, and the components and raw materials used in each parallel experiment are all of the same kind.

[0105] The ionizable lipids in the embodiments can be conveniently represented as a hydrophobic tail connected to a head group containing an ionizable tertiary amine group. For example, the ionizable lipid compound:

[0106]

[0107] It can be thought of as a combination of a head group and two hydrophobic tails as follows:

[0108]

[0109] Suitable ionizable lipids include compounds consisting of any combination of head groups and hydrophobic tails listed below.

[0110] Some suitable head groups include those shown in Table 1:

[0111] Table 1

[0112]

[0113]

[0114] Some suitable hydrophobic tails include those shown in Table 2:

[0115] Table 2

[0116]

[0117]

[0118]

[0119]

[0120]

[0121] These compounds can be prepared by at least one of the techniques described herein or known organic synthesis techniques. Several examples of compound syntheses are shown below.

[0122] Example 1

[0123] Synthesis route of compound 1:

[0124]

[0125] Step 1: Synthesis of compound 1

[0126] To 4-aminobenzoic acid-2-(diethylamino)ethyl ester (4mmol, 1eq, 945.28mg) was added THF (30mL), RT, 300rpm to dissolve; octadecanal (2.5eq, 10mmol, 2.69g) was added, high-purity nitrogen was introduced, 400rpm, RT to react for 30min; STAB (2.5eq, 10mmol, 2.119g) was added, THF (20mL) was added, RT, 500rpm high-purity nitrogen was protected to react for 24h; saturated NaHCO3 aqueous solution (150mL) was added to the solution, R The reaction mixture was stirred at 400 rpm for 45 minutes to quench unreacted STAB. The solution was transferred to a separatory funnel, and DCM (150 mL) was added. The mixture was shaken vigorously for 5 minutes, allowed to stand for 10 minutes, and the lower organic phase was collected. This process was repeated three times. The mixture was dried over anhydrous NaSO. Insoluble salts were removed by vacuum filtration, and the filtrate was rotary evaporated to obtain a crude product. The crude product was purified on a silica gel column using a DCM:ultra (85:15) solvent, with the ultra being a mixture (mixing ratio of DCM / MeOH / NH4OH (aq) = 75 / 22 / 3) to obtain Compound 1 as a colorless, transparent, oily liquid. High-resolution mass spectrometry (HRMS) m / z (ESI): 742.38 [M+H]+ for Compound 1.

[0127] Example 2

[0128] Synthesis route of compound 2:

[0129]

[0130] Step 1: Synthesis of compound 2-1

[0131] THF (190 mL) was added to oleic acid (4.71 g, 15 mmol, 1.0 eq) and the mixture was cooled to 0°C. LAH solution in THF (1 mol / L, 22.5 mmol, 1.5 eq, 22.5 mL) was added dropwise with stirring at 500 rpm. After the addition of the LAH solution, the reaction solution was returned to room temperature (RT) and stirred overnight. The reaction solution was cooled to 0°C and ultrapure water (0.85 mL) - NaOH solution (1 N, 1 mol / L, 0.85 mL) - ultrapure water (2.6 mL) were slowly added dropwise in the following order. Filter through celite and collect the filtrate; the filtrate is dried by rotary evaporation to remove the solvent to obtain a colorless oily fatty alcohol crude product 2-1;

[0132] Step 2: Synthesis of compound 2-2

[0133] Compound 2-1 (5.753 g, 1 eq, 15 mmol,) was dissolved in DCM (160 mL) at RT, 300 rpm; NaHCO3 (8.821 g, 105 mmol, 7 eq) was added, followed by DMP (7.634 g, 18 mmol, 1.2 eq), and the reaction solution was reacted at RT, 500 rpm for 1 h; the insoluble matter was removed by filtration; the filtrate was diluted with petroleum ether (160 mL); the petroleum ether organic phase was washed three times with saturated NaHCO3 solution (300 mL / time), and the organic phase was collected; the organic phase was washed three times with saturated NaCl solution (300 mL / time), and the organic phase was collected; anhydrous Na2SO4 was added to the organic phase, shaken and allowed to stand for 30 min to absorb residual moisture; the dried organic phase was filtered to remove Na2SO4, and the organic phase filtrate was collected; the organic phase was transferred to a rotary evaporator to remove the solvent to obtain the crude product compound 2-2.

