Ionizable lipid compound and uses thereof

Abstract

The application provides an ionizable lipid compound as shown in formula (I) and application thereof. The lipid compound, as a key component of a lipid nanoparticle (LNP), can significantly improve the encapsulation rate and delivery efficiency of mRNA. The mRNA-LNP system exhibits excellent transfection performance and biological safety in in vitro cells and in vivo animal models, and has a broad clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an ionizable lipid compound and its applications. Background Technology

[0002] Compared to traditional small-molecule chemical drugs and antibody drugs, nucleic acid drugs theoretically overcome the limitation of the "drugifiability" of target proteins. With the advancement of clinical trials and the maturation of related technologies, nucleic acid drugs are showing a broader application prospect. However, as exogenous drugs, their entry into the body to exert their effects requires overcoming multiple obstacles: instability, immunogenicity, low cellular uptake efficiency, and difficulty in endosome escape. Therefore, an efficient and safe delivery system is crucial to ensuring that nucleic acid drugs overcome these bottlenecks and achieve stable targeted therapeutic effects.

[0003] Currently, lipid nanoparticles (LNPs), with their unique structure and physicochemical properties, exhibit high delivery efficiency and good safety in vivo, and have become one of the mainstream delivery systems for nucleic acid drugs. Their successful application in COVID-19 mRNA vaccines has further promoted the expansion of LNPs in fields such as tumor vaccines, cell therapy, and gene editing. In the LNP system, ionizable lipid compounds serve as the core component, undertaking important functions such as binding negatively charged nucleic acids, promoting cellular uptake and endosome escape, and enhancing the in vivo transfection effect of nucleic acid drugs.

[0004] Although various ionizable lipid compounds have been disclosed in existing technologies, most still suffer from limited transfection efficiency, insufficient stability, or potential safety hazards. Especially in applications requiring the delivery of large doses of nucleic acid drugs, safety becomes a crucial factor that cannot be ignored, in addition to efficient transfection.

[0005] Therefore, there is an urgent need in this field to provide an ionizable lipid compound with high transfection efficiency, good stability, and high safety. Summary of the Invention

[0006] The present invention aims to provide an ionizable lipid compound with high transfection efficiency, good stability and high safety, specifically relating to an ionizable lipid compound and its application.

[0007] In a first aspect of the invention, an ionizable lipid compound is provided, characterized in that the compound has a chemical structure as shown in formula (I): (I) In this context, a and b are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, or 8; G1 and G2 are each independently selected from C2~C10 alkylene groups; G3 is selected from C4-C8 alkylene groups; or G3 is selected from (CH2). d -O-(CH2) e Where d and e are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, or 9, and d+e is an integer from 3 to 10; or G3 is selected from , where g is selected from 1, 2, 3 or 4; L1 is selected from -(C=O)O-, -O(C=O)-, -(C=O)S- or -S(C=O)-; L2 is selected from -(C=O)O- or -O(C=O)-; R1 and R2 are each independently selected from the following group: C1~C20 straight-chain saturated hydrocarbon groups or C2~C20 straight-chain unsaturated hydrocarbon groups; R3 and R4 are each independently selected from the following groups: H, C1~C20 straight-chain saturated hydrocarbon groups or C2~C10 straight-chain unsaturated hydrocarbon groups.

[0008] In another preferred embodiment, G1 and G2 are each independently selected from C4 to C10 hydrocarbon groups.

[0009] In another preferred embodiment, G3 is selected from the group consisting of: C4-C8 alkylene groups, (CH2) groups, etc. d -O-(CH2) e , where d and e are each independently selected from 2, 3, 4, 5, or 6.

[0010] In another preferred embodiment, G3 is a C4 alkylene group or (CH2)2-O-(CH2)2; In another preferred embodiment, L1 is selected from -(C=O)O-, -O(C=O)-, or -S(C=O)-; L2 is selected from -(C=O)O- or -O(C=O)-; R1 and R2 are each independently selected from the following group: C6~C20 straight-chain saturated hydrocarbon groups; preferably, R2 is a C7 straight-chain saturated hydrocarbon group; In another preferred embodiment, the chemical structure of the compound is shown in formula (II): (II) Wherein, r, s, and t are each independently selected from 4, 5, 6, 7, 8, or 9; The definitions of a, b, L1, L2, R3, and R4 are as described above.

[0011] In another preferred embodiment, the ionizable lipid compound is selected from the group consisting of: .

[0012] A second aspect of the present invention provides a lipid nanoparticle, characterized in that it comprises one or more combinations of an ionizable lipid compound, its stereoisomers, its tautomers, or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention.

[0013] In another preferred embodiment, the lipid nanoparticles have a particle size of 60-120 nm.

[0014] In another preferred embodiment, the lipid nanoparticles further comprise any one or more combinations of structural lipids, auxiliary lipids, PEG-lipids, and polymers.

[0015] In another preferred embodiment, the structural lipid is selected from one or more combinations of sterols, nonsterols, or their respective derivatives; and / or The auxiliary lipid is selected from one or more combinations of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, myristoyl phosphatidylglycerol, DOTAP, DODAP, 18:1 PA, HS15, and GL67; and / or The PEG-lipid is selected from one or more combinations of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and methoxy polyethylene glycol bis(tetradecyl)acetamide.

[0016] In another preferred embodiment, the molar ratio of the ionizable lipid compound: auxiliary lipid: structural lipid: PEG-lipid is (20-65): (3-50): (15-60): (0.1-10).

[0017] In another preferred embodiment, the molar ratio of the ionizable lipid compound to the polymer is 0.5:1 to 100:1.

[0018] In another preferred embodiment, the molar ratio of the ionizable lipid compound to the polymer is 10:1 to 80:1.

[0019] In another preferred embodiment, the molar ratio of the ionizable lipid compound to the polymer is 40:1 to 80:1.

[0020] In another aspect, the present invention provides a composition comprising the aforementioned lipid nanoparticles.

[0021] In another preferred embodiment, the composition further comprises the drug contained thereon and / or pharmaceutically acceptable excipients.

[0022] In another preferred embodiment, the drug contained includes one or more of nucleic acid molecules, small molecule compounds, and proteins.

[0023] In another preferred embodiment, the drug contained is mRNA.

[0024] In another preferred embodiment, the mRNA coding region (CDS) is selected from any one of CDS sequence 1 (SEQ ID No. 5), CDS sequence 2 (SEQ ID No. 6), CDS sequence 3 (SEQ ID No. 7), CDS sequence 4 (SEQ ID No. 8), and CDS sequence 5 (SEQ ID No. 9); preferably, the mRNA sequence includes a 5' untranslated region (5'UTR), a coding region (CDS), and a 3' untranslated region (3'UTR). The 5'UTR is selected from any one of the sequences 5'UTR-1 (SEQ ID No. 1), 5'UTR-2 (SEQ ID No. 2), 5'UTR-3 (SEQ ID No. 3), and 5'UTR-4 (SEQ ID No. 4); CDS is selected from any one of CDS sequence 1 (SEQ ID No. 5), CDS sequence 2 (SEQ ID No. 6), CDS sequence 3 (SEQ ID No. 7), CDS sequence 4 (SEQ ID No. 8), and CDS sequence 5 (SEQ ID No. 9); The 3'UTR is selected from any one of the sequences 3'UTR-1 (SEQ ID No. 10), 3'UTR-2 (SEQ ID No. 11), 3'UTR-3 (SEQ ID No. 12), and 3'UTR-4 (SEQ ID No. 13); In another preferred embodiment, the 5'UTR is the sequence shown in 5'UTR-1 (SEQ ID No. 1).

[0025] In another preferred embodiment, the CDR is the sequence shown in CDS sequence 2 (SEQ ID No. 6).

