Ionizable lipid molecules, method for producing the same, and application in the production of lipid nanoparticles

Abstract

The present invention discloses an ionizable lipid molecule and a method for producing the same, a composition containing the same, an application in the production of a carrier for delivering nucleic acid to cells, and an application in the production of lipid nanoparticles LNP, which improve the action and effect of an mRNA-LNP formulation by improving the structure of the ionizable lipid molecule and adjusting the LNP composition scheme. The ionizable lipid molecule in the present invention has a structure represented by formula (I). The ionizable lipid molecule, phospholipid, cholesterol, and polyethylene glycol are synthesized by a microfluidic control synthesis method to obtain LNP, and the obtained LNP can improve the translation expression level of the carrier-mRNA in cells and enhance the action and effect of the mRNA-LNP formulation, providing a theoretical basis for theoretical treatment with an individualized mRNA-LNP formulation.

Description

Technical Field

[0001] This application claims the priority of CN202110159969.3, and the full text of the priority document is incorporated herein by reference.

[0002] The present invention relates to the field of biotechnology, and in particular, to ionizable lipid molecules, methods for producing the same, and applications in the production of lipid nanoparticles.

Background Art

[0003] Nucleic acid therapeutic substances include small interfering RNA (siRNA), microRNA (miRNA), and messenger RNA (mRNA). Such nucleic acids function by multiple mechanisms. In the case of siRAN or miRNA, the intracellular level of a specific protein may be reduced by the RNA interference (RNAi) method. After introducing siRAN or miRNA into the cytoplasm, siRAN or miRNA binds to the RISC protein, and its sense strand is removed by the RISC complex. Thus, in RISC, a template is provided that is recognizable and has a complementary sequence to the bound siRAN or miRNA sequence and binds to the mRNA. After binding to the complementary mRNA, the RISC complex cleaves the mRNA and releases the cleaved strand. RNAi can down-regulate a specific protein by target-specific destruction of the mRNA synthesized by the corresponding encoded protein.

[0004] Therapeutic applications using RNAi technology are widely carried out because corresponding siRNAs or miRNAs can be constructed using any nucleotide sequence against a target protein. Currently, siRNA or miRNA constructs have demonstrated the ability to specifically downregulate target proteins in in vitro and in vivo models. However, when siRNAs or miRNAs are used as systemic therapeutic agents, they face the following two problems. One is that they are very sensitive to nucleases in plasma, and the other is that when siRNAs or miRNAs are administered systemically, they have limited ability to enter the intracellular compartments where they can bind to RISC. To stabilize such double-stranded molecules, chemically modified nucleotide linkers, such as phosphorothioate groups, can be incorporated into the molecule. However, such chemical modifications can only provide limited protection and may also reduce the activity of the molecule. Other methods include using carrier delivery systems such as polymers and ionizable lipid molecules, or chemically modifying such double-stranded molecules (e.g., by covalently binding ionizable lipid molecules) to facilitate the intracellular delivery of siRNAs or miRNAs.

[0005] mRNA controls protein synthesis necessary for living cells by transcribing the instructions stored in DNA. In mRNA-based therapy, chemically modified mRNA molecules are introduced into the cytoplasm, and transcription and expression are carried out using self-nucleotides in the cytoplasm to produce the proteins required by the body. mRNA vaccines are used to modify the relevant mRNA in vitro and then transfer it into living cells for expression, producing protein antigens to induce an immune response against the antigen in the body and further expand the body's immune capacity. In theory, since mRNA can express any protein, it is possible to treat various diseases and produce various vaccines.

[0006] mRNA can be classified into two types: nonreplicating mRNA and self-amplifying mRNA. Nonreplicating mRNA encodes only the target protein and contains 5’ and 3’ untranslated regions (UTRs). Self-amplifying mRNA encodes not only the target protein but also a replication mechanism that enables intracellular RNA amplification and protein expression. Compared with nonreplicating mRNA, self-amplifying mRNA can continuously express large amounts of the target protein, that is, it can be administered at a lower dose under the premise of obtaining the same therapeutic or immune effect.

