Alcohol amine multi-tail lipid as well as preparation method and application thereof

Abstract

The invention discloses alcohol amine multi-tail lipid as well as a preparation method and application thereof. The structural formula of the lipid is a compound as shown in a formula I, a compound as shown in a formula II, or a stereoisomer, a tautomer or pharmaceutically acceptable salt thereof: # imgabs0. The ionizable lipid compound disclosed by the invention can maintain the stability and delivery efficiency of nanoparticles while simplifying LNP components; the method has the advantages of high efficiency, low toxicity and the like, and the ionizable lipid compound can still realize mRNA adsorption by virtue of interaction of hydrogen bonds and Van der Waals force even under a neutral condition. The synthesis method of the diethanolamine multi-tailed ionizable lipid is simple, a large amount of ionizable lipid can be prepared through several steps of addition reactions, and high-throughput screening of materials is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of drug delivery, and particularly relates to an alkanolamine multi-tailed lipid, a preparation method thereof, and an application thereof. Background Art

[0002] RNA therapy is an emerging technology that uses RNA molecules to treat or prevent diseases. RNA therapies mainly include types such as mRNA, antisense oligonucleotides (ASOs), and small interfering RNAs (siRNAs). These therapies achieve the treatment of diseases through different mechanisms. For example, mRNA therapy regulates the immune response or directly produces therapeutic proteins by encoding specific proteins. In recent years, RNA therapy has made remarkable progress in preclinical and clinical studies. Especially after the successful development of the COVID-19 mRNA vaccine, this field has received extensive attention. The success of the mRNA vaccine not only demonstrates the potential of RNA therapy in the prevention and control of infectious diseases but also promotes its application in the treatment of other diseases. Although RNA therapy has many advantages, such as high specificity, modular development, and relative safety, its research and development still face some challenges. For example, RNA molecules are easily degraded in vivo, resulting in a short half-life, so chemical modification is required to improve their stability. In addition, the optimization of the delivery system is also one of the key factors for the success of RNA therapy because an effective delivery system can improve the targeting of RNA drugs and reduce immunogenicity. Lipid nanoparticles (LNPs), as a delivery system that has successfully entered clinical research, especially LNPs-mRNA vaccines are currently used in clinical treatment, which is an important milestone for the lipid nanoparticle delivery system.

[0003] Compared with traditional cationic liposomes, ionizable lipids have greatly improved stability and transfection efficiency in vivo, and are electrically neutral during in vivo transportation, thus having low biotoxicity. Traditional LNPs usually contain four components: ionizable lipids, phospholipids, cholesterol, and PEG lipids. However, reducing the components of LNPs has always been a difficult point in the delivery field. The three-component LNP simplifies the preparation process of LNPs by reducing the number of components while maintaining its high efficiency and specificity in gene delivery. This design not only improves the delivery efficiency but also provides new possibilities for precision medicine. In terms of the delivery mechanism, LNPs help RNA cross the cell membrane and be released into the cytoplasm by forming a lipid bilayer structure that encapsulates RNA. However, the process of RNA escaping from endosomes to the cytoplasm remains a challenge and further research and technological improvements are needed to improve the delivery efficiency. Summary of the Invention

[0004] The present invention aims to at least solve one of the above technical problems existing in the prior art. To this end, the object of the present invention is to provide an alkanolamine multi-tailed lipid, a preparation method thereof, and an application thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect of the present invention, there is provided an ionizable lipid compound, and the structural formula of the ionizable lipid compound is a compound of formula I, a compound of formula II, or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof:

[0007]

[0008] Wherein, L0, L1, L2, L3, L4, L5, L6, L7, L8, L9 are each independently selected from C1-C8 alkyl;

[0009] n1, n2, n3, n4, n5, n6, m1, m2, m3, m4, m5, m6 are all natural numbers, and are each independently selected from natural numbers from 1 to 20;

[0010] k1, k2, k3, k4 are all natural numbers, and are each independently selected from natural numbers from 0 to 4.

[0011] In a second aspect of the present invention, there is provided a method for preparing the ionizable lipid compound, comprising the following steps:

[0012] React the compound of formula 1 with N,N'-carbonyldiimidazole, then react with the compound of formula 3, and then react with a C1-C8 alkenyl acyl chloride to obtain intermediate A; react intermediate A with the compound of formula 6 to obtain the compound of formula I;

[0013] React intermediate A with the compound of formula 7 to obtain the compound of formula II;

[0014]

[0015] Wherein, the definitions of n1, n3, n4, k1, L2, L3, L4, L7, L8, L9 are as described above.

[0016] In a third aspect of the present invention, there is provided a lipid nanoparticle comprising the ionizable lipid compound.

[0017] In a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising the lipid compound and / or the lipid nanoparticle.

[0018] In a fifth aspect of the present invention, there is provided the use of the lipid compound and the lipid nanoparticle in the preparation of a drug delivery carrier.

[0019] The beneficial effects of the present invention are:

[0020] The ionizable lipid compound of the present invention is a diethanolamine multi-tail ionizable lipid, which is an ionizable lipid with multiple tails and is commonly used for RNA delivery.

[0021] In the ionizable lipid compound of the present invention, the cross-section of the tail region of the multi-tail ionizable lipid increases, which can produce a more conical structure and has stronger endosome disruption ability. In addition, the head contains a special diethanolamine group, which enables it to construct a new three-component mRNA delivery system formulation. This formulation does not contain co-lipids and can encapsulate mRNA and form nanoparticles under neutral pH conditions. Its chemical backbone contains more ester bonds, which can be rapidly hydrolyzed by enzymes after effectively releasing RNA in vivo and is easily metabolized and cleared in vivo, having biodegradability.

[0022] The synthesis method of the diethanolamine multi-tail type ionizable lipid of the present invention is simple, and a large amount of ionizable lipid can be prepared through several addition reactions, facilitating high-throughput screening of materials.