[0134] Step 3: Synthesis of compound 2

[0135] To 2-(diethylamino)ethyl 4-aminobenzoate (4mmol, 1eq, 945.28mg) was added THF (30mL), RT, 300rpm to dissolve; compound 2-2 (2.5eq, 10mmol, 2.66g) was added, high-purity nitrogen was introduced, 400rpm, RT to react for 30min; STAB (2.5eq, 10mmol, 2.119g) was added, THF (20mL) was added, RT, 500rpm high-purity nitrogen was protected to react for 24h; saturated NaHCO3 aqueous solution (150mL) was added to the solution, The unreacted STAB was quenched at 400 rpm for 45 minutes at room temperature. The solution was transferred to a separatory funnel, and DCM (150 mL) was added. The mixture was shaken vigorously for 5 minutes, allowed to stand for 10 minutes, and the lower organic phase was collected. This was repeated three times. The mixture was dried over anhydrous NaSO. Insoluble salts were removed by vacuum filtration, and the filtrate was rotary evaporated to obtain the crude product. The crude product was purified on a silica gel column using a DCM:ultra (85:15) solvent, with the ultra being a mixture (mixture ratio of DCM / MeOH / NH4OH (aq) = 75 / 22 / 3) to obtain Compound 2 as a colorless, transparent, oily liquid. High-resolution mass spectrometry (HRMS) m / z (ESI): 739.47 [M+H]+ for Compound 2.

[0136] Example 3

[0137] Synthesis route of compound 3:

[0138]

[0139] Step 1: Synthesis of compound 3-1

[0140] To linoleic acid (4.21 g, 15 mmol, 1.0 eq) was added THF (190 mL) and cooled to 0°C. LAH solution in THF (1 mol / L, 22.5 mmol, 1.5 eq, 22.5 mL) was added dropwise with stirring at 500 rpm. After the addition of the LAH solution was completed, the reaction solution was returned to room temperature (RT) and stirred overnight. The reaction solution was cooled to 0°C and ultrapure water (0.85 mL) - NaOH solution (1 N, 1 mol / L, 0.85 mL) - ultrapure water (2.6 mL) were slowly added dropwise in the order of: Filter through celite and collect the filtrate; the filtrate is dried by rotary evaporation to remove the solvent to obtain a colorless oily fatty alcohol crude product 3-1;

[0141] Step 2: Synthesis of compound 3-2

[0142] Compound 3-1 (4 g, 1 eq, 15 mmol) was dissolved in DCM (160 mL) at RT and 300 rpm; NaHCO3 (8.821 g, 105 mmol, 7 eq) was added, followed by DMP (7.634 g, 18 mmol, 1.2 eq), and the reaction solution was reacted at RT and 500 rpm for 1 h; the insoluble matter was removed by filtration; the filtrate was diluted into petroleum ether (160 mL); the petroleum ether organic phase was washed three times with a saturated NaHCO3 solution (300 mL / time), and the organic phase was collected; the organic phase was washed three times with a saturated NaCl solution (300 mL / time), and the organic phase was collected; anhydrous Na2SO4 was added to the organic phase, shaken and allowed to stand for 30 min to absorb residual moisture; the dried organic phase was filtered to remove Na2SO4, and the organic phase filtrate was collected; the organic phase was transferred to a rotary evaporator to remove the solvent to obtain a crude product compound 3-2.

[0143] Step 3: Synthesis of compound 3

[0144] To 4-aminobenzoic acid-2-(diethylamino)ethyl ester (4mmol, 1eq, 945.28mg) was added THF (30mL), RT, 300rpm to dissolve; compound 3-2 (2.5eq, 10mmol, 2.64g) was added, high-purity nitrogen was introduced, 400rpm, RT to react for 30min; STAB (2.5eq, 10mmol, 2.119g) was added, THF (20mL) was added, RT, 500rpm high-purity nitrogen was protected to react for 24h; saturated NaHCO3 aqueous solution (150mL) was added to the solution, The unreacted STAB was quenched at RT and 400 rpm for 45 minutes. The solution was transferred to a separatory funnel, and DCM (150 mL) was added. The mixture was shaken vigorously for 5 minutes, allowed to stand for 10 minutes, and the lower organic phase was collected. This was repeated three times. The mixture was dried over anhydrous Na2SO4. Insoluble salts were removed by vacuum filtration, and the filtrate was rotary evaporated to obtain a crude product. The crude product was purified by silica gel column with a solvent of DCM:ultra (85:15), where ultra was a mixture (mixing ratio of DCM / MeOH / NH4OH (aq) = 75 / 22 / 3) to obtain compound 3 as a colorless, transparent, oily liquid. High-resolution mass spectrum of compound 3: HRMS m / z (ESI): 734.37 [M+H] + .