[0026] In another preferred embodiment, the 3'UTR is the sequence shown in 3'UTR-3 (SEQ ID No. 12).

[0027] In another aspect of the present invention, the use of the aforementioned composition in the preparation of medicaments for the prevention and / or treatment of diseases is provided.

[0028] In another preferred embodiment, the composition is used in the preparation of a medicament for treating hepatic propionic acidemia.

[0029] In another preferred embodiment, the composition is used to express PCCA and PPCB proteins in a subject after administration; more preferably, the composition is used to express PCCA and PPCB proteins in the liver of a subject after administration.

[0030] In another preferred embodiment, the object is a mammal, preferably a human.

[0031] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0032] Figure 1 Particle size distribution of LNPs prepared from C4-25 lipids; Figure 2 The study showed that different 5'UTR sequences regulated luciferase expression activity in HEK293 cells; Figure 3 The study demonstrated the luciferase expression activity regulated by different 3'UTR sequences in HEK293 cells. Figure 4 The study demonstrated the luciferase expression activity regulated by different combinations of UTR sequences in HEK293 cells; Figure 5The protein expression levels of CDS sequences with different codon optimizations are shown in HEK293 cells (*p<0.05, **p<0.01, ***p<0.001). Detailed Implementation

[0033] Through systematic and in-depth research, and extensive experimental design and screening, the inventors have successfully developed a novel ionizable lipid compound. This compound can form structurally stable LNPs with other lipids, exhibiting excellent physicochemical properties and significantly improving the encapsulation efficiency and delivery efficiency of mRNA. The mRNA-LNP system has demonstrated excellent transfection performance and biosafety in both in vitro cell models and in vivo animal models, showing broad prospects for clinical application.

[0034] Specifically, the present invention adopts the following technical solution: This invention provides an ionizable lipid compound with the chemical structure shown in formula (Ⅰ): (I) Wherein, a and b are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7 or 8; G1 and G2 are each independently selected from C2~C10 alkylene groups; G3 is selected from C1-C10 alkylene groups; or G3 is selected from (CH2). d -O-(CH2) e Where d and e are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, or 9, and d+e is an integer from 2 to 10; or G3 is selected from , where g is selected from 1, 2, 3 or 4; L1 is selected from -(C=O)O-, -O(C=O)-, -(C=O)S- or -S(C=O)-; L2 is selected from -(C=O)O- or -O(C=O)-; R1 and R2 are each independently selected from C1~C20 straight-chain saturated hydrocarbon groups or C2~C20 straight-chain unsaturated hydrocarbon groups; R3 and R4 are each independently selected from H, C1~C20 straight-chain saturated hydrocarbon groups or C2~C20 straight-chain unsaturated hydrocarbon groups; The term "hydrocarbon group" includes saturated hydrocarbon groups and unsaturated hydrocarbon groups. Preferred saturated hydrocarbon groups are C1-C20 (i.e., 1-20 carbon atoms, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms) hydrocarbon groups, further preferred are C4-C10 hydrocarbon groups, and most preferably are C5-C8 hydrocarbon groups. Preferred unsaturated hydrocarbon groups are C2-C20 (i.e., 2-20 carbon atoms) olefin groups and C2-C20 (i.e., 2-20 carbon atoms) alkyne groups. The unsaturated hydrocarbon group can be an unsaturated polyalkyl group or an unsaturated monoalkyl group.

[0035] When describing "C2~C10 alkylene group", it means that the group can be an alkylene group (such as alkylene group, alkenylene group, ynylene group) with 2 to 10 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms).

[0036] In some embodiments of the present invention, G1 and G2 are each independently selected from C5-C10 alkylene groups; preferably, G1 is a C5-C9 alkylene group and G2 is a C5-C8 alkylene group.

[0037] In some embodiments of the present invention, G3 is selected from C1~C5 alkylene groups, preferably C2~C4 alkylene groups, and more preferably C4 alkylene groups; or G3 is selected from (CH2)2-O-(CH2)2.

[0038] In some embodiments of the present invention, R1 and R2 are each independently selected from C6 to C10 straight-chain saturated hydrocarbon groups, preferably C7 straight-chain saturated hydrocarbon groups.

[0039] A second aspect of the present invention provides a lipid nanoparticle comprising one or more combinations of the above-described ionizable lipid compounds, their stereoisomers, their tautomers, or pharmaceutically acceptable salts thereof.

[0040] The term "stereoisomer" refers to isomers that have the same atomic connection order but different spatial arrangements of atoms.

[0041] The term "tautomer" refers to the phenomenon where the structure of a compound undergoes an equilibrium interconversion between two functional group isomers, and the corresponding isomers are called tautomers.

[0042] The term "pharmaceutically acceptable salt" refers to an acid addition salt or a base addition salt. All compounds of the present invention existing as free bases or free acids can be converted into pharmaceutically acceptable salts by treatment with appropriate inorganic or organic bases or acids according to methods known to those skilled in the art. Salts of the compounds of the present invention can be converted into their free base or acid forms using standard techniques.

[0043] Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by other methods used in the art, such as ion exchange, and containing an amino group. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, citrate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptate, glyceryl phosphate, gluconate, hemisulfate, heptaate, hydroiodate, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, and ammonium salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates. Other pharmaceutically acceptable salts include those formed by the quaternization of amines using suitable electrophilic agents (e.g., alkyl halides) to form quaternized alkylated amino salts.

[0044] The lipid nanoparticles provided by this invention further comprise one or more combinations of structural lipids, auxiliary lipids, PEG-lipids, and polymers.

[0045] In some specific embodiments of the present invention, the molar ratio of ionizable lipid compound: auxiliary lipid: structural lipid: PEG-lipid in the lipid nanoparticles is (20-65): (3-50): (15-60): (0.1-10).

[0046] In some other embodiments of the present invention, the molar ratio of the ionizable lipid compound to the polymer in the lipid nanoparticles is 0.5:1-100:1, preferably 10:1-80:1, and more preferably 40:1-80:1.

[0047] In some specific embodiments of the present invention, the molar ratio of ionizable lipid compound: auxiliary lipid: structural lipid: PEG-lipid in the lipid nanoparticles is (20-65): (3-40): (20-60): (0.1-10).

[0048] In some specific embodiments of the present invention, the molar ratio of ionizable lipid compound: auxiliary lipid: structural lipid: PEG-lipid in the lipid nanoparticles is (35-50): (5-20): (35-50): (1-2).

[0049] The term "structural lipid" refers to a structure that can stabilize the composition, including but not limited to one or more combinations of sterols and their derivatives and non-sterols and their derivatives.

[0050] In some specific embodiments, the structural lipids include, but are not limited to, one or more combinations of sterols and their derivatives, nonsterols, sitosterol, ergosterol, cholesterol, cholesterolenone, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, coprosterol, α-tocopherol, or corticosteroids. Sterols are preferably cholesterol and its derivatives; non-limiting examples of cholesterol derivatives include: polar analogs such as 5α-cholesterol, 5α-coprosterol, cholesterolyl-(2'-hydroxy)ethyl ether, cholesterolyl-(4'-hydroxy)butyl ether, and 6-ketocholesterol; non-polar analogs such as 5α-cholesterol, cholesterolenone, 5α-cholesterone, and decanoic acid cholesterol ester; and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog such as cholesterolyl-(4'-hydroxy)butyl ether. This is not an exhaustive list; the choice of structural lipids is not limited, and any structural lipid can be used in this invention.

[0051] In some specific embodiments, the structural lipid is one or more of cholesterol, sitosterol, ergosterol, corticosteroids and their derivatives.