[0023] The ionizable lipid compound of the present invention can maintain the stability and delivery efficiency of nanoparticles while simplifying the LNP components, and has advantages such as high efficiency and low toxicity. Even under neutral conditions, the ionizable lipid compound can still rely on hydrogen bond and van der Waals force interactions to achieve mRNA adsorption. Compared with the standard formulation, the diethanolamine multi-tail ionizable lipid with optimized structure can significantly improve the delivery efficiency, and its delivery efficiency is better than that of the marketed lipid. Description of the Drawings

[0024] Figure 1 It is the hydrogen spectrum of compound 21-5-C8 in Example 1 of the present invention.

[0025] Figure 2 It is the hydrogen spectrum of compound 21-5-C8C10 in Example 1 of the present invention.

[0026] Figure 3 It is the relative luciferase activity result of Example 2 of the present invention.

[0027] Figure 4 It is the relative luciferase activity result of Example 3 of the present invention.

[0028] Figure 5 It is the relative luciferase activity result of Example 4 of the present invention.

[0029] Figure 6 It is the relative luciferase activity result of Example 4 of the present invention.

[0030] Figure 7 It is the relative luciferase activity result of Example 5 of the present invention.

[0031] Figure 8Results of relative luciferase activity of LNP-saRNA delivery in Example 6 of the present invention.

[0032] Figure 9 Results of relative luciferase activity of LNP-modRNA delivery in Example 6 of the present invention.

[0033] Figure 10 Results of relative luciferase activity of LNP-circRNA delivery in Example 6 of the present invention.

[0034] Figure 11 Results of IVIS on the seventh day of in vivo delivery of ionizable lipid bodies in Example 7 of the present invention.

[0035] Figure 12 Results of IVIS of in vivo delivery of ionizable lipid bodies in Example 7 of the present invention.

[0036] Figure 13 Results of IVIS on the seventh day of in vivo delivery of ionizable lipid bodies in Example 8 of the present invention; wherein A is 21-5-C8C10:Cholesterol:DMG-PEG2000 = 30:48:1; B is 21-5-C8C10:Cholesterol:DMG-PEG2000 = 30:48:2; C is 21-5-C8C10:Cholesterol:DMG-PEG2000 = 30:48:2.5; D is 21-5-C8C10:Cholesterol:DMG-PEG2000 = 30:48:5; ALC-O315 group.

[0037] Figure 14 Results of IVIS of in vivo delivery of ionizable lipid bodies in Example 8 of the present invention; wherein A is 21-5-C8C10:Cholesterol:DMG-PEG2000 = 30:48:1; B is 21-5-C8C10:Cholesterol:DMG-PEG2000 = 30:48:2; C is 21-5-C8C10:Cholesterol:DMG-PEG2000 = 30:48:2.5; D is 21-5-C8C10:Cholesterol:DMG-PEG2000 = 30:48:5; ALC-O315 group. Detailed Description of the Invention

[0038] The content of the present invention will be further described in detail through specific examples below. The raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial channels or can be obtained by existing technical methods without special instructions. Unless otherwise specified, the test or measurement methods are conventional methods in the art.

[0039] In the first aspect of the present invention, an ionizable lipid compound is provided, and the structural formula of the ionizable lipid compound is a compound of Formula I, a compound of Formula II, or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof:

[0040]

[0041] Wherein, L0, L1, L2, L3, L4, L5, L6, L7, L8, L9 are each independently selected from C1-C8 alkyl groups;

[0042] n1, n2, n3, n4, n5, n6, m1, m2, m3, m4, m5, m6 are all natural numbers, and are each independently selected from natural numbers from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20;

[0043] k1, k2, k3, k4 are all natural numbers, and are each independently selected from natural numbers from 0 to 4; such as 0, 1, 2, 3, 4.

[0044] The "stereoisomer" refers to a constitutional isomer with the same atomic connection order but different atomic spatial arrangements.

[0045] The "tautomer" refers to the phenomenon of equilibrium mutual conversion of the structure of a compound between two functional group isomers, and the corresponding isomers are called tautomers.

[0046] The "pharmaceutically acceptable salt" refers to an acid addition salt or a base addition salt. All compounds of the present invention in the form of free base or free acid can be converted into their pharmaceutically acceptable salts by treatment with appropriate inorganic or organic bases or acids according to methods known to those skilled in the art. The salts of the compounds of the present invention can be converted into their free base or acid forms by standard techniques.

[0047] Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts 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 using other methods such as ion exchange used in the art for salts having an amino group. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, citrates, dodecyl sulfates, ethanesulfonates, formates, fumarates, glucoheptanoates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxyethanesulfonates, lactates, lactobionates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, etc. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts. Representative alkali metal salts or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, etc. In appropriate cases, additional pharmaceutically acceptable salts contain non-toxic ammonium, quaternary ammonium and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates and arylsulfonates. Additional pharmaceutically acceptable salts include salts formed by quaternization of amines, which quaternization is carried out using appropriate electrophiles (e.g., alkyl halides) to form quaternized alkylated ammonium salts.

[0048] In some embodiments of the present invention, the alkyl is selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl.

[0049] In some embodiments of the present invention, the compound of formula I has a structural formula of formula I-1 or formula I-2:

[0050]

[0051] In some embodiments of the present invention, the compound of formula II has a structural formula of formula II-1 or formula II-2:

[0052]

[0053]

[0054] In some embodiments of the present invention, the compound of formula I is selected from the following compounds:

[0055]

[0056] In some embodiments of the present invention, the compound of formula II is selected from the following compounds:

[0057]

[0058]

[0059] In a second aspect of the present invention, there is provided a method for preparing the ionizable lipid compound as described above, comprising the following steps:

[0060] Reacting the compound of formula 1 with N,N'-carbonyldiimidazole, then reacting with the compound of formula 3, and then reacting with a C1-C8 alkenyl acyl chloride to obtain intermediate A; reacting intermediate A with the compound of formula 6 to obtain the compound of formula I;

[0061] Reacting intermediate A with the compound of formula 7 to obtain the compound of formula II;

[0062]

[0063] wherein the definitions of n1, n3, n4, k1, L2, L3, L4, L7, L8, L9 are as described above.