[0145] Example 4

[0146] Synthesis route of compound 4:

[0147]

[0148] Step 1: Synthesis of compound 4-1

[0149] To linolenic acid (4.18 g, 15 mmol, 1.0 eq) was added THF (190 mL) and cooled to 0°C. LAH solution in THF (1 mol / L, 22.5 mmol, 1.5 eq, 22.5 mL) was added dropwise with stirring at 500 rpm. After the addition of the LAH solution was completed, the reaction solution was returned to room temperature and stirred overnight. The reaction solution was cooled to 0°C and ultrapure water (0.85 mL) - NaOH solution (1 N, 1 mol / L, 0.85 mL) - ultrapure water (2.6 mL) were slowly added dropwise in the order of: Filter through celite and collect the filtrate; the filtrate is dried by rotary evaporation to remove the solvent to obtain a colorless oily fatty alcohol crude product 4-1;

[0150] Step 2: Synthesis of compound 4-2

[0151] Compound 4-1 (3.97 g, 1 eq, 15 mmol,) was dissolved in DCM (160 mL) at RT, 300 rpm; NaHCO3 (8.821 g, 105 mmol, 7 eq) was added, followed by DMP (7.634 g, 18 mmol, 1.2 eq), and the reaction solution was reacted at RT, 500 rpm for 1 h; the insoluble matter was removed by filtration; the filtrate was diluted with petroleum ether (160 mL); the petroleum ether organic phase was washed three times with saturated NaHCO3 solution (300 mL / time), and the organic phase was collected; the organic phase was washed three times with saturated NaCl solution (300 mL / time), and the organic phase was collected; anhydrous Na2SO4 was added to the organic phase, shaken and allowed to stand for 30 min to absorb residual moisture; the dried organic phase was filtered to remove Na2SO4, and the organic phase filtrate was collected; the organic phase was transferred to a rotary evaporator to remove the solvent to obtain a crude product compound 4-2.

[0152] Step 3: Synthesis of compound 4

[0153] To 4-aminobenzoic acid-2-(diethylamino)ethyl ester (4mmol, 1eq, 945.28mg) was added THF (30mL), RT, 300rpm to dissolve; compound 4-2 (2.5eq, 10mmol, 2.62g) was added, high-purity nitrogen was introduced, 400rpm, RT to react for 30min; STAB (2.5eq, 10mmol, 2.119g) was added, THF (20mL) was added, RT, 500rpm high-purity nitrogen was protected to react for 24h; saturated NaHCO3 aqueous solution (150mL) was added to the solution, The unreacted STAB was quenched at RT and 400 rpm for 45 minutes. The solution was transferred to a separatory funnel, and DCM (150 mL) was added. The mixture was shaken vigorously for 5 minutes, allowed to stand for 10 minutes, and the lower organic phase was collected. This was repeated three times. The mixture was dried over anhydrous Na2SO4. Insoluble salts were removed by vacuum filtration, and the filtrate was rotary evaporated to obtain a crude product. The crude product was purified by silica gel column with a solvent of DCM:ultra (85:15), where ultra was a mixture (mixing ratio of DCM / MeOH / NH4OH (aq) = 75 / 22 / 3) to obtain compound 4 as a colorless, transparent, oily liquid. High-resolution mass spectrum of compound 4: HRMS m / z (ESI): 730.38 [M+H] + .

[0154] Example 5

[0155] Synthesis route of compound 5:

[0156]

[0157] Step 1: Synthesis of compound 5-1

[0158] 90% oleic acid (5.0 g, 17.7 mmol) was added to a KOH aqueous solution (1.25%, 400 mL) at 50°C with stirring in an oil bath at 300 rpm, and stirred until a transparent clear liquid was formed; another portion of KOH aqueous solution (1.25%, 400 mL) was added to the clear oleic acid solution, and KMnO4 (5.0 g, 31.64 mmol) was added to the solution and stirred for 15 min; sodium thiosulfate (10% aqueous solution) and sodium sulfite Na2SO3 (20% aqueous solution) were added to the reaction solution to quench the reaction; hydrochloric acid solution (37%) was added and acidified for 2-3 h until a white loose precipitate 5-1 was formed, which was filtered to obtain compound 5-1; compound 5-1 was transferred to a vacuum drying oven at a constant temperature of 45°C and dried in a vacuum oven with a yield of approximately 90%; the product 5-1 was purified by recrystallization; after crystallization was complete, compound 5-1 was filtered and dried;

