Cationic lipid compound and its composition and application

Abstract

The present invention discloses a cationic lipid compound and its composition and application, and relates to the field of biomedical technology. The technical key points are as follows: the compound of the present invention is a cationic lipid compound, the center of which is an acrylate monomer containing a degradable group, and the number and length of the peripheral hydrophobic chain are controllable. The preparation method of the cationic lipid compound has the advantages of easy availability of raw materials, mild reaction conditions, high reaction yield, good reaction selectivity, simple and easy operation, and low requirements for instruments and equipment. The cationic lipid compound provided by the present invention is screened by the size and potential of lipid nanoparticles, in vitro cell transfection efficiency, cytotoxicity, in vivo delivery efficiency, lipid ratio regulation in lipid nanoparticle compositions, etc., which significantly improves the effectiveness and safety of nucleic acid delivery. The lipid nanoparticle composition of the present invention can selectively deliver mRNA to different organs and has good in vivo targeting.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a cationic lipid compound and a composition and application thereof. Background Art

[0002] Nucleic acid-based therapeutic approaches have been shown to have great potential for application in multiple fields, including vaccines against SARS-Cov-2 virus, protein replacement therapy, gene editing, cancer immunotherapy, etc. However, the development and application of nucleic acid therapeutic drugs still face many challenges, including the susceptibility of nucleic acid macromolecules to degradation by nucleases in vivo, the difficulty of nucleic acids to spontaneously enter cells, and the lack of efficient and safe nucleic acid delivery vectors. Lipid nanoparticles (LNP), as a non-viral gene carrier, provide a reliable solution for the delivery of nucleic acid drugs. They are a type of nucleic acid carrier that has been successfully transformed into clinical practice and have been successfully applied to mRNA COVID-19 vaccines (mRNA-1273, BNT162b2, etc.) and siRNA drugs (Onpattro).

[0003] LNP nucleic acid delivery systems are usually composed of four components: cationic lipids, phospholipids, cholesterol, and pegylated lipids. Among them, cationic lipids directly determine the delivery efficiency, safety, targeting and other properties of nucleic acid drugs, and become the core key to the development of LNP delivery technology. In order to cope with different application scenarios, such as the different molecular weights of nucleic acid molecules used and the different organs / tissues / cells to be delivered, the required LNP delivery carriers also have large differences in lipid structure. Therefore, it is of great significance to develop cationic lipid compounds and compositions of different chemical structures to achieve safe, effective and precise nucleic acid drug delivery.

[0004] At present, the development of new LNP nucleic acid delivery systems is the key to the development of nucleic acid drugs, and cationic lipids are the core of LNP systems. Designing new cationic lipid structures is a top priority in this field. In the prior art, cationic lipid design and synthesis mostly start from small molecule amines, which are prepared by connecting hydrophobic alkyl chains through linking groups, and ultimately it is difficult to achieve targeted nucleic acid delivery.

[0005] Therefore, in order to obtain a safer, more efficient and targeted cationic lipid and LNP nucleic acid delivery system, the present invention aims to provide a cationic lipid compound and its composition and application to solve the above problems, by taking a small molecule containing a degradable ester group as the center and connecting alkylamine to prepare a new cationic lipid; then, by adjusting the distribution ratio of the four components of LNP, efficient, safe and targeted mRNA delivery can be achieved. Summary of the invention

[0006] The purpose of the present invention is to solve the above problems and to address the deficiencies of existing nucleic acid carriers, and to provide a cationic lipid compound and its composition and application. The present invention enriches the types of existing cationic lipid compounds, and lipid compounds with specific structures can be selected as nucleic acid carriers according to the types of nucleic acid drugs and the organs / tissues / cells that need to be enriched.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] The present invention provides a cationic lipid compound represented by general formula (I), or its stereoisomers, tautomers, pharmaceutically acceptable salts, prodrugs or solvates,

[0009]