[0052] In some specific implementations, the structural lipid is cholesterol.

[0053] There is no limitation on the type of "auxiliary lipids", but phospholipid lipids are preferred, including but not limited to: one or more combinations of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, and myristoyl phosphatidylglycerol.

[0054] In some specific embodiments, the auxiliary lipid may be selected from: 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC). PC), 1,2-enoacyl-sn-glycerol-choline phosphate (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 diether PC), dimyristoyl phosphate ethanolamine (DMPE), distearate phosphatidyl ethanolamine (DSPE), 1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine (DLPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), 1- Oleoyl-2-cholesterolyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-O-hexadecyl-sn-glycerol-3-phosphate choline, 1,2-dilinanoyl-sn-glycerol-3-phosphate choline, 1,2-distearatel-sn-glycerol-3-phosphate choline, 1,2-docohexanoyl-sn-glycerol-3-phosphate choline, 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine, 1,2-distearatel-sn-glycerol-3-phosphate ethanolamine, 1,2-di-di-sinyl ...inyl-sn-glycerol-3-phosphate ethanolamine, 1,2-di-sinyl-sinyl-sn-glycerol-3-phosphate ethanolamine, 1,2-di-sinyl-sinyl-sn-glycerol-3-phosphate ethanolamine, 1,2-di-sinyl-sinyl-sn-glycerol-3-phosphate ethanolamine, 1,2-di-sinyl-sinyl-sn-glycerol-3-phosphate ethanolamine, 1,2- Oleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-disarachidanoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docohexanoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), diacetylphosphatidylethanolamine (DEPE), stearoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine, and sphingomyelin are one or more combinations thereof.

[0055] In some specific implementations, phosphatidylcholine is one or a combination of DSPC, DPPC, DMPC, DOPC, and POPC.

[0056] In some specific implementations, the auxiliary lipid is phosphatidylcholine, specifically DSPC.

[0057] In some specific implementations, the auxiliary lipid is phosphatidylcholine, specifically DPPC.

[0058] In some specific embodiments, the auxiliary lipid is phosphatidylcholine, specifically a combination of DSPC and DPPC.

[0059] In some specific embodiments, the auxiliary lipid is phosphatidylethanolamine, specifically DOPE.

[0060] In some specific embodiments, the auxiliary lipid is selected from one or more combinations of DOTAP ((1,2-dioleopropyl)trimethylammonium chloride), DODAP (1,2-dioleoyl-3-dimethylammonium-propane), 18:1 PA (1,2-DI(cis-9-octadecenoyl)-SN-glycerol-3-phosphate sodium salt), HS15 (polyethylene glycol (15)-hydroxystearate), and GL67 (N4-arginine cholesterol carbonyl amide).

[0061] The "PEG-lipid" mentioned in this invention generally refers to a conjugate formed by linking PEG (polyethylene glycol) and lipid molecules through chemical bonds. This includes, but is not limited to, PEG-modified phospholipids and derived lipids, exemplified by one or more combinations of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and methoxy polyethylene glycol bis(tetradecyl)acetamide.

[0062] In some specific embodiments, the PEG lipids include, but are not limited to, PEG-C-DMG, PEG-C-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, PEG-DPPC, PEG-distearate phosphatidylethanolamine (PEG-DSPE), PEG-DS, Chol (cholesterol)-PEG, 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol (PEG-DMG), PEG-S-DMG, polyethylene glycol phosphatidylethanolamine, polyethylene glycol ceramide, and polyethylene glycol. Dimethicone (PEG-DMA), PEG distearate glycerol, PEG dipalmitoyl, PEG dioleyl, PEG distearate, PEG diacyl glycamide, PEG dipalmitoyl phosphatidylethanolamine, PEG phosphatidylethanol, PEG phosphatidyl ethylenedimyristyloxypropyl-3-amine, PEG oxypropyl alcoholamine, 1,2-distearate oxypropyl-3-amine-N[methoxy(polyethylene glycol)] (PEG-DSA), methoxypolyethylene glycol lauric acid, methoxypolyethylene glycol bis(tetradecyl acetamide) (ALC0159) or a combination thereof.

[0063] In some specific implementations, the PEG-lipid is PEG-DMG.

[0064] There is no limitation on the type of "polymer". The polymer may include, but is not limited to, amphiphilic block copolymers. Amphiphilic block copolymers are block copolymers composed of hydrophobic polymers and hydrophilic compounds, including but not limited to polylactic acid (PLA), polylactic acid-polyglycolic acid copolymer (PLGA), glycolide-lactide copolymer (PLCG), polycaprolactone (PCL), polyorthoester, polyanhydride (PAH), polyphosphazene, polyβ-polyurethane (PBAE), poly(α-hydroxy acid), and lactide / glycolic acid copolymer (PLGA or PLG). (This includes lactide / glycolic acid copolymers, D-lactide / glycolic acid copolymers, L-lactide / glycolic acid copolymers, and D,L-lactide / glycolic acid copolymers), polyglycolic acid (PGA), polyorthoester (POE), linear or branched polyethylene glycol (PEG), poly(α-hydroxy acid) couplings, polyaspirins, polyphosphazenes, D-lactide, D,L-lactide-caprolactone, D,L-lactide-glycolic acid-caprolactone, dextran, and vinylpyrrole. Alkyl ketones, polyvinyl alcohol (PVA), methacrylates, poly-N-isopropylenamide, SAIB (sucrose acetate isocyanate), hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, carboxymethyl cellulose or its salts, polycarboxyethylene, poly(hydroxyethyl methacrylate), poly(methoxyethyl methacrylate), poly(methoxyethoxy-ethyl methacrylate), polymethyl methacrylate (PMMA), methyl isobutyl acrylate (MMA), PVA-g-PLGA, PEGT-PBT copolymer, PEO-PPO-PEO (pluronics), PEO-PPO-PAA copolymer, PLGA-PEO-PLGA, PEG-PLGA, PLA-PLGA, PEG-PLA, PEG-PCL, poloxamer 407, PEG-PLGA-PEG triblock copolymer, PEG-PLA-PEG triblock copolymer, PEG-PCL-PEG triblock copolymer or their block copolymers with polyethylene glycol (PEG), or one or more combinations of the above polymers or copolymers.

[0065] In some embodiments of the present invention, the weight-average molecular weight of PEG in the PEG-lipid is 1000 to 10000, for example 1000 to 2000, 2000 to 4000, 4000 to 6000, 6000 to 8000, 8000 to 10000, preferably 2000.

[0066] The present invention also provides the use of the lipid nanoparticles in the preparation of pharmaceutical compositions, the pharmaceutical compositions further comprising a drug and / or pharmaceutically acceptable excipients.

[0067] The "drugs" described in this invention include, but are not limited to, any one or a combination of multiple of nucleic acids, small molecules, and proteins.

[0068] The "nucleic acid" described in this invention can be a nucleotide polymer of any length. This includes, but is not limited to, single-stranded DNA, double-stranded DNA, plasmid DNA, short isoforms, mRNA, tRNA, rRNA, long non-coding RNA (lncRNA), micro-non-coding RNA (miRNA and siRNA), telomerase RNA, small RNA (snRNA and scRNA), circular RNA (circRNA), synthetic miRNA (miRNA mimics, miRNA agomir, miRNA antagomir), antisense oligonucleotides (ASO), ribozymes, asymmetric interfering RNA (aiRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), guide RNA (gRNA), small guide RNA (sgRNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholine antisense oligonucleotides, morpholine-substituted oligonucleotides, or bio-customized oligonucleotides, or one or more combinations thereof.

[0069] In some embodiments of the present invention, the nucleic acid is mRNA. mRNA is a single-stranded ribonucleic acid transcribed from one strand of DNA as a template, carrying genetic information and directing protein synthesis. mRNA can be monocistronic or polycistronic.