[0064] As is well known to those skilled in the art, in the compound of formula 1, k1 can also be k2, k3, k4; n3 can also be n5, m3; n4 can also be n6, m6; in the compound of formula 3, n1 can also be n2, m1, m2; and they are each independent of one another.

[0065] In some embodiments of the present invention, the C1-C8 alkenyl acyl chlorides include acetyl chloride, acryloyl chloride, but-3-enoyl chloride, 4-pentenoyl chloride, 6-chloro-1-hexene, 7-chloro-1-heptene, 8-chloro-1-octene, etc.

[0066] In some embodiments of the present invention, intermediate A and the compound of formula 6 are reacted at 80°C to 100°C for 12 to 48 h.

[0067] In some embodiments of the present invention, the method for preparing the ionizable lipid compound as described above comprises the following steps:

[0068] The compound of formula 1 is reacted with N,N'-carbonyldiimidazole at 30°C to 50°C for 12 to 48 hours, then reacted with the compound of formula 3 at 30°C to 50°C for 12 to 48 hours, and further reacted with C1-C8 alkenyl acyl chloride at 10°C to 30°C for 6 to 24 hours to obtain intermediate A; intermediate A is reacted with the compound of formula 6 at 80°C to 100°C for 12 to 48 hours to obtain the compound of formula I;

[0069] Intermediate A is reacted with the compound of formula 7 at 80°C to 100°C for 12 to 48 hours to obtain the compound of formula II.

[0070] In some embodiments of the present invention, when the C1-C8 alkenyl acyl chloride is acryloyl chloride, intermediate A is the compound of formula 5

[0071]

[0072] Those skilled in the art are well aware that in the compound of formula 5, k1 can also be k2, k3, k4; n3 can also be n5, m3; n4 can also be n6, m6; n1 can also be n2, m1, m2; and they are each independent of each other.

[0073] The third aspect of the present invention provides a lipid nanoparticle comprising the ionizable lipid compound described above.

[0074] In some embodiments of the present invention, the lipid nanoparticle further comprises at least one of a structural lipid, a helper lipid, a polyethylene glycol-modified lipid, and a polymer.

[0075] In some embodiments of the present invention, the ionizable lipid compound and at least one of the structural lipid, the helper lipid, and the polyethylene glycol-modified lipid are used as a drug carrier to deliver a drug active ingredient. The lipid nanoparticle of the present invention may not include a helper lipid and still be able to efficiently deliver the drug active ingredient.

[0076] In some embodiments of the present invention, the molar percentages of the ionizable lipid compound, the structural lipid, the helper lipid, and the polyethylene glycol-modified lipid are 10-100:0-90:0-90:0-90, and at least any one of the helper lipid, the structural lipid, and the polyethylene glycol-modified lipid is not 0; such as (20-65):(0-60):(0-60):(0-10), (20-65):(3-50):(0-60):(0.1-10).

[0077] In some embodiments of the present invention, calculated by molar percentage, the lipid nanoparticle contains 20% to 65% ionizable lipid compound, 0% to 40% helper lipid, 20% to 60% structural lipid, and 0.1% to 10% polyethylene glycol-modified lipid.

[0078] In some embodiments of the present invention, the lipid nanoparticles comprise 35% to 49% ionizable lipid compounds, 5% to 20% helper lipids, 35% to 50% structural lipids, and 1% to 3% polyethylene glycol-modified lipids, where % refers to mole percentage.

[0079] In some embodiments of the present invention, the lipid nanoparticles comprise 35% to 49% ionizable lipid compounds, 35% to 50% structural lipids, and 1% to 3% polyethylene glycol-modified lipids, where % refers to mole percentage.

[0080] The "structural lipid" refers to a structure that can stabilize the composition, including but not limited to one or a combination of sterols and their derivatives and non-sterols and their derivatives.

[0081] In some embodiments of the present invention, the structural lipids include but are not limited to: one or a combination of sterols and their derivatives, non-sterols, sitosterol, ergosterol, cholestanone, cholestenone, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, coprostanol, α-tocopherol, or corticosteroids. Sterols are preferably cholesterol and its derivatives; non-limiting examples of cholesterol derivatives include: polar analogs such as 5α-cholestanol, 5α-coprostanol, cholesteryl-(2'-hydroxy)ethyl ether, cholesteryl-(4'-hydroxy)butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestane, cholestenone, 5α-cholestenone, and cholesteryl caprate; and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)butyl ether. This is not an exhaustive list, and the choice of structural lipids is not limited, and any structural lipid can be applied to the present invention.

[0082] In some embodiments of the present invention, the structural lipid is one or a combination of cholesterol, sitosterol, ergosterol, corticosteroids, and their derivatives.

[0083] In some embodiments of the present invention, the structural lipid is cholesterol.

[0084] The types of the "helper lipids" are not limited, and phospholipid-like lipids are preferred, including but not limited to: one or a combination of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, and dimyristoyl phosphatidylglycerol.

[0085] In some embodiments of the present invention, the helper lipid can be selected from one or more combinations of: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-docosanoyl-sn-glycerophosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenoyl-sn-glycero-3-phosphocholine (18:0 diether PC), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), dipalmitoylphosphatidylethanolamine (DPPE), 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-O-hexadecyl-sn-glycerophosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-docosahexaenoyl-sn-glycero 3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docosahexaenoyl-sn-glycero 3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), diacetyl-phosphatidylethanolamine (DEPE), stearoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, sphingomyelin.

[0086] In some embodiments of the present invention, the phosphatidylcholine is one or more combinations of DSPC, DPPC, DMPC, DOPC, POPC.

[0087] In some embodiments of the present invention, the helper lipid is phosphatidylcholine, specifically DSPC.

[0088] In some embodiments of the present invention, the helper lipid is phosphatidylethanolamine, specifically DOPE.