[0010] Wherein, R1 is selected from the following structures: all groups except acrylate groups in acrylate monomers or C1-C 26 Alkyl or C1-C 26 Alkenyl, one or more C in the alkyl or alkenyl is optionally replaced by O or N, and the alkyl or alkenyl structure substituent group is C1-C 18 One or more of an alkyl group, an ester group, a carbonate group, a carbonyl group, an amide group, an ether group, an amine group, a hydrocarbon group, an olefin group, a carboxyl group, an acyl group, and an alkoxy group, wherein the C1-C 18 The alkyl group is unsubstituted or optionally substituted with one or more of the following substituents: ester group, carbonate group, carbonyl group, amide group, ether group, amine group, hydrocarbon group, olefin group, carboxyl group, acyl group, alkoxy group;

[0011] R2 is selected from one or more of the following structures: H, methyl, saturated alkyl chain, unsaturated alkyl chain;

[0012] L1 is selected from one or more of the following structures: -O-, -S-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -C(=O)NH-, -NHC(=O)-, -C(=S)O-, -OC(=S)-, -OC(=S)O-, -C(=O)S-, -SC(=O)-, -OC(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=S)O-;

[0013] L2 is a linear or branched, saturated or unsaturated alkyl or alkenyl structure containing 1 to 18 carbon atoms, wherein the alkyl or alkenyl structure is optionally substituted with one or more of the following substituents: C1-C6 alkyl, ester, carbonate, carbonyl, amide, ether, amine, hydrocarbon, olefin, carboxyl, acyl, alkoxy;

[0014] y is an integer from 0 to 6.

[0015] Furthermore, the cationic lipid compound is one or more of the compounds shown in the following structures:

[0016]

[0017]

[0018] Furthermore, in formula (II): R1 is selected from the following structures: all groups except acrylate groups in acrylate monomers or C1-C 26 Alkyl or C1-C 26 Alkenyl, one or more C in the alkyl or alkenyl is optionally replaced by O or N, and the substituent group of the alkyl or alkenyl structure is C1-C 18 One or more of an alkyl group, an ester group, a carbonate group, a carbonyl group, an amide group, an ether group, an amine group, a hydrocarbon group, an olefin group, a carboxyl group, an acyl group, and an alkoxy group, wherein the C1-C 18 The alkyl group is unsubstituted or optionally substituted with one or more of the following substituents: ester group, carbonate group, carbonyl group, amide group, ether group, amine group, hydrocarbon group, olefin group, carboxyl group, acyl group, alkoxy group;

[0019] R2 is selected from one or more of the following structures: H, methyl, saturated alkyl chain, unsaturated alkyl chain;

[0020] n' is an integer from 0 to 16.

[0021] The present invention also provides a composition comprising a therapeutic or preventive agent and a carrier for delivering the therapeutic or preventive agent, wherein the carrier comprises a cationic lipid compound, and the cationic lipid compound comprises one or more of the general formula (I) or (II) according to any one of claims 1 to 3, or its stereoisomers, or its tautomers, or its pharmaceutically acceptable salts, prodrugs, or solvates thereof.

[0022] Further, the cationic lipid is used with one or more auxiliary components to form a lipid nanoparticle system for delivering a therapeutic or prophylactic agent.

[0023] Furthermore, the auxiliary components include sterols, charged lipids, neutral lipids, and pegylated lipids.

[0024] Furthermore, the sterol includes any one or more of cholesterol, coproposterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, and α-tocopherol.

[0025] Furthermore, the sterol is cholesterol.

[0026] Furthermore, the charged lipid is any lipid molecule that exists in a positively or negatively charged form at a selected pH value or range, and the selected pH value or range corresponds to the pH conditions of the intended use environment of the lipid, such as physiological pH and endosomal lysosomal pH 4.5-6.5.

[0027] Furthermore, the neutral lipids include one or more of phospholipids, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and their derivatives.

[0028] Furthermore, the neutral lipid is a phospholipid.

[0029] Furthermore, the PEGylated lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000.

[0030] Furthermore, the therapeutic or preventive agent includes one or more of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein.

[0031] Furthermore, the mass ratio of the carrier to the therapeutic or preventive agent is 5:1 to 50:1.