[0070] The term "small molecule" as used in this invention refers to a compound that is not a protein or nucleic acid molecule. Small molecules can be small molecules of therapeutic and / or preventative agents, such as antibiotics, anti-inflammatory drugs, anticancer drugs, antiviral drugs, immunosuppressants, analgesics, antifungal drugs, antiparasitic drugs, anticonvulsants, antidepressants, anti-anxiety drugs, antipsychotics, etc.

[0071] The term "protein" as used in this invention refers to a molecule or complex comprising one or more polypeptides having secondary, tertiary, and / or quaternary structures. The secondary, tertiary, and / or quaternary structures of proteins are typically stabilized using non-covalent bonds such as ionic bonds, hydrogen bonds, hydrophobic interactions, and / or van der Waals interactions. Additionally, or alternatively, proteins may include disulfide bonds, for example, between thiol groups of cysteine ​​residues. Exemplary proteins include, but are not limited to, antibodies, antigens or fragments thereof, fusion proteins, recombinant proteins, polypeptides, short peptides, enzymes, etc.

[0072] The pharmaceutical compositions of this invention also comprise pharmaceutically acceptable excipients. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 4-8, preferably about 5-7, although the pH value may vary depending on the nature of the formulated substances and the condition to be treated. The formulated pharmaceutical compositions can be administered via conventional routes, including but not limited to: intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, intracavitary injection, inhalation, implantation, etc.

[0073] The term "pharmaceutical acceptable" as used in this invention means that when the drug is properly administered to animals or humans, it does not produce adverse, allergic, or other adverse reactions.

[0074] "Pharmaceutical-acceptable excipients" should be compatible with the active ingredient, meaning they can be mixed with it without significantly reducing the drug's efficacy under normal circumstances. Specific examples of substances that can be used as pharmaceutically acceptable excipients include sugars such as glucose, mannitol, sucrose, lactose, trehalose, and maltose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; alcohols such as ethanol, propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; surfactants; lyophilization protectants; colorants; flavoring agents; tableting agents; stabilizers; diluents; excipients; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; buffer solutions, and combinations thereof. These substances are used as needed to improve the stability of the formulation or to help improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0075] The pharmaceutical compositions of the present invention can be formulated into inhaled nebulizer formulations (such as dry powder formulations, aerosol formulations, inhaled droplet formulations, etc.), implantable gel formulations, microneedle formulations, and can also be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or aqueous solutions containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions should preferably be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount.

[0076] In some embodiments of the present invention, the pharmaceutical composition comprises LNPs, the LNPs having an average particle size of 60-120 nm, which may be 60-70 nm, 70-80 nm, 80-90 nm, 90-100 nm, 100-110 nm, 110-120 nm, or 60-80 nm, 80-100 nm, 100-120 nm.

[0077] In some embodiments of the present invention, the pharmaceutical composition comprises nucleic acid lipid nanoparticles, wherein the nucleic acid is encapsulated in the nanolipid nanoparticles at a rate greater than 80%, and may be 80%~85%, 85%~90%, 90%~95%, 95%~97%, 97%~99%, or above 99%.

[0078] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0079] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, this invention can be implemented using any prior art methods, apparatus, and materials similar to or equivalent to those described in the embodiments of this invention, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention.

[0080] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. All materials and equipment used in this invention are commercially available products.

[0081] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Significantly Enhanced Physicochemical Properties: The ionizable lipid compounds of this invention can efficiently assemble with other lipids to form structurally stable, uniformly sized LNPs, exhibiting a high mRNA encapsulation efficiency. The LNP formulation maintains its intact nanostructure and encapsulation integrity even under long-term storage conditions, demonstrating excellent physicochemical stability.

[0082] 2. High transfection efficiency: In in vitro cell models, the transfection efficiency of the mRNA-LNP is significantly better than that of conventional reagents; in in vivo animal experiments, the system can also achieve efficient and durable protein expression, and its overall efficacy is comparable to that of existing mainstream commercial products, showing strong potential for therapeutic applications.

[0083] 3. Reliable biocompatibility and safety: Comprehensive in vitro cytotoxicity assessments and in vivo animal safety studies have confirmed that the LNP system has good biocompatibility, with no significant toxic reactions observed, and has broad clinical application value.

[0084] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0085] Example 1: Synthesis of ionizable lipid compounds 1. Synthesis of ionizable lipid compound C4-25 The synthesis route is as follows: (1) Synthesis of compound A-1 Octanoic acid (4.08 g, 28.27 mmol), 2-hydroxymethyl-1,3-propanediol (1.00 g, 9.42 mmol), and DMAP (0.35 g, 2.83 mmol) were dissolved in 40 mL of dichloromethane. The mixture was cooled to 0 °C, and DCC (4.86 g, 23.56 mmol) was added. The mixture was gradually heated to room temperature and stirred for 12 h. After the reaction was completed by TLC monitoring, the reaction solution was filtered, the filtrate was collected and concentrated, and purified by column chromatography (silica gel column, eluent: petroleum ether (PE): ethyl acetate (EA) = 10:1) to give 1.82 g of product, yield: 54%.

[0086] (2) Synthesis of compound A-2 6-Bromohexanoic acid (0.98 g, 5.02 mmol), compound A-1 (1.80 g, 5.02 mmol), and DMAP (0.18 g, 1.51 mmol) were dissolved in 10 mL of dichloromethane. The mixture was cooled to 0 °C, and DCC (1.24 g, 6.02 mmol) was added. The mixture was gradually heated to room temperature and stirred for 12 h. After the reaction was completed by TLC monitoring, the reaction solution was filtered, the filtrate was collected and concentrated, and purified by column chromatography (silica gel column, eluent:PE:EA = 100:1) to give 1.93 g of product, yield: 72%.

[0087] (3) Synthesis of compound A-3 6-Bromohexanol (5.00 g, 27.61 mmol), 2-hexyldecanoic acid (7.08 g, 27.61 mmol), and DMAP (1.01 g, 8.28 mmol) were dissolved in 50 mL of dichloromethane. The mixture was cooled to 0 °C, and DCC (6.84 g, 30.14 mmol) was added. The mixture was gradually heated to room temperature and stirred for 12 h. After the reaction was completed as monitored by TLC, the reaction solution was filtered, the filtrate was collected and concentrated, and purified by column chromatography (silica gel column, eluent:PE:EA = 50:1) to give 10.12 g of product, yield: 87%.

[0088] (4) Synthesis of compound A-4 A-3 (10.00 g, 23.84 mmol), 4-amino-1-butanol (21.25 g, 238.39 mmol), and 100 mL of ethanol were added to a reaction flask. The reaction mixture was heated to 90 °C and stirred for 12 h. After the reaction was completed by TLC, the reaction mixture was concentrated and then poured into 1000 mL of ethyl acetate / water (1:1) and extracted. The organic phase was collected and washed once with an equal volume of pure water and twice with an equal volume of saturated sodium chloride aqueous solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated to give 9.57 g of product, yield: 94%.

[0089] (5) Synthesis of ionizable lipid compound C4-25 Add A-2 (1.25 g, 2.34 mmol), A-4 (1.00 g, 2.34 mmol), DIPEA (0.39 g, 3.04 mmol), and 10 mL of acetonitrile to a reaction flask. Heat the reaction mixture to 80 °C and stir for 12 h. After the reaction is complete as monitored by TLC, concentrate the reaction mixture and pour it into 50 mL of ethyl acetate / water (1:1). Extract the mixture, collect the organic phase, and wash once with an equal volume of pure water and twice with an equal volume of saturated sodium chloride aqueous solution. Collect the organic phase, dry it with anhydrous sodium sulfate, filter and concentrate it. Purify the crude product by column chromatography (PE:EA = 2:1) to obtain 1.53 g of product, yield: 74%.