[0089] In some embodiments of the present invention, the auxiliary lipid is selected from one or more combinations of DOTAP ((1,2-dioleoylpropyl) trimethylammonium chloride), DODAP (1,2-dioleoyl-3-dimethylammonium-propane), 18:1PA (1,2-DI(cis-9-octadecenoyl)-sn-glycero-3-phosphate sodium salt), HS15 (polyethylene glycol (15)-hydroxystearate), GL67 (N4-arginine cholesterol carbonylamide).

[0090] The "polyethylene glycol-modified lipid" in the present invention generally refers to a conjugate formed by chemically linking polyethylene glycol (PEG) with a lipid molecule. It includes but is not limited to PEG-modified phospholipids and derived lipids, such as 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 methoxypolyethylene glycol ditetradecylacetamide.

[0091] In some embodiments of the present invention, the polyethylene glycol-modified lipid includes but is not limited to PEG-C-DMG, PEG-C-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, PEG-DPPC, PEG-distearoyl phosphatidylethanolamine (PEG-DSPE), PEG-DS, Chol (cholesterol)-PEG, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (PEG-DMG), PEG-S-DMG, polyethylene glycol phosphatidylethanolamine, polyethylene glycol ceramide, polyethylene glycol dimethacrylate (PEG-DMA), PEG distearyl glycerol, PEG dipalmitoleyl, PEG dioleyl, PEG-based distearyl, PEG-based diacylglycamide, PEG dipalmitoyl phosphatidylethanolamine, PEG-based phosphatidylethanol, PEG-based phosphatidylethylene myristyloxypropyl-3-amine, PEG-based oxypropylolamine, 1,2-distearyloxypropyl-3-amine-N[methoxy(polyethylene glycol)] (PEG-DSA), methoxypolyethylene glycol lauric acid, and methoxypolyethylene glycol ditetradecylacetamide (ALC0159).

[0092] In some embodiments of the present invention, the polyethylene glycol-modified lipid is PEG-DMG.

[0093] In some embodiments of the present invention, the polyethylene glycol-modified lipid has a weight-average molecular weight of 1000 to 10000 for the polyethylene glycol, such as 1000 to 2000, 2000 to 4000, 4000 to 6000, 6000 to 8000, 8000 to 10000, and preferably 2000.

[0094] There is no limitation on the type of the "polymer". The polymer may include, but is not limited to, amphiphilic block copolymers. The amphiphilic block copolymer is a block copolymer composed of a hydrophobic polymer and a hydrophilic compound, including but not limited to polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(lactide-co-caprolactone) (PLCG), polycaprolactone (PCL), polyorthoester, polyanhydride (PAH), polyphosphazene, poly(β - amino ester) (PBAE), poly(α - hydroxy acid), lactide / glycolide copolymer (PLGA or PLG) (which includes lactide / glycolide copolymer, D - lactide / glycolide copolymer, L - lactide / glycolide copolymer, and D,L - lactide / glycolide copolymer), polyglycolide (PGA), polyorthoester (POE), linear or branched polyethylene glycol (PEG), conjugate of poly(α - hydroxy acid), polyaspirins, polyphosphazenes, D - lactide, D,L - lactide - caprolactone, D,L - lactide - glycolide - caprolactone, dextran, vinylpyrrolidone, polyvinyl alcohol (PVA), methacrylate, poly(N - isopropylacrylamide), SAIB (sucrose acetate isobutyrate), hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose, carboxymethyl cellulose or its salts, carbopol, poly(2 - hydroxyethyl methacrylate), poly(2 - methoxyethyl methacrylate), poly(2 - methoxyethoxy - ethyl methacrylate), polymethyl methacrylate (PMMA), methyl methacrylate (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 a combination of one or more of the above polymers or copolymers.

[0095] In the fourth aspect of the present invention, there is provided a pharmaceutical composition comprising the ionizable lipid compound and / or the lipid nanoparticle described above.

[0096] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically active ingredient and / or a pharmaceutically acceptable excipient.

[0097] In some embodiments of the present invention, the pharmaceutically active ingredient includes at least one of nucleic acid, small molecule, and protein drugs.

[0098] In some embodiments of the present invention, the "nucleic acid" can be a nucleotide polymer of any length, including but 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 nuclear RNA (snRNA and scRNA), circular RNA (circRNA), synthetic miRNA (miRNA mimics, miRNA agomir, miRNA antagonist), antisense oligonucleotide (ASO), ribozyme, 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), morpholino antisense oligonucleotide, morpholino oligonucleotide, or a combination of one or more of bio-customized oligonucleotides.

[0099] In some embodiments of the present invention, the nucleic acid is mRNA. The mRNA is a type of single-stranded ribonucleic acid transcribed from one strand of DNA as a template and carrying genetic information to guide protein synthesis. The mRNA can be monocistronic mRNA or polycistronic mRNA.

[0100] In some embodiments of the present invention, the "small molecule" refers to a compound that is not a protein or nucleic acid molecule. The small molecule can be a small molecule of a therapeutic agent and / or a prophylactic agent, such as an antibiotic, an anti-inflammatory drug, an anti-cancer drug, an antiviral drug, an immunosuppressant, an analgesic, an anti-fungal drug, an anti-parasitic drug, an anti-convulsant drug, an anti-depressant drug, an anti-anxiety drug, an antipsychotic drug, etc.

[0101] In some embodiments of the present invention, the protein drugs include colony-stimulating factors, interleukins, lymphotoxins, interferon-like proteins, tumor necrosis factors, antibodies, and protein antigens.

[0102] The pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable excipient. Generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 4-8, preferably about 5-8, 5-7.5, although the pH value may vary depending on the nature of the substance being formulated and the disease to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (such as intraperitoneal), intracranial injection, intracavitary injection, inhalation administration, implantation administration, etc.

[0103] "Pharmaceutically acceptable" as used herein means that when the drugs are properly administered to animals or humans, they do not produce adverse, allergic or other adverse reactions.