[0032] Furthermore, the nucleic acid molecule is a linear mRNA or a circular mRNA.

[0033] Furthermore, the nucleic acid molecule is RNA, mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitor, microRNA activator or shRNA, DNA, antisense nucleic acid, aptamer, ribozyme, immunostimulatory nucleic acid or PNA.

[0034] The present invention also provides a lipid nanoparticle composition, which is composed of a cationic lipid compound, a phospholipid, cholesterol and a pegylated lipid, wherein the cationic lipid compound accounts for 14.5% to 37.9%, the phospholipid accounts for 14.9% to 37.9%, the cholesterol accounts for 22.1% to 44.5%, and the pegylated lipid accounts for 1.5% to 8.2%.

[0035] Further, the composition is composed of a cationic lipid compound, a phospholipid, cholesterol and a pegylated lipid, and the molar ratio of the cationic lipid compound, the phospholipid, cholesterol and the pegylated lipid is: 15 / 20 / 25 / 2 or 10 / 10 / 15 / 1 or 10 / 15 / 20 / 2 or 10 / 20 / 25 / 3 or 10 / 25 / 30 / 4 or 15 / 10 / 20 / 3 or 15 / 15 / 15 / 4 or 15 / 20 / 30 / 1 or 15 / 25 / 25 / 2 or 20 / 10 / 25 / 4 or 20 / 15 / 30 / 3 or 20 / 20 / 15 / 2 or 20 / 25 / 20 / 1 or 25 / 10 / 30 / 2 or 25 / 15 / 25 / 1 or 25 / 20 / 20 / 4 or 25 / 25 / 15 / 3.

[0036] The present invention also provides the cationic lipid compound represented by the above-mentioned general formula (I), or its stereoisomers, tautomers, pharmaceutically acceptable salts, prodrugs or solvates, or the above-mentioned composition, or the use of the above-mentioned lipid nanoparticle composition in the preparation of drugs that can deliver nucleic acid drugs targeted to different organs or locations.

[0037] Further, the different organs or locations include spleen, liver and muscle.

[0038] The difficulty and significance of the technical problem solved by the present invention are:

[0039] To meet the needs of nucleic acid therapy, LNP delivery systems are suitable for the delivery of a variety of nucleic acid molecules (mRNA, siRNA, sgRNA, microRNA, etc.), showing excellent in vivo RNA delivery performance and clinical transformation potential, and have been successfully applied to mRNA COVID-19 vaccines (BNT162b2 and mRNA-1273, etc.) and siRNA drugs (Onpattro). LNP nucleic acid delivery systems are usually composed of four components: cationic lipids, phospholipids, cholesterol, and pegylated lipids. Among them, ionizable cationic lipids are the key components of LNP. Many scientists at home and abroad have done a lot of work on LNP delivery systems and designed many cationic lipids such as DLin-MC3-DMA (Onpattro core component), ALC-0315 (BNT162b2 core component), and SM-102 (mRNA-1273 core component). In order to develop precision nucleic acid drugs, we still need a safer, more efficient, and more precise LNP nucleic acid delivery system. The key to achieving this goal lies in the development of new cationic lipid chemical structures (new chemical structures) and the optimization of LNP multi-component distribution ratios (new LNP formulas).

[0040] Compared with the prior art, this solution has the following beneficial effects:

[0041] 1. The compound provided by the scheme of the present invention is a cationic lipid compound, the core of which is an acrylate monomer containing a degradable group, and the number and length of the peripheral hydrophobic chains are controllable;

[0042] 2. The method for preparing the cationic lipid compound of the present invention has the advantages of readily available raw materials, mild reaction conditions, high reaction yield, good reaction selectivity, simple and easy operation and low requirements for instruments and equipment;

[0043] 3. The cationic lipid compounds of the present invention are screened by the size and potential of lipid nanoparticles, in vitro cell transfection efficiency, cytotoxicity, in vivo delivery efficiency, lipid ratio regulation in lipid nanoparticle compositions, etc., to significantly improve the effectiveness and safety of nucleic acid delivery;