[0090] Using the above method, by replacing the corresponding starting materials, compounds C4-2~C4-4, C4-6~C4-8, C4-25~C4-32, C4-49~C4-54, C4-67~C4-68, C4O-2~C4O-4, C4O-6~C4O-8, C4O-25~C4O-32, C4O-49~C4O-54, C4O-67~C4O-68, and B-01 can be prepared. For example, replacing 4-amino-1-butanol with diethylene glycolamine can prepare C4O-25; replacing 2-hexyldecanoic acid with 2-hexyldecanol can prepare C4-27.

[0091] 2. Synthesis of ionizable lipid compound C4-41 (1) Synthesis of compound A-9 Under nitrogen protection, DCM (200 mL), 1,3-dihydroxyacetone (10.00 g, 111.01 mmol), and triphenylphosphoacetate tert-butyl ester (41.79 g, 111.01 mmol) were added sequentially to a 500 mL reaction flask, and the reaction was carried out at room temperature for 20 h. After the reaction was completed by TLC monitoring, the reaction solution was concentrated and purified by column chromatography (DCM:EtOH = 40:1) to obtain 15.29 g of product, yield: 73%.

[0092] (2) Synthesis of compound A-10 EtOH (80 mL), A-9 (8.32 g, 44.20 mmol), Pd / C (3.76 g, 1.77 mmol), and Et3N (670.9 mg, 6.63 mmol) were added sequentially to a 250 mL reaction flask. Nitrogen gas was replaced with a hydrogen balloon, and the reaction was carried out at room temperature for 24 h. After the reaction was completed as monitored by TLC, the reaction solution was concentrated and purified by column chromatography (DCM:EtOH = 40:1) to obtain 7.66 g of product, yield: 91%.

[0093] (3) Synthesis of compound A-11 A-10 (5.00 g, 26.28 mmol), octanoic acid (7.58 g, 52.57 mmol), and DMAP (0.96 g, 7.88 mmol) were dissolved in 50 mL of dichloromethane. The mixture was cooled to 0 °C, and DCC (11.39 g, 55.19 mmol) was added. The mixture was gradually heated to room temperature and stirred for 12 h. After the reaction was completed by TLC monitoring, the reaction solution was filtered, the filtrate was collected and concentrated, and purified by column chromatography (silica gel column, eluent:PE:EA = 100:1) to give 9.56 g of product, yield: 82%.

[0094] (4) Synthesis of compound A-12 Under nitrogen protection, DCM (30 mL), A-1 (3.28 g, 7.41 mmol), and TFA (9.84 mL, 3V) were added sequentially to a 100 mL reaction flask, and the reaction was carried out at room temperature for 24 h. After the reaction solution was concentrated, DCM (30 mL) was added, and the DCM was washed with water (30 mL × 2), then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give 2.71 g of product, yield: 94.8%.

[0095] (5) Synthesis of compound A-13 Referring to the synthesis method of compound A-2 in (2), compound A-13 can be synthesized.

[0096] (6) Synthesis of ionizable lipid compound C4-41 Compound C4-41 can be synthesized by referring to the synthetic method of ionizable lipid compound C4-25.

[0097] Using the above method, by replacing the corresponding starting materials, compounds C4-18~C4-20, C4-22~C4-24, C4-42~C4-48, C4-61~C4-66, C4-71~C4-72, C4O-18~C4O-20, C4O-22~C4O-24, C4O-41~C4O-48, C4O-61~C4O-66, and C4O-71~C4O-72 can be prepared. For example, replacing 4-amino-1-butanol with diethylene glycolamine can prepare C4O-41.

[0098] 3. Synthesis of ionizable lipid compound C4-33 The synthesis route is as follows: (1) Synthesis of compound A-14 Water (200 mL), NaOH (10.1 g, 251.99 mmol), benzyl alcohol (10.9 g, 100.80 mmol), 1,4-dibromobutane (50.1 g, 231.83 mmol), and tetrabutylammonium hydrogen sulfate (855.6 mg, 2.52 mmol) were added sequentially to a 500 mL reaction flask. The mixture was reacted in an oil bath at 80 °C for 24 h. After cooling to room temperature, the mixture was extracted with ethyl acetate (200 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was purified by column chromatography (n-hep:EA = 50:1) to give 23.3 g of colorless liquid, yield: 95.1%.

[0099] (2) Synthesis of compound A-15 Under nitrogen protection, THF (400 mL), NaH (4.60 g, 114.99 mmol), dimethyl malonate (25.32 g, 191.66 mmol), and A-14 (23.30 g, 95.83 mmol) were added sequentially to a 3 L reaction flask, and the reaction was carried out at 76 °C for 12 h. The reaction solution was poured into water (200 mL), extracted with ethyl acetate (200 mL × 3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was purified by column chromatography (n-hep:EA = 5:1) to obtain 19.0 g of colorless liquid. Yield: 67.4%.

[0100] (3) Synthesis of compound A-16 Under nitrogen protection, THF (100 mL), 1 M LiAlH4 (142.0 mL, 142.0 mmol), and A-15 (19.0 g, 64.55 mmol) were added sequentially to a 500 mL reaction flask, and the reaction was carried out at room temperature for 22 h. After the reaction was completed, water (5.4 mL), 10% NaOH (5.4 mL), and water (16.2 mL) were added sequentially under ice bath conditions, and the mixture was filtered through a Buchner funnel using diatomaceous earth. The filter cake was washed with ethyl acetate, and the filtrate was concentrated to obtain 13 g of crude product. The crude product was purified by column chromatography (n-hep:EA = 1:2) to obtain 4.28 g of colorless liquid, yield: 27.8%.

[0101] (4) Synthesis of compound A-17 Referring to the synthesis method of compound A-2 in (2), compound A-17 can be synthesized.

[0102] (5) Synthesis of compound A-18 Referring to the synthesis method of compound A-10 in (2), compound A-18 can be synthesized.

[0103] (6) Synthesis of compound A-19 Referring to the synthesis method of compound A-2 in (2), compound A-19 can be synthesized.

[0104] (7) Synthesis of ionizable lipid compound C4-33 Compound C4-33 can be synthesized by referring to the synthetic method of ionizable lipid compound C4-25.

[0105] Using the above method, by replacing the corresponding starting materials, compounds C4-10~C4-12, C4-14~C4-16, C4-34~C4-40, C4-55~C4-60, C4-69~C4-70, C4O-10~C4O-12, C4O-14~C4O-16, C4O-33~C4O-40, C4O-55~C4O-60, and C4O-69~C4O-70 can be prepared. For example, replacing 4-amino-1-butanol with diethylene glycolamine can prepare C4O-33.

[0106] 4. Synthesis of C4-1, an ionizable lipid compound (1) Synthesis of compound A-5 1,6-Dibromohexane (85.45 g, 350.25 mmol) was dissolved in 400 mL of tetrahydrofuran, cooled to 0 °C, and potassium thioacetate (20.00 g, 175.13 mmol) was added in six portions over 1 h. The mixture was then brought to room temperature and stirred for 12 h. After the reaction was complete as monitored by TLC, the reaction solution was filtered, the filtrate was collected and concentrated, and purified by column chromatography (silica gel column, eluent:PE:EA = 500:1) to give 28.15 g of product, yield: 67%.