[0159] Step 2: Synthesis of compound 5-2

[0160] DCM (45 mL) was added to compound 5-1 (1.2 g, 1 eq, 3.792 mmol); NaIO4 aqueous solution (3 eq, 11.376 mmol, 2.433 g, 54.072 mg / mL, 45 mL) was added to the DCM solution and stirred at 300 rpm at RT; tetrabutylammonium hydrogen sulfate TBA-HS aqueous solution (0.5 eq, 1.896 mmol, 643.75 mg, 5 mL) was added to the two-phase solution and stirred at 500 rpm at RT for 10 h; the reaction solution was separated and the organic phase was collected; the organic phase was dried over anhydrous Na2SO4; the solvent was removed by filtration and rotary evaporation, and the product 5-2 was obtained by purification on a silica gel column (hexane:ethyl acetate = 1:1);

[0161] Step 3: Synthesis of compound 5-4

[0162] Compound 5-2 (516 mg, 172 g / mol, 3 mmol, 1 eq) and EDCI (632.61 mg, 1.1 eq, 3.3 mmol) were dissolved in anhydrous DCM (15 mL) at RT and 300 rpm; DMAP (10 mg, 0.082 mmol) was added to the reaction solution; compound 5-3 (1.5 eq, 4.5 mmol, 586.04 mg) was added to the reaction system, and the reaction solution was stirred at 0°C and 500 rpm for 2 h; the reaction solution was warmed to RT and stirred at 500 rpm for 24 h; ultrapure water was added to the reaction product, and the organic phase was collected and washed repeatedly with ultrapure water; the organic phase was dried over anhydrous Na2SO4; the solvent was removed by suction filtration and rotary evaporation, and the product 5-4 was obtained by purification on a silica gel column (hexane:ethyl acetate = 95:5);

[0163] Step 4: Synthesis of compound 5

[0164] To 4-aminobenzoic acid-2-(diethylamino)ethyl ester (4mmol, 1eq, 945.28mg) was added THF (30mL), RT, 300rpm to dissolve; compound 5-4 (2.5eq, 10mmol, 2.84g) was added, high-purity nitrogen was introduced, 400rpm, RT to react for 30min; STAB (2.5eq, 10mmol, 2.119g) was added, THF (20mL) was added, RT, 500rpm high-purity nitrogen was protected to react for 24h; saturated NaHCO3 aqueous solution (150mL) was added to the solution, The unreacted STAB was quenched at RT and 400 rpm for 45 minutes. The solution was transferred to a separatory funnel, and DCM (150 mL) was added. The mixture was shaken vigorously for 5 minutes, allowed to stand for 10 minutes, and the lower organic phase was collected. This was repeated three times. The mixture was dried over anhydrous Na2SO4. Insoluble salts were removed by vacuum filtration, and the filtrate was rotary evaporated to obtain a crude product. The crude product was purified by silica gel column with a solvent of DCM:ultra (85:15), where ultra was a mixture (mixing ratio of DCM / MeOH / NH4OH (aq) = 75 / 22 / 3) to obtain compound 5 as a colorless, transparent, oily liquid. High-resolution mass spectrum of compound 5: HRMS m / z (ESI): 774.87 [M+H] + .

[0165] Example 6

[0166] Synthesis route of compound 6:

[0167]

[0168] Step 1: Synthesis of compound 6-1

[0169] To a solution of 2-hexyldecanol (1.0 g, 4.13 mmol, 1.0 eq) and 6-bromohexanoic acid (0.96 g, 5 mmol, 1.2 eq) in DCM (15 mL) were added diisopropylethylamine (133.45 mg, 1.04 mmol, 0.25 eq) and DMAP (100.9 mg, 0.835 mmol, 0.2 eq). The mixture was stirred at room temperature for 5 min, followed by the addition of EDCI (1.425 g, 7.435 mmol, 1.8 eq). The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was diluted with DCM (150 mL), washed with saturated NaHCO , ultrapure water, and saturated NaCl solution. The combined organic layers were dried over Na 2 SO 4 and the solvent was removed by rotary evaporation to obtain a crude product. The crude product was purified by column chromatography (eluent: n-hexane solution containing 0.1% EA (volume percentage)), and the pure product fractions were evaporated to obtain compound 6-1 as a colorless oil.