[0044] 4. The lipid nanoparticle composition of the present invention can selectively deliver mRNA to different organs and has good in vivo targeting. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The invention relates to the design and preparation of the cationic lipid in Example 1;

[0046] Figure 2 is the H NMR spectrum of the cationic lipid 3Ac1-C18 in Example 2 of the present invention;

[0047] Figure 3 is the H NMR spectrum of the cationic lipid 3Ac1-C212 in Example 3 of the present invention;

[0048] Figure 4 is the H NMR spectrum of the cationic lipid 5Ac1-C12 in Example 4 of the present invention;

[0049] Figure 5 is the H NMR spectrum of the 5-cationic lipid 6Ac1-C12 in the embodiment of the present invention;

[0050] Figure 6 is the H NMR spectrum of 6 cationic lipid 6Ac1-C210 in the embodiment of the present invention;

[0051] Figure 7 is the particle size of the representative mRNA lipid nanoparticle composition in Example 8 of the present invention;

[0052] Figure 8 is the zeta potential of the representative mRNA lipid nanoparticle composition in Example 8 of the present invention;

[0053] Fig. 9 is the cell survival rate of the mRNA lipid nanoparticle composition (1Ac1-C6 to 1Ac5-C212) in Example 9 of the present invention;

[0054] Fig.10 is the cell survival rate of the mRNA lipid nanoparticle composition (2Ac1-C6 to 3Ac2-C212) in Example 9 of the present invention;

[0055] Fig.11 is the cell survival rate of the mRNA lipid nanoparticle composition (4Ac1-C6 to 6Ac1-C212) in Example 9 of the present invention;

[0056] Fig.12 is the mRNA delivery efficiency of the mRNA lipid nanoparticle composition (1Ac1-C6 to 1Ac5-C212) in Example 10 of the present invention;

[0057] Fig.13 is the mRNA delivery efficiency of the mRNA lipid nanoparticle composition (2Ac1-C6 to 3Ac2-C212) in Example 10 of the present invention;

[0058] Fig.14 is the mRNA delivery efficiency of the mRNA lipid nanoparticle composition (4Ac1-C6 to 6Ac1-C212) in Example 10 of the present invention;

[0059] Fig.15 The orthogonal method and multi-factor and multi-level experimental design in Example 11 of the present invention are used to optimize the lipid ratio in the mRNA lipid nanoparticle composition ((a) cationic lipid / phospholipid / cholesterol / pegylated lipid four-component molar ratio design; the effect of cationic lipid (b), phospholipid (c), cholesterol (d), and pegylated lipid (e) content on mRNA delivery efficiency);

[0060] Fig.16 The lipid ratio in the mRNA lipid nanoparticle composition of Example 11 of the present invention is optimized ((a) chemical structure of cationic lipid 6Ac1-C12; (b) molar ratio design of the four components of 6Ac1-C12 / DOPE / cholesterol / DMG-PEG2000; (c) mRNA delivery efficiency of lipid nanoparticle compositions with different ratios; (d) cell survival rate);

[0061] Fig.17 The effect of the lipid ratio of the lipid nanoparticle composition in Example 12 of the present invention on the in vivo delivery of mRNA ((a) the design of the molar ratio of the four components 6Ac1-C12 / DOPE / cholesterol / DMG-PEG2000 and the mass ratio of 6Ac1-C12 / mRNA; (b) the in vivo mRNA delivery efficiency);

[0062] Fig.18 is the in vivo mRNA delivery efficiency of the lipid nanoparticle composition based on different cationic lipids in Example 12 of the present invention;

[0063] Fig.19 is the in vivo organ-selective mRNA delivery of lipid nanoparticle compositions based on different cationic lipids after intravenous administration in Example 12 of the present invention;

[0064] Fig. 20 : This is the in vivo mRNA delivery efficiency of lipid nanoparticle compositions of different cationic lipids using intramuscular injection in Example 13 of the present invention. DETAILED DESCRIPTION

[0065] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0066] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0067] Embodiment 1:

[0068] Synthesis of the novel cationic lipid nAcx-Cm:

[0069]

[0070] Add acrylate monomer (nAcx) and amine small molecule (Cm) to a dry reaction bottle, wherein the molar amount of the conjugated double bond of the acrylate is equal to the molar amount of the secondary amine group of the amine small molecule. Stir at 50-80°C for 48-72h to obtain a crude product, which is then purified by silica gel column chromatography to obtain the final product cationic lipid nAcx-Cm (such as Figure 1 shown).