[0104] "Pharmaceutically acceptable excipients" should be compatible with the active ingredient, i.e., they can be blended with it without significantly reducing the efficacy of the drug under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable excipients can be sugars, such as glucose, mannitol, sucrose, lactose, trehalose, maltose, etc.; starches, such as corn starch and potato starch, etc.; cellulose and its derivatives, such as sodium methylcellulose, ethyl cellulose and methyl cellulose, etc.; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate, etc.; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter, etc.; alcohols, such as ethanol, propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol, etc.; alginic acid; emulsifiers, such as Tween, etc.; wetting agents, such as sodium lauryl sulfate, etc.; surfactants; lyoprotectants; colorants; flavorants; tabletting agents; stabilizers; diluents; excipients; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; buffer solutions, etc., and their combinations. These substances are used as needed to improve the stability of the formulation or to help improve the activity or its bioavailability or to produce an acceptable taste or odor in the case of oral administration.

[0105] The pharmaceutical composition of the present invention can be made into inhalation aerosol preparations (such as dry powder preparations, aerosol preparations, inhaled aerosol droplet preparations, etc.), implantable gel preparations, microneedle preparations, and can also be made into injection forms, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day.

[0106] In some embodiments of the present invention, the pharmaceutical composition contains lipid nanoparticles (Lipid Nanoparticle, LNP), and the average particle size of the lipid nanoparticles is 30 to 150 nm, and can be 60 to 120 nm, 60 to 70 nm, 70 to 80 nm, 80 to 90 nm, 90 to 100 nm, 100 to 110 nm, 110 to 120 nm, or can also be 60 to 80 nm, 80 to 100 nm, 100 to 120 nm.

[0107] In some embodiments of the present invention, the ratio of the protonated amino group of the ionizable lipid compound to the nitrogen and phosphorus of the nucleic acid drug in the pharmaceutical composition is 1 to 100:1, such as 1 to 60:1, 1 to 50:1, 5 to 30:1.

[0108] In some embodiments of the present invention, the pharmaceutical composition comprises nucleic acid lipid nanoparticles, and the encapsulation efficiency of the nucleic acid in the nano lipid particles is greater than 80%, and can be 80% - 85%, 85% - 90%, 90% - 95%, 95% - 97%, 97% - 99% or more than 99%.

[0109] Example 1

[0110] In this example, an alcohol amine multi-tail lipid was prepared. The specific process is as follows:

[0111]

[0112] S1: Synthesis of compound A: In a 100 mL reaction tube, 10 mmol of 9-heptadecanol, 30 mmol of N,N'-carbonyldiimidazole, 20 mmol of triethylamine (TEA), 30 mL of dichloromethane (DCM), and a magnetic stir bar were added in sequence. The reaction tube was placed in a heating mantle at 40 °C and reacted for 24 h. The reaction progress was detected by Thin Layer Chromatography (TLC). After the reaction was completed, the reaction mixture was transferred to a separatory funnel, and extracted with DCM (2 x 100 mL) and saturated brine (2 x 100 mL), and washed with 1M HCl (2 x 20 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The solvent was removed using a rotary evaporator. Product A could be used for the next reaction without purification.

[0113] S2: Synthesis of compound B: In a 100 mL reaction tube equipped with a magnetic stir bar, 10 mmol of intermediate product A, 20 mmol of 5-amino-1-pentanol, and 30 mL of dichloromethane were added in sequence. The reaction tube was placed in a heating mantle at 40 °C and reacted for 24 h. The reaction progress was detected by TLC. After the reaction was completed, it was extracted with dichloromethane and saturated brine. The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The solvent was removed using a rotary evaporator, and the product was separated by a thin layer chromatography column.

[0114] S3: Synthesis of compound C: In a three-necked flask equipped with a magnetic stir bar, 10 mmol of intermediate product B, 15 mmol of triethylamine, and 30 mL of dichloromethane were added in sequence. The three-necked flask was pre-cooled in an ice bath for 30 min, and 13 mmol of acryloyl chloride (premixed in 10 mL of dichloromethane) was slowly added dropwise using a constant pressure funnel. After the addition of acryloyl chloride was completed, the ice bath was removed. The reaction was carried out overnight at room temperature, then diluted with dichloromethane (30 mL) and washed with 1M HCl (50 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed using a rotary evaporator. The product was separated by a thin layer chromatography column.

[0115] S4: Synthesis of Compound 21-5-C8: Add 50 mg of N,N’-bis(2-hydroxyethyl)ethylenediamine and 2.2-fold chemical equivalent of Intermediate C into a 5 mL reaction flask equipped with a magnetic stir bar (the bottle cap is lined with tetrafluoroethylene), and react at 90 °C for 24 h. After the reaction is completed, the product is separated by a thin-layer chromatography column (methylene chloride:methanol = 20:1) to obtain the ionizable lipid 21-5-C8.

[0116] The 1H NMR spectrum of the obtained product is as shown in Figure 1 the following, and the 1H NMR data are as follows:

[0117] 1 H NMR (400 MHz, CDCl3): 4.74 - 4.58 (m, 4H), 4.15 - 4.05 (m, 4H), 3.66 - 3.59 (m, 4H), 3.19 - 3.15 (m, 4H), 2.92 - 2.49 (m, 12H), 1.62 - 1.25 (m, 72H), 0.87 (t, J = 8.0 Hz, 12H).

[0118] The difference in the synthesis of Compound 21-5-C8C10 from the above steps is that in S1, the branched-chain tail is replaced by 2-octyldodecanol, and the remaining steps are the same, thus obtaining the ionizable lipid 21-5-C8C10.

[0119] The 1H NMR spectrum of the obtained product is as shown in Figure 2 the following, and the 1H NMR data are as follows:

[0120] 1 H NMR (400 MHz, CDCl3): 4.81 - 4.66 (m, 2H), 4.15 - 3.93 (m, 8H), 3.66 - 3.60 (m, 4H), 3.19 - 3.14 (m, 4H), 2.90 - 2.48 (m, 12H), 1.67 - 1.25 (m, 84H), 0.87 (t, J = 8.0 Hz, 12H).