[0170] Step 2: Synthesis of compound 6-2

[0171] THF (190 mL) was added to oleic acid (4.71 g, 15 mmol, 1.0 eq) and the mixture was cooled to 0°C. LAH solution in THF (1 mol / L, 22.5 mmol, 1.5 eq, 22.5 mL) was added dropwise with stirring at 500 rpm. After the addition of the LAH solution, the reaction solution was returned to room temperature and stirred overnight. The reaction solution was cooled to 0°C and ultrapure water (0.85 mL) - NaOH solution (1 N, 1 mol / L, 0.85 mL) - ultrapure water (2.6 mL) were slowly added dropwise in the following order. Filter through celite and collect the filtrate; the filtrate is dried by rotary evaporation to remove the solvent to obtain a colorless oily fatty alcohol crude product 6-2;

[0172] Step 3: Synthesis of compound 6-3

[0173] Compound 6-2 (5.753 g, 1 eq, 15 mmol) was dissolved in DCM (160 mL) at RT and 300 rpm; NaHCO3 (8.821 g, 105 mmol, 7 eq) was added, followed by DMP (7.634 g, 18 mmol, 1.2 eq), and the reaction solution was reacted at RT and 500 rpm for 1 h; the insoluble matter was removed by filtration; the filtrate was diluted with petroleum ether (160 mL); the petroleum ether organic phase was washed three times with a saturated NaHCO3 solution (300 mL / time), and the organic phase was collected; the organic phase was washed three times with a saturated NaCl solution (300 mL / time), and the organic phase was collected; anhydrous Na2SO4 was added to the organic phase, shaken and allowed to stand for 30 minutes to absorb residual moisture; the dried organic phase was filtered to remove Na2SO4, and the organic phase filtrate was collected; the organic phase was transferred to a rotary evaporator to remove the solvent to obtain the crude product compound 6-3.

[0174] Step 4: Synthesis of compound 6-4

[0175] THF (30 mL) was added to abacavir (4 mmol, 1 eq, 1.145 g) and dissolved at RT, 300 rpm; compound 6-3 (1 eq, 4 mmol, 1.136 g) was added, and high-purity nitrogen was introduced at 400 rpm and RT for 30 min; STAB (1.25 eq, 5 mmol, 1.06 g) was added, and THF (20 mL) was added, and high-purity nitrogen was protected at RT, 500 rpm for 24 h; saturated NaHCO3 aqueous solution (150 mL) was added to the solution, and the mixture was stirred at RT, 400 rpm. The unreacted STAB was quenched after 45 min; the solution was transferred to a separatory funnel, DCM (150 mL) was added, and the mixture was shaken vigorously for 5 min. The mixture was allowed to stand for 10 min and the lower organic phase was collected. The reaction was repeated three times; the mixture was dried over anhydrous Na2SO4; the insoluble salts were removed by vacuum filtration, and the filtrate was rotary evaporated to obtain a crude product. The crude product was purified by silica gel column with a solvent of DCM:ultra (85:15), where ultra was a mixture (mixing ratio of DCM / MeOH / NH4OH (aq) = 75 / 22 / 3) to obtain compound 6-4 as a colorless, transparent, oily liquid.

[0176] Step 5: Synthesis of compound 6

[0177] Compound 6-4 (397.24 mg, 0.74 mmol, 1.0 eq), compound 6-1 (620.8 mg, 1.47 mmol, 2.0 eq), K2CO3 (306.3 mg, 2.22 mmol, 3.0 eq), Cs2CO3 (71.7 mg, 0.22 mmol, 0.3 eq) and NaI (32.9 mg, 0.22 mmol, 0.3 eq) in acetonitrile (15 mL) were stirred at 90 ° C overnight. The reaction mixture was diluted with EA, washed with water and concentrated. The residue was purified by chromatography to obtain the target product, compound 6. High-resolution mass spectrum HRMS m / z (ESI) of compound 6: 876.77 [M+H] + .