[0071] Embodiment 2:

[0072] Synthesis of cationic lipid compound 3Ac1-C18:

[0073]

[0074] Trimethylolpropane triacrylate (3Ac1, 0.2 mmol, 1.0 eq) and N-methyl-1-octadecylamine (C18, 0.6 mmol, 3.0 eq) were added to a dry reaction bottle, and the mixture was stirred magnetically at 60°C for 72 h. The crude product was purified by silica gel column chromatography to obtain the product 3Ac1-C18. The hydrogen spectrum of cationic lipid 3Ac1-C18 is shown in Figure 2 .

[0075] Embodiment 3:

[0076] Synthesis of cationic lipid compound 3Ac1-C212:

[0077]

[0078] Trimethylolpropane triacrylate (3Ac1, 0.2 mmol, 1.0 eq) and didodecylamine (C212, 0.6 mmol, 3.0 eq) were added to a dry reaction bottle, and the mixture was stirred magnetically at 60°C for 72 h. The crude product was purified by silica gel column chromatography to obtain the product 3Ac1-C212. The hydrogen spectrum of cationic lipid 3Ac1-C212 is shown in Figure 3 .

[0079] Embodiment 4:

[0080] Synthesis of cationic lipid compound 5Ac1-C12:

[0081]

[0082] Dipentaerythritol pentaacrylate (5Ac1, 0.2 mmol, 1.0 eq) and N-dodecylmethylamine (C12, 1.0 mmol, 5.0 eq) were added to a dry reaction bottle, and the mixture was stirred magnetically at 60°C for 72 h. The crude product was purified by silica gel column chromatography to obtain the product 5Ac1-C12. The hydrogen spectrum of the cationic lipid 5Ac1-C12 is shown in Figure 4 .

[0083] Embodiment 5:

[0084] Synthesis of cationic lipid compound 6Ac1-C12:

[0085]

[0086] Dipentaerythritol hexaacrylate (6Ac1, 0.2 mmol, 1.0 eq) and N-dodecylmethylamine (C12, 1.2 mmol, 6.0 eq) were added to a dry reaction bottle, and the mixture was stirred magnetically at 60°C for 72 h. The crude product was purified by silica gel column chromatography to obtain the product 6Ac1-C12. The hydrogen spectrum of the cationic lipid 6Ac1-C12 is shown in Figure 5 .

[0087] Embodiment 6:

[0088] Synthesis of cationic lipid compound 6Ac1-C210:

[0089]

[0090] Dipentaerythritol hexaacrylate (6Ac1, 0.2 mmol, 1.0 eq) and didecylamine (C210, 1.2 mmol, 6.0 eq) were added to a dry reaction bottle, and the mixture was stirred magnetically at 60°C for 72 h. The crude product was purified by silica gel column chromatography to obtain the product 6Ac1-C210. ​​The hydrogen spectrum of cationic lipid 6Ac1-C210 is shown in Figure 6 .

[0091] Embodiment 7:

[0092] Preparation of lipid nanoparticles (LNP formulations):

[0093] Cationic lipid compounds, phospholipid DOPE (purchased from Avanti, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), cholesterol (purchased from Sigma), DMG-PEG2000 (purchased from Avanti, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000) were dissolved in ethanol at a molar ratio of 15 / 20 / 25 / 2 or other specified molar ratios to prepare an ethanol lipid solution, and luciferase mRNA (Fluc mRNA) was diluted in 10mM citrate buffer (pH=4) to obtain an aqueous solution of mRNA. The ethanol phase and the aqueous phase solution were quickly mixed at a volume ratio of 1:3, and allowed to stand for 10-30min to prepare LNP, and finally 1×PBS was added to the required volume, wherein the mass ratio of cationic lipid to mRNA was 5 / 1 to 30 / 1. LNP used in animal experiments needs to be dialyzed in 1×PBS in advance before injection.