[0121] Using the above method and replacing the corresponding starting materials, Compound 21-6-C8, Compound 21-5-C6C8, Compound 21-6-C6C8, Compound 21-6-C8C10, Compound 22-5-C8, Compound 22-6-C8, Compound 22-5-C6C8, Compound 22-5-C8C10, and Compound 22-6-C8C10 can be prepared; if N,N’-bis(2-hydroxyethyl)ethylenediamine (CAS No.: 4439-20-7) in S4 is replaced by N,N-bis(2-hydroxyethyl)ethylenediamine (CAS No. 3197-06-6) and the remaining steps are the same, the ionizable lipid 22-6-C8 is obtained.

[0122] Example 2

[0123] In this example, ionizable lipids are used to deliver mRNA. The specific process is as follows:

[0124] Verify the efficiency of ionizable lipid-mediated delivery of self-replicating mRNA (saRNA-Luc) encoding firefly luciferase (Luc) in the 293T cell line. Use ionizable lipids 21-5-C8, 22-5-C8, 21-6-C8, 22-6-C8, 21-5-C6C8, 22-5-C6C8, 21-6-C6C8, 21-6-C6C8, 21-5-C8C10, 22-5-C8C10, 21-6-C8C10, 21-6-C8C10 and the commercial material ALC-0315 to deliver saRNA intracellularly, respectively.

[0125] Specific steps:

[0126] 1. Cell culture

[0127] One day before the experiment, seed the cultured 293T cells in a 96-well cell culture plate (at a density of 50%). When the cell density grows to about 70-80%, perform cell transfection experiments.

[0128] 2. Prepare lipid nanoparticles LNP-saRNA-Luc for cell transfection

[0129] Dissolve ionizable lipids 21-5-C8, 22-5-C8, 21-6-C8, 22-6-C8, 21-5-C6C8, 22-5-C6C8, 21-6-C6C8, 21-6-C6C8, 21-5-C8C10, 22-5-C8C10, 21-6-C8C10, 21-6-C8C10 with distearoyl phosphatidylcholine (DSPC), cholesterol, and dimyristoyl glycerol-polyethylene glycol 2000 (PEG-DMG2000) in absolute ethanol at a certain concentration, and mix them evenly according to the molar ratio of ionizable lipid:Cholesterol:DSPC:PEG-DMG2000 = 40:48:10:2. At the same time, pipette 150 ng of saRNA-Luc into sodium acetate buffer (the volume of sodium acetate buffer is twice the total volume of the lipid mixture, pH = 5.2), and then quickly mix the saRNA buffer with the lipid mixture solution, and incubate at room temperature for 20 min to assemble into stable lipid nanoparticles. Dilute with 2 volumes of sterile PBS and add them to a 96-well cell culture plate for transfection, respectively. The optimal ratio of the nitrogen-to-phosphorus ratio (N / P ratio) of ionizable lipid to saRNA is 18:1, that is, the molar ratio between the protonatable amino group and the phosphate group on saRNA.

[0130] Positive control group: The commercial lipid ALC-0315 was assembled into lipid nanoparticles according to its disclosed preparation method.

[0131] Negative control group: 293T cells were cultured normally without transfection.

[0132] 3. Analysis of cell transfection efficiency

[0133] At 36 hours after cell transfection, the medium in the 96-well cell culture plate was aspirated, and cell lysate was added to lyse the cells on ice for 25 min. After centrifugation, the supernatant was taken and transferred to a white 96-well detection plate. Firefly luciferase substrate was added, and the content of firefly luciferase (chemiluminescence) was detected using a microplate reader.

[0134] The relative luciferase activity results are as Figure 3 shown. The results show that the ionizable lipid synthesized in the present invention can greatly enhance the transfection efficiency of self-replicating mRNA. When the head of the ionizable lipid is 21, the RNA expression efficiency is the highest. The transfection efficiency of lipids represented by 21-5-C8 and 21-5-C8C10 needs to optimize the commercial lipid ALC-0315, with an increase in efficiency of about 3 times, indicating the rationality and high efficiency of the overall chemical structure of the ionizable lipid designed in the present invention.

[0135] Example 3

[0136] In this example, ionizable lipid was used to deliver mRNA. The specific process was as follows:

[0137] The specific operation of the experiment referred to Example 2. Ionizable lipid 21-5-C8C10 was used, and in the LNP four-component ratio, other components remained unchanged, and the distearoylphosphatidylcholine (DSPC) was 20, 15, 10, 5, 0 (mol%) in turn.

[0138] The relative luciferase activity results are as Figure 4 shown. The results show that the content of DSPC will greatly affect the RNA delivery efficiency. When the DSPC for assembling LNP is 0%, the ionizable lipid can still efficiently deliver self-replicating mRNA, indicating that the ionizable lipid compound of the present invention, namely diethanolamine lipid, can efficiently deliver self-replicating mRNA in three components.

[0139] Example 4

[0140] In this example, ionizable lipid was used to deliver mRNA. The specific process was as follows:

[0141] The specific operation of the experiment refers to Example 2. Using ionizable lipid 21-5-C8C10, in the LNP four-component ratio, first keep the ratio of DMG-PEG2000 and cholesterol unchanged, and optimize the ratio of 21-5-C8C10 (30%, 35%, 40%, 45%, 50%) and DSPC (0%, 5%, 10%) (as Figure 5 shown); then keep the ratio of 21-5-C8C10 and cholesterol unchanged, and optimize the ratio of DSCP (0%, 5%) and DMG-PEG2000 (1%, 1.5%, 2%, 2.5%, 5%) (as Figure 6 shown).

[0142] The results of relative luciferase activity are as Figure 5 、 Figure 6 shown. The molar ratio between the components of LNP will also affect the RNA delivery efficiency to a certain extent. The optimal ratio is ionizable lipid: Cholesterol: DMG-PEG2000 = 30:48:2.5 (equal to 37.3:59.6:3.1).