[0178] At least one of the organic synthesis techniques of Examples 1 to 6 above is used to prepare the ionizable lipid compounds of the present invention. Some typical ionizable lipid compounds and their structures are shown in Table 3:

[0179] Table 3

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] Preparation of nanolipid particles (LNP formulation):

[0193] Cationic lipid compounds were dissolved in ethanol with DSPC, cholesterol, and DMG-PEG2000 at a molar ratio of 50:10:38.5:1.5, respectively, to prepare an ethanolic lipid solution. Mus ATP7B mRNA was then diluted in 10 to 50 mM citrate buffer (pH = 4) to obtain an mRNA aqueous solution. Liposomes were prepared by mixing the ethanolic lipid solution and the mRNA aqueous solution at a volume ratio of 1:3 using a microfluidic device, with a total lipid to mRNA weight ratio of approximately 15 to 30:1. The ethanol was removed by static dialysis for 12-24 hours and replaced with DPBS. Finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter to obtain a LNP formulation encapsulating Mus ATP7B mRNA using ionizable lipid / DSPC / cholesterol / DMG-PEG2000 (50 / 10 / 38.5 / 1.5 mol%).

[0194] Characterization of Lipid Nanoparticles (LNP Formulations):

[0195] The microstructure of lipid nanoparticles was observed by transmission electron microscopy. The nanoparticles prepared by the microfluidic mixer were dropped onto a 200-mesh carbon film, and 2% (w / v) phosphotungstic acid was added to the copper mesh. The mesh was stained for 2 minutes and dried at room temperature. Subsequently, the mesh was placed under a transmission electron microscope for observation and photography. The micromorphology of lipid nanoparticles is shown in FIG. Figure 1 shown.

[0196] The size and polydispersity index (PDI) of lipid nanoparticles were determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) in 173° backscattering detection mode. The test results are shown in Table 4.

[0197] The encapsulation efficiency of lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantification kit (Thermo Fisher Scientific, UK). The test results are shown in Table 4.

[0198] Table 4

[0199]

[0200]

[0201]

[0202] The above results show that the head group is The particle size of the prepared lipid nanoparticles is about 100-200nm, and the encapsulation efficiency of mRNA is generally 80%-95%, which is a low encapsulation efficiency. The prepared lipid nanoparticles have a particle size of approximately 100-130 nm and generally achieve a high mRNA encapsulation efficiency of 97%-99%. This difference is hypothesized to be related to the hydrophilicity of the head group and the number of tertiary amines. A higher number of tertiary amines or the presence of hydroxyl groups in the head group results in a stronger hydrophilicity, resulting in a tighter binding to mRNA in an acidic environment and a higher encapsulation efficiency.

[0203] Stability testing of lipid nanoparticles:

[0204] The lipid nanoparticles were incubated at 4°C in PBS supplemented with 10% FBS for 2 h. Figure 2 As shown, the results show that the lipid nanoparticles prepared from the ionizable lipid compound of the present invention can still maintain a stable particle size in a 10% FBS PBS solution within 20 hours, indicating that the lipid nanoparticles are relatively stable, providing a basis for their wider application.

[0205] In vitro cytotoxicity test of nanolipid particles:

[0206] The CCK-8 assay was used to test the toxicity of different lipid nanoparticles on ATP7B knockout hepatocytes at different concentrations. 4 Cells were evenly seeded in a 96-well plate (100 μL / well). The corresponding concentration of lipid nanoparticles was added to each well and cultured for 48 h. CCK-8 solution (10 μL / well) was then added and cultured for another 4 h. The absorbance (OD) at 450 nm was measured using a microplate reader. The experiment was repeated five times, and the cell viability of each group was calculated according to the following formula:

[0207] Cell viability (100%) = 100% × (ODtreated -OD blank ) / (OD control -OD blank )

[0208] Among them, OD treated is the OD value of cells treated with lipid nanoparticles, OD control is the OD value of normal cells, OD blank is the OD value of CCK-8 solution, and the results are as follows Figure 3 shown.

[0209] In vitro cell proliferation assay of nanolipid particles:

[0210] The CCK-8 method was used to test the effects of different lipid nanoparticles on the proliferation of ATP7B-knockout hepatocytes at different concentrations. ATP7B-knockout hepatocytes (1x104 cells / well) in the logarithmic growth phase were evenly inoculated into a 96-well plate, each well containing 200 μL of complete culture medium, and cultured statically. Lipid nanoparticles of corresponding concentrations were added to each well and cultured for 48 hours. Then, CCK-8 solution (10 μL / well) was added and cultured for another 4 hours. The absorbance value (OD) at 450 nm was measured using a microplate reader. The experiment was repeated 5 times, and the cell survival rate of each group was calculated according to the following formula:

[0211] Cell viability (100%) = 100% × (ODtreated - ODblank) / (ODcontrol - ODblank)

[0212] Among them, ODtreated is the OD value of cells treated with lipid nanoparticles, ODcontrol is the OD value of normal cells, and ODblank is the OD value of CCK-8 solution. The results are as follows Figure 4 shown.