[0107] (2) Synthesis of compound A-6 A-5 (20.00 g, 83.62 mmol) and 13.94 mL of concentrated hydrochloric acid (12 M, 167.24 mmol) were dissolved in 200 mL of methanol, and the mixture was heated to 60 °C and stirred for 12 h. After the reaction was complete as monitored by TLC, the reaction solution was poured into 600 mL of ethyl acetate / water (1:1) and extracted. The organic phase was collected and washed once with an equal volume of pure water and once with an equal volume of saturated sodium bicarbonate aqueous solution. The organic phase was collected, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (silica gel column, eluent PE:EA = 20:1) to give 13.26 g of product, yield: 80%.

[0108] (3) Synthesis of compound A-7 A-6 (10.00 g, 50.73 mmol), 2-hexyldecanoic acid (13.01 g, 50.73 mmol), and DMAP (1.86 g, 15.22 mmol) were dissolved in 200 mL of dichloromethane. The mixture was cooled to 0 °C, and DCC (12.56 g, 60.87 mmol) was added. The mixture was then heated to room temperature and stirred for 12 h. After the reaction was completed as monitored by TLC, the reaction solution was filtered, the filtrate was collected and concentrated, and purified by column chromatography (silica gel column, eluent:PE:EA = 100:1) to give 17.51 ​​g of product, yield: 79%.

[0109] (4) Synthesis of compound A-8 A-7 (10.00 g, 22.96 mmol), 4-amino-1-butanol (20.47 g, 229.60 mmol), and 20.0 mL of ethanol were added to a reaction flask. The reaction mixture was heated to 80 °C and stirred for 12 h. After the reaction was completed by TLC monitoring, the reaction mixture was concentrated and then poured into 600 mL of ethyl acetate / water (1:1) and extracted. The organic phase was collected and washed once with an equal volume of pure water and twice with an equal volume of saturated sodium chloride aqueous solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated to give 8.94 g of product, yield: 88%.

[0110] (5) Synthesis of ionizable lipid compound C4-1 Referring to the synthesis method of ionizable lipid compound C4-25 in (5), compound C4-1 can be synthesized.

[0111] Using the above method, by replacing the corresponding starting materials, compounds C4-5, C4-9, C4-13, C4-17, C4-21, C4O-1, C4O-5, C4O-9, C4O-13, C4O-17, and C4O-21 can be prepared. For example, replacing 4-amino-1-butanol with diethylene glycolamine can prepare C4O-33.

[0112] 5. Synthesis of ionizable lipid compound B-02 The synthesis route is as follows: (1) Synthesis of compound A-20 Referring to the synthesis method of compound A-4 in (4), compound A-20 can be synthesized.

[0113] (2) Synthesis of compound A-21 6-Bromohexanol (5.00 g, 27.61 mmol) and 4-dimethylaminopyridine (5.06 g, 41.42 mmol) were dissolved in 100 mL of dichloromethane (DCM) under nitrogen protection and stirred in an ice bath for 10 min. Then, phenyl p-nitrochloroformate (8.35 g, 41.42 mmol) was added in portions, and the mixture was gradually brought to room temperature. After stirring at room temperature for 3 h, nonanol (3.98 g, 27.61 mmol) was added under ice bath conditions, and the reaction was stirred at room temperature for 12 h. After the reaction was complete as monitored by TLC, the solvent was removed by rotary evaporation under reduced pressure. 200 mL of ethyl acetate was added, followed by washing twice with an equal volume of saturated sodium bicarbonate solution, and once with an equal volume of saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate for 30 min, and the solvent was removed by rotary evaporation under reduced pressure. The solution was purified by column chromatography (silica gel column, eluent:PE:EA = 50:1 (v / v)) to give 7.12 g of a colorless liquid, with a yield of 73%.

[0114] (3) Synthesis of ionizable lipid compound B-02 Following the synthetic method for ionizable lipid compound C4-25, compound B-02 can be synthesized. NMR verification confirmed the successful synthesis of the desired compound, as shown in Table 1.

[0115] Table 1. NMR data of some compounds Example 2: Preparation and Screening of Nucleic Acid Lipid Nanoparticles (mRNA-LNP) 1. Preparation of nucleic acid lipid nanoparticles (1) The ionizable lipid compounds (Lipid), DSPC, cholesterol, and PEG-lipid (PEG2000-DMG) prepared in Example 1 were dissolved in ethanol according to the molar relationship shown in the ratio of 46.3 / 9.4 / 42.7 / 1.6 (molar ratio) to prepare different lipid ethanol solutions (Lipid concentration 20 mg / mL).

[0116] (2) Prepare mRNA with an LNP to mRNA mass ratio of 10:1 to 30:1 (the ratio used in this example is 15:1), and dilute the mRNA to 0.2 mg / mL with citrate or sodium acetate buffer (pH=3 or 5) to obtain an mRNA solution.

[0117] (3) The lipid ethanol solution and mRNA solution were thoroughly mixed at a volume ratio of 1:5 to 1:1 (the ratio used in this example was 1:3). The obtained lipid nanoparticles were purified by ultrafiltration and dialysis. After filtration and sterilization, the average particle size and PDI (polydispersity index) of mRNA-LNP (lipid nanoparticles encapsulating mRNA) were characterized, and the encapsulation efficiency of mRNA was determined using a quantitative assay kit. The results are shown in Table 2 and... Figure 1 As shown.

[0118] Table 2 Characterization data of mRNA-LNP in each group The results showed that all the ionizable lipid compounds provided in Example 2 of this invention could form stable nanostructures, and the resulting mRNA-LNPs had a narrow size distribution, with the size varying depending on the structure of the different mRNA-LNPs, ranging from 60 to 120 nm. Under the same conditions, using the comparative samples shown in Table 3, commercially available SM-102, comparative samples B-01 (prepared in Example 1 above), and B-02 (prepared in Example 1 above), respectively, to replace the aforementioned ionizable lipid compounds in the preparation of mRNA-LNPs, the average particle size, PDI, and encapsulation efficiency were tested. The structural formulas of SM-102, B-01, and B-02 are shown in Table 3. The test results are shown in Table 4.

[0119] Table 3 Structural formulas of comparative samples Table 4 Characterization data of mRNA-LNP in the comparison samples The comparison of data in Tables 2 and 4 shows that the mRNA-LNPs provided in the embodiments of the present invention can form stable nanostructures with narrow size distribution. The size varies with the structure of different mRNA-LNPs. In the range of 60-120 nm, the encapsulation efficiency is above 90%, and some compounds are greater than 95%, exhibiting excellent physicochemical properties.

[0120] 2. Low-temperature storage stability The mRNA-LNP provided by C4-25 prepared in Example 2 was stored in a dry environment at 4°C. The average particle size, PDI, and encapsulation efficiency of the mRNA-LNP were tested at different time points (0 days, 15 days, 30 days, and 60 days). The results are shown in Table 5.

[0121] Table 5. Comparison of the effects of low-temperature preservation at different times. As shown in Table 5, the average particle size, PDI and encapsulation efficiency of the mRNA-LNP provided by group C4-25 in this embodiment remained basically unchanged within 60 days, demonstrating good low-temperature storage stability and facilitating storage and transportation.

[0122] Following the aforementioned method, mRNA-LNPs prepared from other compounds in this example group were measured, and the average particle size, PDI, and encapsulation efficiency remained essentially unchanged over 60 days.