[0094] Embodiment 8:

[0095] LNP Particle Size and Zeta Potential Characterization:

[0096] Lipid nanoparticles (LNPs) were prepared with a molar ratio of 15 / 20 / 25 / 2 for cationic lipid / DOPE / cholesterol / DMG-PEG2000 and diluted to the required volume. The particle size and polydispersity coefficient of LNPs were determined by dynamic light scattering using a Malvern Zetasizer NanoZS. The test results are shown in Figure 7 At the same time, the Zeta potential of LNP was measured using a Malvern Zetasizer NanoZS. The test results are shown in Figure 8 .

[0097] Embodiment 9:

[0098] Cytotoxicity Evaluation of Lipid Nanoparticle Compositions:

[0099] Human ovarian cancer cells IGROV1 in the logarithmic growth phase were digested, and the cells after digestion and centrifugation were resuspended in 1640 medium containing 10% fetal bovine serum and 1% double antibody, and inoculated in a white 96-well plate at a cell density of 10,000 to 15,000 cells / well, and placed in a 37°C constant temperature incubator containing 5% CO2 for 24 hours. Then, the prepared LNPs encapsulating FlucmRNA were added, the molar ratio of cationic lipid / DOPE / cholesterol / DMG-PEG2000 was 15 / 20 / 25 / 2, and the mRNA was set to 25ng per well, and incubated at 37°C for 24 hours. The cell viability was determined by the Alamarblue detection kit method, the culture medium in the white 96-well plate was discarded, 100μL of 1640 culture medium without fetal bovine serum and 10μL of Alamarblue detection reagent were added to each well, and the wells were incubated for 2 hours at 37°C, and the color in the wells changed from indigo blue to pink. The detection was performed using a multifunctional microplate reader, with the excitation wavelength set at 530 nm and the emission wavelength set at 590 nm. Figure 9-11 The results show that the lipid nanoparticle composition in the embodiment of the present invention has almost no inhibitory effect on cells, and the cell survival rate can reach more than 80%.

[0100] Embodiment 10:

[0101] In vitro evaluation of lipid nanoparticle compositions for mRNA delivery:

[0102] Human ovarian cancer cells IGROV1 in the logarithmic growth phase were digested, and the cells after digestion and centrifugation were resuspended in 1640 medium containing 10% fetal bovine serum and 1% double antibody, and inoculated in a white 96-well plate at a cell density of 10,000 to 15,000 cells / well, and placed in a 37°C constant temperature incubator containing 5% CO2 for 24 hours. Then, the prepared LNPs encapsulating FlucmRNA were added, with a cationic lipid / DOPE / cholesterol / DMG-PEG2000 molar ratio of 15 / 20 / 25 / 2, and the mRNA per well was set to 25ng, and the incubation continued at 37°C. After 24 hours, Bio-Lumi TM The luciferase reporter gene detection kit and multifunctional microplate reader were used to detect the Fluc mRNA expression efficiency. The results of the in vitro mRNA delivery efficiency evaluation of the lipid nanoparticle composition determined by the above method are shown in Figure 12-14 shown.

[0103] Embodiment 11:

[0104] Screening of lipid component ratios of lipid nanoparticle compositions:

[0105] Human ovarian cancer cells IGROV1 in the logarithmic growth phase were digested, and the cells after digestion and centrifugation were resuspended in 1640 medium containing 10% fetal bovine serum and 1% double antibody, and inoculated into a white 96-well plate at a cell density of 10,000 to 15,000 cells / well, and placed in a 37°C constant temperature incubator containing 5% CO2 for 24 hours. Then, the prepared LNPs encapsulated with Fluc mRNA were added, and the mRNA was set to 25 ng per well, and the incubation continued at 37°C. Cationic lipid / DOPE / cholesterol / DMG-PEG2000 molar ratio was used in an orthogonal method for multi-factor and multi-level experimental design ( Fig.15 a), 6Ac1-C12 / DOPE / cholesterol / DMG-PEG2000 molar ratios included 10 / 10 / 15 / 1, 10 / 15 / 20 / 2, 10 / 20 / 25 / 3, 10 / 25 / 30 / 4, 15 / 10 / 20 / 3, 15 / 15 / 15 / 4, 15 / 20 / 30 / 1, 15 / 25 / 25 / 2, 20 / 10 / 25 / 4, 20 / 15 / 30 / 3, 20 / 20 / 15 / 2, 20 / 25 / 20 / 1, 25 / 10 / 30 / 2, 25 / 15 / 25 / 1, 25 / 20 / 20 / 4, 25 / 25 / 15 / 3. After 24 h, the Bio-Lumi TM The luciferase reporter gene detection kit and multifunctional microplate reader were used to detect the expression efficiency of Fluc mRNA. The results of the in vitro mRNA delivery efficiency evaluation of lipid nanoparticle compositions with different lipid ratios are shown in Figure 2. Figure 15-16 .

[0106] Embodiment 12:

[0107] Evaluation of mRNA delivery after intravenous injection of lipid nanoparticle compositions in vivo:

[0108] Female C57BL / 6 mice aged about 6 weeks were selected, and the prepared lipid nanoparticle composition LNP was injected into the tail vein at a dosage of 0.25 mg / kg of Fluc mRNA. The molar ratio of cationic lipid (nAcx-Cm) / DOPE / cholesterol / DMG-PEG2000 is 15 / 20 / 25 / 2, unless otherwise agreed; the mass ratio of cationic lipid (nAcx-Cm) / mRNA is 10 / 1 or 20 / 1. After 6 hours, 100 μL of D-luciferin potassium salt with a concentration of 30 mg / mL was injected intraperitoneally into the mice. After 5 minutes, the mice were placed under an in vivo imaging system to observe and take pictures to record the chemiluminescence intensity of the mouse body and isolated organs. Figure 17-Figure 19 The figure shows the delivery efficiency of Fluc mRNA in vivo of a representative lipid nanoparticle composition, and DLin-MC3-DMA LNP is used as a control. The formula used in DLin-MC3-DMA LNP is the same as the FDA-approved Onpattro drug, that is, the molar ratio of DLin-MC3-DMA / DSPC / cholesterol / DMG-PEG2000 is 50 / 10 / 38.5 / 1.5. The cationic lipids described in the present invention have multiple expression intensities similar to those of DLin-MC3-DMA, and multiple expression intensities are significantly better than DLin-MC3-DMA. In addition, 2Ac3-C18 LNP can specifically deliver mRNA to the spleen for expression, and multiple other cationic lipid LNPs can specifically deliver mRNA to the liver for expression (such as Fig.19 shown), Fig.19 Among them, 2Ac3-C18 LNP can deliver mRNA to the spleen for expression, and other multiple cationic lipid LNPs can deliver mRNA to the liver for expression. The mRNA delivery efficiency of multiple lipid nanoparticle compositions is better than that of DLin-MC3-DMALNP.

[0109] Embodiment 13:

[0110] Evaluation of mRNA delivery after intramuscular injection of lipid nanoparticle compositions in vivo:

[0111] Female C57BL / 6 mice aged about 6 weeks were selected and intramuscularly injected with the prepared lipid nanoparticle composition LNP. The molar ratio of cationic lipid (nAcx-Cm) / DOPE / cholesterol / DMG-PEG2000 was 15 / 20 / 25 / 2. After 6 hours, 100 μL of D-luciferin potassium salt with a concentration of 30 mg / mL was injected intraperitoneally into the mice. After 5 minutes, the mice were placed under a living imaging system to observe and take pictures to record the chemiluminescence intensity of the mice. Fig. 20The delivery efficiency of Fluc mRNA in vivo by a representative lipid nanoparticle composition is shown, with SM-102 and ALC-0315LNPs used as controls. The formula used in SM-102LNP is the same as the FDA-approved new crown mRNA vaccine mRNA-1273, that is, the molar ratio of SM-102 / DSPC / cholesterol / DMG-PEG2000 is 50 / 10 / 38.5 / 1.5; the formula used in ALC-0315LNP is the same as the FDA-approved new crown mRNA vaccine BNT162b2, that is, the molar ratio of ALC-0315 / DSPC / cholesterol / ALC-0159 is 46.3 / 9.4 / 42.7 / 1.6. The cationic lipids described in the present invention have multiple expression intensities that are significantly better than SM-102 and ALC-0315LNPs.