[0143] Example 5

[0144] In this example, ionizable lipid is used to deliver mRNA. The specific process is as follows:

[0145] The specific operation of the experiment refers to Example 2. Using ionizable lipid 21-5-C8C10, the buffer for preparing nanoparticles has changed to "dissolve 150 ng of saRNA-Luc in sodium acetate buffer (the volume of sodium acetate buffer is twice the total volume of the lipid mixture, pH = 7.2)".

[0146] The results of relative luciferase activity are as Figure 7 shown. The results show that LNP can still normally encapsulate self-replicating mRNA under the condition of buffer pH = 7.2 and can efficiently express the target protein. This indicates that the ionizable lipid compound diethanolamine lipid of the present invention can not only encapsulate RNA by electrostatic attraction, but can adsorb self-replicating mRNA to the surface of LNP by hydrogen bond and van der Waals force interaction under neutral conditions.

[0147] Example 6

[0148] In this example, the efficiency of ionizable lipid-mediated delivery of self-replicating mRNA encoding firefly luciferase (saRNA-Luc), modified mRNA encoding firefly luciferase (modRNA-Luc), and circular mRNA encoding firefly luciferase (circRNA-Luc) was verified in 293T cell line. Ionizable lipids 21-5-C8, 22-5-C8, 21-6-C8, 22-6-C8, 21-5-C6C8, 22-5-C6C8, 21-6-C6C8, 21-6-C6C8, 21-5-C8C10, 22-5-C8C10, 21-6-C8C10, 21-6-C8C10 and commercial material ALC-0315 were used to deliver saRNA, modRNA and circRNA into cells respectively.

[0149] Specific steps:

[0150] 1. Cell culture

[0151] One day before the experiment, the cultured 293T cells were seeded in a 96-well cell culture plate (at 50% density). When the cell density grew to about 70-80%, the cell transfection experiment was carried out.

[0152] 2. Prepare lipid nanoparticles LNP-saRNA-Luc, LNP-modRNA-Luc, LNP-circRNA-Luc for cell transfection. The following steps take LNP-saRNA-Luc as an example.

[0153] The ionizable lipids 21-5-C8, 22-5-C8, 21-6-C8, 22-6-C8, 21-5-C6C8, 22-5-C6C8, 21-6-C6C8, 21-6-C6C8, 21-5-C8C10, 22-5-C8C10, 21-6-C8C10, 21-6-C8C10 and cholesterol, and dimyristoyl glycerol-polyethylene glycol 2000 (PEG-DMG2000) were dissolved in absolute ethanol at a certain concentration, and uniformly mixed according to the molar ratio of ionizable lipid:Cholesterol:PEG-DMG2000 = 30:48:2.5. At the same time, 150 ng of saRNA-Luc was absorbed and dissolved in sodium acetate buffer (the volume of sodium acetate buffer was twice the total volume of the lipid mixture, pH = 7.2), and then the saRNA buffer was quickly mixed with the lipid mixture solution, and incubated at room temperature for 20 min to assemble into stable lipid nanoparticles. Dilute with 2 volumes of sterile PBS and add them to 96-well cell culture plates for transfection respectively. Among them, the nitrogen-phosphorus ratio (N / P ratio) of ionizable lipid to saRNA of 18:1 was the optimal ratio, that is, the molar ratio between the protonable amino group and the phosphate group on saRNA.

[0154] Positive control group: The commercial lipid ALC-0315 was assembled into lipid nanoparticles according to its publicly reported preparation method.

[0155] Negative control group: 293T cells were cultured normally without transfection.

[0156] 3. Analysis of cell transfection efficiency

[0157] 36 hours after cell transfection, the medium in the 96-well cell culture plate was aspirated, cell lysate was added to lyse the cells on ice for 25 min, the supernatant was taken after centrifugation, transferred to a white 96-well detection plate, firefly luciferase substrate was added, and the content of firefly luciferase (chemiluminescence) was detected by an enzyme-linked immunosorbent assay. The results of relative luciferase activity are as Figure 8 (LNP-saRNA), Figure 9 (LNP-modRNA), Figure 10(LNP-circRNA) as shown. The results show that the ionizable lipids synthesized in the present invention can greatly enhance the transfection efficiency of self-replicating mRNA, modified mRNA, and circular RNA. They can efficiently deliver three different RNAs. When the head of the ionizable lipid is 21, the RNA expression efficiency is the highest. Lipids represented by 21-5-C8, 21-6-C8, and 21-5-C8C10 have extremely high transfection efficiency in all three RNAs and are superior to the commercial lipid ALC-0315, with a 2- to 3-fold increase. This indicates that the ionizable lipid compound of the present invention, namely diethanolamine lipid, can efficiently deliver self-replicating mRNA, modRNA, and circRNA in neutral buffer under three-component conditions.

[0158] Example 7

[0159] In this example, the ionizable lipid compound 21-5-C8C10 was used to deliver self-replicating mRNA (saRNA-Luc) encoding firefly luciferase (Luc) in Balb / c6 mice. After intramuscular injection, the in vivo imaging system (IVIS) was used to detect on the 2nd, 5th, 6th, 8th, 10th, 12th, 15th, and 20th days after injection.

[0160] Specific steps:

[0161] The experimental groups were: 21-5-C8C10 (pH = 7.2), 21-5-C8C10 (pH = 5.2). The ionizable lipid, cholesterol, and dimyristoyl glycerol-polyethylene glycol 2000 (PEG-DMG2000) were dissolved in absolute ethanol at a certain concentration. 1.5 μg of saRNA-Luc was taken and dissolved in sodium acetate buffer (the volume of sodium acetate buffer was twice the total volume of the lipid mixture, pH = 7.2 or 5.2). The saRNA-Luc dissolved in the buffer was taken and added to the lipid mixture solution, and quickly mixed evenly to assemble into lipid nanoparticles. The mixed solution was incubated at room temperature for 10 min, and then dialyzed in PBS using a dialysis bag (14,000 MW) for 1 hour, followed by intramuscular injection (the number of mice in each group was n = 4). The ratio used was ionizable lipid compound 21-5-C8C10:Cholesterol:DMG-PEG2000 = 30:48:2.5. The solution for pre-mixing RNA was sodium acetate, and the nitrogen-to-phosphorus ratio (N / P ratio) for preparing the nanoparticles was 18:1.