[0213] like Figure 3 and Figure 4 As shown, lipid nanoparticles prepared from different ionizable lipid compounds have similar toxicity to cells, and with the increase of lipid nanoparticle concentration, the cells show slight toxicity.

[0214] In vivo testing of nanolipid particles:

[0215] Lipid nanoparticles prepared in some of the examples were systemically administered via tail vein injection to 6-8 week-old male ATP7B knockout BALB / c mice at a dose of 0.5 mg / kg. Six hours after administration, the mice were euthanized and liver tissue was harvested. The tissue was ground and centrifuged at 10,000 g for 10 minutes at 4°C. The supernatant was then snap-frozen and stored at -80°C for analysis. ELSA analysis was performed using a commercially available kit. The ATP7B expression levels (ng / mL) measured for each test group are shown in Table 5.

[0216] Table 5

[0217]

[0218]

[0219]

[0220] The above results indicate that the lipid nanoparticles prepared from the ionizable lipids of the present invention can carry MusATP7B mRNA into the liver tissue of mice and express it. The prepared lipid nanoparticles carrying Mus ATP7B mRNA had low expression efficiency in the liver tissue of mice. The prepared lipid nanoparticles loaded with MusATP7B mRNA showed high expression efficiency in mouse liver tissue. Furthermore, for the same head group, the more branched the hydrophobic tail, the higher the expression efficiency of MusATP7B mRNA in mouse liver tissue. This difference is speculated to be due to the fact that a highly branched hydrophobic tail facilitates the endosomal escape of lipid nanoparticles in hepatocytes, thereby facilitating the intracellular release of MusATP7B mRNA and increasing its expression.

[0221] In summary, the ionizable lipid compounds of the present invention can deliver nucleic acid molecules, small molecule compounds, polypeptides or proteins, etc. The vectors prepared using the ionizable lipid compounds of the present invention have high encapsulation efficiency for nucleic acid molecules and can successfully transport nucleic acid molecules into cells for expression.

[0222] 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 the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An ionizable lipid compound, characterized in that The ionizable lipid compound is at least one of the compound of formula (I), its pharmaceutically acceptable salts, stereoisomers, and solvates; In formula (I), G1 and G2 are each independently selected from a bond, a hydroxyl substituted or unsubstituted C 10 -C 18 Straight chain alkyl, C 10 -C 26 Branched alkyl, hydroxy substituted or unsubstituted C4-C 20 Straight chain alkenyl, hydroxy substituted or unsubstituted C4-C 20 Branched alkenyl, hydroxy substituted or unsubstituted C2-C 16 Straight chain alkylene, hydroxy substituted or unsubstituted C2-C 16 Straight chain alkylene, branched chain alkylene, hydroxy substituted or unsubstituted C2-C 16 Straight chain alkenylene, hydroxy substituted or unsubstituted C2-C 16 branched alkenylene; Said X1 and X2 are each independently selected from a bond, -O-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(O)S-; The R1 and R2 are each independently selected from C6-C 14 Straight chain alkyl, C6-C 14 Branched alkyl, C6-C 14 Straight chain alkenyl or C6-C 14 branched alkenyl; The group represented by formula (II) is a group containing a protonatable tertiary amine; The group containing a protonatable tertiary amine may be selected from one of the following groups:

2. The ionizable lipid compound according to claim 1, characterized in that Satisfy at least one of the following (a) to (d): (a) when X1 and X2 are a bond, R1 and R2 are alkyl or alkenyl; (b) X1 and X2 are each independently a bond, -S-, -C(O)O-, -OC(O)-, -C(O)NH- or -OC(O)O-; (c) X3 is selected from a bond, -C(O)O- or -OC(O)-; (d) R7 and R8 are each independently selected from H or alkoxy.

3. The ionizable lipid compound according to claim 1, characterized in that said R3 and R4 together with the nitrogen atom to which they are attached form a heterocyclic ring; or One of R3 and R4 is selected from an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a heterocyclic ring or a substituted heterocyclic compound, and the other forms a heterocyclic ring or a heteroaryl group with the nitrogen atom to which they are attached and R5.