[0112] Through the above embodiments of the present invention, the compound provided by the scheme of the present invention is a cationic lipid compound, the center of which is an acrylate monomer containing a degradable group, and the number and length of the peripheral hydrophobic chain are controllable. The preparation method of the cationic lipid compound has the advantages of easy availability of raw materials, mild reaction conditions, high reaction yield, good reaction selectivity, simple and easy operation, and low requirements for instruments and equipment. The cationic lipid compound provided by the present invention is screened by the size and potential of lipid nanoparticles, in vitro cell transfection efficiency, cytotoxicity, in vivo delivery efficiency, lipid ratio regulation in lipid nanoparticle compositions, etc., which significantly improves the effectiveness and safety of nucleic acid delivery. The lipid nanoparticle composition of the present invention can selectively deliver mRNA to different organs and has good in vivo targeting.

[0113] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A cationic lipid compound represented by general formula (II), or a stereoisomer or tautomer thereof, characterized in that , The cationic lipid compound represented by the general formula (II) is one or more of the compounds represented by the following structures:

2. A composition, characterized in that: The composition includes a therapeutic or preventive agent and a carrier for delivering the therapeutic or preventive agent, wherein the carrier includes a cationic lipid compound, wherein the cationic lipid compound is the cationic lipid compound according to claim 1 or one or more of its stereoisomers or tautomers; and the therapeutic or preventive agent is an mRNA nucleic acid molecule.

3. A composition as claimed in claim 2, characterized in that: The cationic lipids are used with one or more auxiliary components to form lipid nanoparticle systems for delivery of therapeutic or prophylactic agents.

4. A composition as claimed in claim 3, characterized in that: The auxiliary component is selected from cholesterol, phospholipids, and PEGylated lipids; The PEGylated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000.

5. A composition as claimed in claim 2, characterized in that: The mass ratio of the carrier to the therapeutic or preventive agent is 5:1 to 50:

1.

6. A lipid nanoparticle composition, characterized in that: The composition is composed of a cationic lipid compound, a phospholipid, cholesterol and a pegylated lipid, wherein the cationic lipid compound accounts for 14.5% to 37.9%, the phospholipid accounts for 14.9% to 37.9%, the cholesterol accounts for 22.1% to 44.5%, and the pegylated lipid accounts for 1.5% to 8.2%; The molar ratio of the cationic lipid compound, phospholipid, cholesterol and pegylated lipid is: 15 / 20 / 25 / 2 or 10 / 10 / 15 / 1 or 10 / 15 / 20 / 2 or 10 / 20 / 25 / 3 or 10 / 25 / 30 / 4 or 15 / 10 / 20 / 3 or 15 / 15 / 15 / 4 or 15 / 20 / 30 / 1 or 15 / 25 / 25 / 2 or 20 / 10 / 25 / 4 or 20 / 15 / 30 / 3 or 20 / 20 / 15 / 2 or 20 / 25 / 20 / 1 or 25 / 10 / 30 / 2 or 25 / 15 / 25 / 1 or 25 / 20 / 20 / 4 or 25 / 25 / 15 / 3; The cationic lipid compound in the composition is the cationic lipid compound according to claim 1 or one or more of its stereoisomers or tautomers.

7. Use of a cationic lipid compound represented by the general formula (II) as claimed in claim 1, or its stereoisomers, tautomers or the composition as claimed in any one of claims 2 to 5, or the lipid nanoparticle composition as claimed in claim 6 in the preparation of a drug that can deliver nucleic acid drugs targeted to the spleen, liver and muscle.

Images