[0162] Analysis of in vivo imaging results

[0163] The IVIS results show that ( Figure 11 , Figure 12)All lipid nanoparticles were successfully expressed on the second day after intramuscular injection of saRNA-Luc. As time increased, the expression values increased successively. The expression value of self-replicating mRNA reached its peak 7 to 10 days after injection, while that of modRNA reached its peak 48 hours after injection, indicating that the ionizable lipid delivery of this self-replicating mRNA in the present invention has a longer expression time and a higher expression level, bringing a more persistent immune effect in the later application of mRNA vaccines.

[0164] Example 8

[0165] In this example, the ionizable lipid compound 21-5-C8C10 and the commercial lipid ALC-0315 were used to deliver self-replicating mRNA (saRNA-Luc) encoding firefly luciferase (Luc) in Balb / c6 mice. After intramuscular injection, the mice were detected using an in vivo imaging system (IVIS) at 2 days, 5 days, 7 days, 10 days, and 14 days after injection.

[0166] Specific steps

[0167] The experimental groups were: four different components of 21-5-C8C10 (named A, B, C, and D). Among them, group A was 21-5-C8C10: Cholesterol: DMG-PEG2000 = 30:48:1; group B was 21-5-C8C10: Cholesterol: DMG-PEG2000 = 30:48:2; group C was 21-5-C8C10: Cholesterol: DMG-PEG2000 = 30:48:2.5; group D was 21-5-C8C10: Cholesterol: DMG-PEG2000 = 30:48:5. The ionizable lipid, cholesterol, and dimyristoyl glycerol-polyethylene glycol 2000 (PEG-DMG2000) were dissolved in absolute ethanol at a certain concentration. 1.5 μg of saRNA-Luc was dissolved in sodium acetate buffer (the volume of sodium acetate buffer was twice the total volume of the lipid mixture, pH = 7.2). The saRNA-Luc dissolved in the buffer was pipetted into the lipid mixture solution and quickly mixed evenly to assemble into lipid nanoparticles. The mixed solution was incubated at room temperature for 10 minutes, and then dialyzed in PBS using a dialysis bag (14000 MW) for 1 hour, followed by intramuscular injection (the number of mice in each group was n = 4). The solution of premixed RNA was sodium acetate, and the nitrogen-to-phosphorus ratio (N / P ratio) for preparing the nanoparticles was 18:1.

[0168] Positive control group: The commercial lipid ALC-0315 was assembled into lipid nanoparticles according to its publicly reported preparation method.

[0169] Analysis of in vivo imaging results

[0170] The IVIS results showed that ( Figure 13 , Figure 14 ) the variation of the LNP components would affect the in vivo expression efficiency of self-replicating mRNA. All lipid nanoparticles were successfully expressed after intramuscular injection of saRNA-Luc. The expression value of self-replicating mRNA reached the peak 7 days after injection. Among them, the efficiency of experimental group D of 21-5-C8C10 was better than that of ALC-O315, and the peak expression value was about five times that of ALC-O315.

[0171] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An ionizable lipid compound, characterized in that: The structural formula of the ionizable lipid compound is as follows: the compound of Formula I, the compound of Formula II, or their stereoisomers, tautomers, or pharmaceutically acceptable salts thereof: Wherein, L0, L1, L2, L3, L4, L5, L6, L7, L8, L9 are each independently selected from C1-C8 alkyl groups; n1, n2, n3, n4, n5, n6, m1, m2, m3, m4, m5, m6 are all natural numbers, and are each independently selected from natural numbers from 1 to 20; k1, k2, k3, k4 are all natural numbers, and are each independently selected from natural numbers from 0 to 4.

2. The ionizable lipid compound according to claim 1, wherein: The compound of Formula I has the structural formula of Formula I-1 or Formula I-2:

3. The ionizable lipid compound according to claim 1, wherein: The compound of Formula II has the structural formula of Formula II-1 or Formula II-2:

4. The ionizable lipid compound according to claim 1, wherein: The ionizable lipid compound described above is selected from the following compounds:

5. A method for preparing an ionizable lipid compound according to any one of claims 1 to 4, characterized in that: Comprising the following steps: After reacting the compound of Formula 1 with N,N'-carbonyldiimidazole, reacting with the compound of Formula 3, and then reacting with C1-C8 alkenyl acyl chloride, Intermediate A is prepared; Intermediate A is reacted with the compound of Formula 6 to prepare the compound of Formula I; Reacting Intermediate A with the compound of Formula 7 to prepare the compound of Formula II; Wherein, the definitions of n1, n3, n4, k1, L2, L3, L4, L7, L8, L9 are as described in any one of Claims 1 to 4.

6. A lipid nanoparticle, characterized in that: Comprising the ionizable lipid compound described in any one of Claims 1 to 4.

7. The lipid nanoparticle according to claim 6, characterized in that: The lipid nanoparticle further comprises at least one of a structural lipid, a helper lipid, a polyethylene glycol-modified lipid, and a polymer.

8. The lipid nanoparticle according to claim 7, wherein: The lipid nanoparticle contains 20% - 65% ionizable lipid compound, 0% - 40% helper lipid, 20% - 60% structural lipid, and 0.1% - 10% polyethylene glycol-modified lipid.

9. A pharmaceutical composition, characterized in that: Comprising the ionizable lipid compound described in any one of Claims 1 to 4 and / or the lipid nanoparticle described in any one of Claims 6 to 8.

10. The pharmaceutical composition according to claim 9, characterized in that: The pharmaceutical composition further comprises a pharmaceutically active ingredient and / or a pharmaceutically acceptable excipient; preferably, the pharmaceutically active ingredient comprises at least one of a nucleic acid, a small molecule, and a protein drug.

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