4. The ionizable lipid compound according to claim 1, characterized in that The G1 and G2 can be independently selected from one of the following groups:

5. The ionizable lipid compound according to claim 1, characterized in that The R1 and R2 can be independently selected from one of the following groups:

6. The ionizable lipid compound according to claim 1, characterized in that The ionizable lipid compound is at least one of the compounds of formula (Ii), formula (I-ii), formula (I-iii), and pharmaceutically acceptable salts, stereoisomers, and solvates thereof; In formula (Ii), formula (I-ii), and formula (I-iii); The z, p, q, t, and u are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8; The R 11 , R 12 , R 100 , R 200 Each independently selected from C5-C 15 Straight chain alkyl, C5-C 15 Branched alkyl, C5-C 15 Straight chain alkenyl, C5-C 15 Branched alkenyl, C5-C 15 Straight chain alkynyl or C5-C 15 branched chain alkynyl; The R 300 Selected from C 12 -C 24 Straight chain alkyl, C 12 -C 24 Branched alkyl, C 12 -C 24 Straight chain alkenyl, C 12 -C 24 Branched alkenyl, C 12 -C 24 Straight chain alkynyl or C 12 -C 24 branched chain alkynyl; The W is a group containing a protonatable tertiary amine, and the W is selected from one of the following groups:

7. The ionizable lipid compound according to claim 6, characterized in that Satisfy at least one of the following (1) to (4): (1) in formula (Ii), p and t are each independently 4, 5 or 6; (2) q and u in formula (Ii) are each independently 0, 1, 2, 3 or 4; (3) in formula (I-ii), z and t are each independently 0, 1, 2, 3, 4, 5 or 6; (4) Formula (I-iii) and t is 0, 1, 2, 3, 4, 5 or 6.

8. The ionizable lipid compound according to claim 1, characterized in that The ionizable lipid compound is selected from at least one of the following compounds:

9. Use of the ionizable lipid compound according to any one of claims 1 to 8 in preparing a bioactive substance delivery system, characterized in that: The delivery system is a microparticle, a nanoparticle, a liposome, a lipid nanoparticle or a microbubble.

10. Use of the ionizable lipid compound according to any one of claims 1 to 8 in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

11. An ionizable lipid composition, characterized in that The invention comprises a carrier and an active agent, wherein the carrier comprises an ionizable lipid compound, and the ionizable lipid compound is the ionizable lipid compound according to any one of claims 1 to 8.

12. The ionizable lipid composition according to claim 11, characterized in that The molar percentage of the ionizable lipid compound in the carrier is 20-60%.

13. The ionizable lipid composition according to claim 11, characterized in that The carrier also includes neutral lipids, PEG lipids, and sterols.

14. The ionizable lipid composition according to claim 13, characterized in that The molar percentage of the neutral lipid in the carrier is 5-25%; and / or The molar percentage of the PEG lipid in the carrier is 0.5-15%; and / or The molar percentage of the sterol in the carrier is 25-55%.

15. The ionizable lipid composition according to claim 13, characterized in that The neutral lipids include at least one of 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), oleoylphosphatidylcholine, and 1-palmitoyl-2-oleoylphosphatidylethanolamine.

16. The ionizable lipid composition according to claim 13, characterized in that The PEG lipid includes at least one of 2-[(polyethylene glycol)-2000]-N,N-tetracosyl acetamide, 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)], PEG-disterol glycerol, PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide, PEG-dipalmitoyl phosphatidylethanolamine, and PEG-1,2-dimyristoyloxypropyl-3-amine.

17. The ionizable lipid composition according to claim 13, characterized in that The sterol includes at least one of cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and corticosteroids.

18. The ionizable lipid composition according to claim 11, characterized in that The active agent includes at least one of a nucleic acid molecule, a small molecule compound, a polypeptide, and a protein.

19. The ionizable lipid composition according to claim 11, characterized in that The nucleic acid includes at least one of single-stranded DNA, double-stranded DNA, short isomers, agomir, antagomir, antisense molecules, small interfering RNA, asymmetric interfering RNA (aiRNA), microRNA, Dicer-substrate RNA, small hairpin RNA, transfer RNA (tRNA), messenger RNA, locked nucleic acid, peptide nucleic acid, morpholino oligonucleotide, aptamer, and ribozyme.

20. The ionizable lipid composition according to claim 11, characterized in that The mass ratio of the carrier to the active agent is (5-50):

1.

21. Use of the ionizable lipid composition according to any one of claims 11 to 20 in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

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