[0123] Example 3: Transfection efficiency and biocompatibility of nucleic acid lipid nanoparticles (mRNA-LNP) 1. Cell transfection efficiency HEK293 cells in the exponential growth phase (100 μL, cell density 6 × 10⁻⁶) were used. 4The cell suspension (cells / ml) was added to a 96-well plate and incubated in a cell culture incubator for 24 h. The complete medium was replaced with 60 μL of DMEM serum-free medium, and 0.4 μg or 0.2 μg of the prepared Luciferase mRNA LNP preparation was added to each well, with three replicates per concentration. The plates were then incubated at 37°C and 6% CO2 for 4 h. Then, 60 μL of complete medium was added, and the plates were incubated for another 24 h. The medium was removed, and the plates were gently washed once with 100 μL of PBS. Then, 30 μL of cell lysis buffer was added to each well, and the plates were shaken at room temperature for 16 min to fully lyse the cells. The lysate was collected, centrifuged at 12000 rpm for 6 min, and the supernatant was collected. 20 μL of the supernatant was added to a new black opaque 96-well plate. The cell lysis buffer served as a blank control. 100 μL of firefly luciferase assay solution was added, and the plates were incubated in the dark for 6 min. The chemiluminescence intensity (RLU) was measured using a multi-mode microplate reader, and the RLU of the blank control was subtracted. The average value was taken. In Table 6, E represents 10 to the power of 10 in scientific notation. For example, "6.41E+04" means 6.41 × 10⁻⁴. 4 .

[0124] Table 6. Transfection efficiency comparison table for HEK293 cells 2. Animal transfection efficiency Male ICR mice (6-8 weeks old) were housed under experimental conditions of 22±2℃ and relative humidity of 45–75%, with a 12-hour light / dark cycle. Luciferase mRNA was used as the reporter gene. Luciferase catalyzes the production of biofluorescence from luciferin; the transfection efficiency of LNP was reflected by detecting the biofluorescence intensity per unit time. Using luciferase mRNA as an example, mRNA-LNP samples obtained in Example 2 were prepared, along with commercially available control samples SM-102, B-01, and B-02. These samples were administered intramuscularly at a dose of 100 μg / kg mRNA, with two mice per group (two legs). At specific time points, luciferin (20 μg / mL) was injected intraperitoneally into the mice. Five minutes later, the mice were placed in a small animal in vivo imaging system to measure the fluorescence intensity. The final results are expressed as average fluorescence intensity. The fluorescence intensity results after intraperitoneal injection in mice are shown in Table 7.

[0125] Table 7 Fluorescence Intensity Comparison Table Compared to commercially available products and control samples, the mRNA-LNP prepared from the ionizable lipid compounds provided in this invention can improve transfection efficiency by 1 to 2 orders of magnitude at the cellular level and in vivo, demonstrating excellent transfection efficiency.

[0126] 3. Biocompatibility Cell viability was determined using a CCK-8 assay kit. HEK 293 cell suspensions in the exponential growth phase (100 μL, cell density 2 × 10⁻⁶ cells / mL) were used. 4 Cells (A1 / mL) were added to 96-well plates and incubated in a cell culture incubator for 24 h. Then, the cell culture medium was removed, and 100 μL of freshly prepared cell culture medium containing 100 μg / mL mRNA of each mRNA-LNP sample obtained in Example 2 was added, and the cells were co-incubated for 24 h. Subsequently, the cell supernatant was removed, fresh cell culture medium was added, and incubation continued for 24 h. The supernatant was removed, and 100 μL of fresh cell culture medium containing 10 μL / mL CCK-8 working solution was added, and incubation was carried out for 2 h. A blank control group was also set up: an equal volume of CCK-8 working solution was used to replace the mRNA-LNP, with all other conditions remaining identical. The absorbance of each well at 450 nm was measured using a multi-functional microplate reader (no air bubbles should appear in the well plate during the measurement process), and the cell viability of the untreated cells (control group) was set to 100%. The cell viability calculation formula for each group is as follows: Cell viability (%) = [A1-A0] / [A2-A0] × 100. Where A1 is the absorbance of the drug-treated group, A0 is the absorbance of the blank group, and A2 is the absorbance of the control group. The experimental results are shown in Table 8.

[0127] Table 8 Comparison of Cell Viability Effects Experimental results show that the LNP prepared from the lipid compounds of this invention has minimal impact on cell viability, with cells maintaining a viability of over 75%. In contrast, at the same high concentration, the viability of the commercially available comparative sample SM-102 cells was only 57%, indicating that its in vitro safety is far inferior to that of the lipid compounds of this invention. Combined with the significantly enhanced nucleic acid delivery capability of the LNP prepared from the lipid compounds of this invention in cells and animals, the application of the lipid compounds of this invention in cell therapy, protein replacement therapy, and gene editing is greatly expanded.

[0128] Example 4: In vivo safety experiment To further verify the safety of the lipid compounds of the present invention, some of the compounds prepared in Example 1 were selected for relevant animal experiments.

[0129] Materials Preparation: 110 six-week-old Balb / c mice (half male, half female, half male), weighing 15-20g, were housed in an experimental environment at 22±2˚C and 45-75% relative humidity, with a 12-hour light / dark cycle. Mice were allowed to acclimatize in the animal facility for one week after purchase before the formal animal experiments. The 110 mice were randomly divided into 11 groups, with half males and half females in each group. Group 1 received an equal volume of PBS intravenously (negative control); Group 2 received an intravenous injection of commercially available control sample SM-102 (260µg mRNA); Group 3 received an intravenous injection of commercially available control sample SM-102 (520µg mRNA); Group 4 received an intravenous injection of control sample B-01 (260µg mRNA); Group 5 received an intravenous injection of control sample B-01 (520µg mRNA); and Group 6 received an intravenous injection of control sample B-02 (260µg mRNA). Group 7: Intravenous injection of control sample B-02 (520 µg mRNA); Group 8: Intravenous injection of control sample C4-25 (260 µg mRNA); Group 9: Intravenous injection of control sample C4-25 (520 µg mRNA); Group 9: Intravenous injection of control sample C4-68 (260 µg mRNA); Group 10: Intravenous injection of control sample C4-68 (520 µg mRNA); All the above mRNAs are full-length Luciferase mRNAs synthesized in vitro through transcription based on a self-designed template.

[0130] The experimental procedure was as follows: at 0h, 24h, 48h and 72h, the number of surviving mice in each group was counted. The number of surviving mice is shown in Table 9.

[0131] Table 9. Statistics on the number of surviving mice Experimental results showed that when mRNA-LNP composed of SM-102 and control sample B-02 was injected intravenously in large doses, it directly caused varying degrees of death in mice (260µg mRNA), or even total death (520µg mRNA). However, no mice died when mRNA-LNP composed of C4-25 and C4-68 was injected in large doses.

[0132] Other compounds in this embodiment also exhibit similar safety effects to C4-25 and C4-68. For example, at higher doses, almost all mice survived with compounds C4-29, C4-49, and C4-52.

[0133] In summary, it can be demonstrated that the ionizable lipids prepared by this invention have extremely high safety and, compared with the already marketed product SM-102 and other similar compounds, have broader clinical application value.

[0134] Example 5: Screening of 5'UTR sequences Different 5'UTR sequences were constructed into mRNA templates containing luciferase coding regions, and the corresponding mRNAs were synthesized via in vitro transcription. Subsequently, different mRNAs were transfected into HEK293 cells using Lipofectamine 3000. Cells were collected at 24 h, 48 h, and 72 h post-transfection, and luciferase activity was measured using a luciferase assay kit. The fluorescence intensity of luciferase reflects its expression level, thus being used to assess the effect of different 5'UTRs on mRNA expression. The results showed that the group containing 5'UTR-1 had the highest luciferase expression level, followed by 5'UTR-4 (see...). Figure 2 ).

[0135] Example 6: Screening of 3'UTR sequences Different 3'UTR sequences were constructed into mRNA templates containing luciferase coding regions, and the corresponding mRNAs were synthesized via in vitro transcription. Subsequently, different mRNAs were transfected into HEK293 cells using Lipofectamine 3000. Cells were collected at 24 h, 48 h, and 72 h post-transfection, and luciferase activity was measured using a luciferase assay kit. The fluorescence intensity of luciferase reflects its expression level, thus being used to assess the effect of different 3'UTRs on mRNA expression. The results showed that the group containing 3'UTR-2 had the highest luciferase expression level, followed by 3'UTR-3 (see...). Figure 3 ).

[0136] Example 7: Screening of mRNA molecules with different UTR structures The candidate sequences 5'UTR-1, 5'UTR-4, 3'UTR-2, and 3'UTR-3 obtained from the first two rounds of screening were combined to construct four groups of mRNA molecules with different UTR structures: UTR1 (5'UTR-1 + 3'UTR-2), UTR2 (5'UTR-1 + 3'UTR-3), UTR3 (5'UTR-4 + 3'UTR-2), and UTR4 (5'UTR-4 + 3'UTR-3). These combinations were then incorporated into mRNA templates containing the luciferase coding region, and the corresponding mRNAs were synthesized via in vitro transcription. Subsequently, different mRNAs were transfected into HEK293 cells using Lipofectamine 3000. Cells were collected at 24, 48, and 72 hours post-transfection, and luciferase activity was measured using a luciferase assay kit. Fluorescence intensity was used to reflect luciferase expression levels, thereby assessing the combined effect of different UTR combinations on mRNA stability and translation efficiency. Experimental results showed that the UTR2 combination (5'UTR-1 and 3'UTR-3) mediated the highest level of luciferase expression (see...). Figure 4 ).

[0137] Example 8: CDS sequence screening with different codon optimizations The selected optimal UTR-2 combination (5'UTR-1 + 3'UTR-3) and five different codon-optimized Merlin protein (NF2 gene-encoded tumor suppressor protein) coding sequences (CDS) were used to construct mRNA templates, and the corresponding mRNAs were synthesized through in vitro transcription. Subsequently, different mRNAs were transfected into HEK293 cells using Lipofectamine 3000. Cells were collected at 24, 48, and 72 hours post-transfection, and the expression level of Merlin protein was detected by Western blotting to assess the impact of different CDS region codon usage on the translation efficiency of the target protein. The results showed that compared with the unoptimized control group, the protein expression levels in all optimized groups were significantly increased, with the CDS-2 group exhibiting the highest Merlin protein expression level (see...). Figure 5 ).

[0138] Example 9: In vivo protein expression experiment To further verify the effectiveness of the lipid compounds of the present invention, some of the compounds prepared in Example 1 were selected for relevant animal experiments.

[0139] Materials Preparation: Fifty six-week-old Balb / c mice (half male and half female, weighing 15-20g) were housed in an experimental environment with a temperature of 22±2˚C and a relative humidity of 45-75%, with a 12-hour light / dark cycle. Mice were allowed to acclimatize in the animal facility for one week after purchase before formal animal experiments. Fifty mice were randomly divided into 5 groups, with half males and half females in each group. Group 1 was intravenously injected with an equal volume of PBS (negative control group). Group 2 was intravenously injected with LNP (mPCCA+mPCCB, referring to mRNA simultaneously expressing PCCA and PCCB, 1.0 mg / kg) prepared from commercially available control sample SM-102. Group 3 was intravenously injected with LNP (mPCCA+mPCCB, 1.0 mg / kg) prepared from control sample B-01. Group 4 was intravenously injected with LNP (mPCCA+mPCCB, 1.0 mg / kg) prepared from control sample B-02. Group 5 was intravenously injected with LNP (mPCCA+mPCCB, 1.0 mg / kg) prepared from sample C4-25. All the above mRNAs were mRNAs expressing full-length human PCCA and PCCB, synthesized in vitro through transcription based on a self-designed template. The LNPs were prepared according to the aforementioned examples.

[0140] The experimental procedure was as follows: On day 2, Western blotting was used to count the protein expression of PCCA and PCCB in the liver of each group of mice. GAPDH was used as an internal control to quantify the protein expression in each group of mice. After deducting the background effect of the negative control group, the data of each group were normalized, and the SM-102 group was set to 1. The results are shown in Table 10.

[0141] Experimental Results: The LNP delivery system prepared from lipid compounds provided in this invention exhibits significantly superior protein expression efficiency compared to existing technologies after encapsulating mRNA encoding the target protein. In vivo protein expression quantification showed that the induced protein expression level was 4-5 times that of mainstream commercially available LNP reagents and more than 10 times that of the control sample.

[0142] Table 10 Statistical table of PCCA and PPCB protein expression Furthermore, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An ionizable lipid compound, characterized in that, The compound has the chemical structure shown in formula (I): （I） In this context, a and b are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, or 8; G1 and G2 are each independently selected from C2~C10 alkylene groups; G3 is selected from C4-C8 alkylene groups; or G3 is selected from (CH2). d -O-(CH2) e Where d and e are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, or 9, and d+e is an integer from 3 to 10; or G3 is selected from , where g is selected from 1, 2, 3 or 4; L1 is selected from -(C=O)O-, -O(C=O)-, -(C=O)S- or -S(C=O)-; L2 is selected from -(C=O)O- or -O(C=O)-; R1 and R2 are each independently selected from the following group: C1~C20 straight-chain saturated hydrocarbon groups or C2~C20 straight-chain unsaturated hydrocarbon groups; R3 and R4 are each independently selected from the following groups: H, C1~C20 straight-chain saturated hydrocarbon groups or C2~C10 straight-chain unsaturated hydrocarbon groups.

2. The ionizable lipid compound according to claim 1, characterized in that, The ionizable lipid compounds are selected from the group consisting of: 。 3. A lipid nanoparticle, characterized in that, It includes one or more combinations of an ionizable lipid compound as described in any one of claims 1-2, its stereoisomers, its tautomers, or its pharmaceutically acceptable salts.

4. The lipid nanoparticles as described in claim 3, characterized in that, It also includes any one or more combinations of structural lipids, auxiliary lipids, PEG-lipids, and polymers.

5. The lipid nanoparticles as described in claim 4, characterized in that, The structural lipid is selected from one or more combinations of sterols, nonsterols, or their respective derivatives; and / or The auxiliary lipid is selected from one or more combinations of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, myristoyl phosphatidylglycerol, DOTAP, DODAP, 18:1 PA, HS15, and GL67; and / or The PEG-lipid is selected from one or more combinations of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and methoxy polyethylene glycol bis(tetradecyl)acetamide.

6. The lipid nanoparticles as described in claim 4, characterized in that, The molar ratio of the ionizable lipid compound: auxiliary lipid: structural lipid: PEG-lipid is (20-65):(3-50):(15-60):(0.1-10); the molar ratio of the ionizable lipid compound to the polymer is 0.5:1-100:

1.

7. A composition comprising the lipid nanoparticles of any one of claims 3-6, the composition further comprising a loaded drug and / or a pharmaceutically acceptable excipient; preferably, the loaded drug comprises one or more of nucleic acid molecules, small molecule compounds, and proteins; more preferably, the loaded drug is mRNA.

8. The composition according to claim 7, characterized in that, The mRNA sequence coding region (CDS) is selected from any one of SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No.

9.

9. The composition according to claim 8, characterized in that, The mRNA sequence includes a 5' untranslated region (5'UTR), a coding region (CDS), and a 3' untranslated region (3'UTR); The 5'UTR is selected from any one of the sequences SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4; CDS is selected from any one of SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, and SEQ ID No. 9; The 3'UTR is selected from any one of the sequences in SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12 and SEQ ID No.

13.

10. Use of the composition according to claims 7-9 in the preparation of medicaments for the prevention and / or treatment of diseases.

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