[0007] In mRNA-based therapy, the short half-life and the ability to be rapidly degraded by a large amount of extracellular RNA enzymes pose a challenge. In addition, its immunogenicity, that is, exogenous RNA, is generally recognized by the body as a signal of viral infection. By performing various chemical modifications on the structure of mRNA, for example, changing the 5’-cap, polyadenylic acid (A) tail, 5’- and 3’-UTRs and coding regions, and using N1-methyladenosine (m1A) or N6-methyladenosine (m6A) instead of natural adenosine, the spatial structure of mRNA can be stabilized, the immunogenicity can be reduced, and a high protein expression level can be achieved. Also, since the size of mRNA is significantly larger than that of siRNA or miRNA constructs, there are many cells with low uptake efficiency for mRNA. So far, researchers have considered delivering chemically modified or unmodified mRNA into cells using carrier delivery systems based on various lipid materials, such as various vesicles, liposomes, lipid nanoparticles (LNP), lipid emulsions, lipid implants, etc.

[0008] Nucleic acids (RNA) have great therapeutic potential in regulating protein expression in the body. However, naked RNA has extremely low efficiency of being internalized by cells, and it is also easily rapidly removed by the kidneys or rapidly degraded by RNA enzymes in the body's delivery process. Therefore, its actual therapeutic effect can only be considered with a large discount. To more safely and effectively exert the therapeutic ability of RNA, scientists use lipid nanoparticles (LNPs) to encapsulate RNA or deliver it to specific sites in the body. Such RNA delivery strategies show great application value in the delivery of double-stranded small interfering RNAs (siRNAs, 21 to 23 nucleotides in length). For example, lipid C12-200 has already been widely used in the manufacture of siRNA-LNP formulations for suppressing protein expression in various therapeutic applications in vivo. After the emergence of artificially synthesized ionizable lipid materials and lipid-like materials, they not only significantly reduce the in vivo toxicity of LNPs but also enable the delivery of large molecular weight RNAs (such as mRNA) in the body. These ionizable lipids or lipid molecules containing amines carry a positive charge and can efficiently bind to negatively charged mRNA by electrostatic attraction and self-assemble to form LNPs. According to LNPs, the blood circulation time of RNA is improved and the cellular uptake rate is also increased. When the RNA in the cell passes through the endosomal escape pathway and is released into the cytoplasm, it can express specific proteins and play a certain therapeutic role.

[0009] The raw material components of LNPs mainly include the following four types: 1) Ionizable lipids or lipid molecules (such as DLin-KC 2-DMA, DLin-MC3-DMA, L319), which are core components enabling the delivery of mRNA in the body. 2) Phospholipid molecules, namely phospholipids, which constitute the LNP bilayer and are also beneficial for the escape of the nuclear bulk. 3) Cholesterol, which improves the stability of LNPs and promotes membrane fusion. 4) Polyethylene glycol, such as DMG-PEG2000, which can control and reduce the particle size of LNPs and "protect" them from the influence of non-specific endocytosis by immune cells.

[0010] In the body, due to changes in the raw materials and composition of LNPs, the physicochemical stability of mRNA-LNP formulations and the effects and efficacy by mRNA are significantly affected. In conventional LNP composition schemes, the effects of mRNA-LNP formulations cannot be fully exerted. Therefore, it is necessary to continuously adjust the structure of ionizable lipid molecules to optimize the design for specific RNAs. The present invention is made to solve such problems.

Summary of the Invention

[0011] In order to make up for the deficiencies of the prior art, the present invention aims to improve the structure of ionizable lipid molecules and adjust the LNP composition scheme to enhance the effects and efficacy of mRNA-LNP formulations, and provides ionizable lipid molecules and their manufacturing methods, as well as their applications in the manufacture of carriers for delivering nucleic acids to cells and in the manufacture of lipid nanoparticles LNPs, and their pharmaceutically acceptable salts and stereoisomers.

[0012] To achieve the above object, the following technical solutions are used in the present invention.

[0013] The present invention provides an ionizable lipid molecule, its pharmaceutically acceptable salt and its stereoisomer, and the ionizable lipid molecule has a structure represented by formula (1). In JPEG0007683017000001.jpg45140, Q is selected from a benzene ring, a cyclopentyl group, a cyclohexyl group, a pyrrole ring or a pyrimidine ring, L 1 and L 4 are each independently selected from -O(C=O)-, -(C=O)O- or a carbon-carbon double bond, L 2 and L 3 are each independently selected from -(CH 2 ) x O- or -O(CH 2 ) x -, provided that x is an integer between 0 and 4, L 5 is selected from -(CH 2 ) y -O(C=O)-, -(CH 2 ) y -(C=O)O- or -(CH 2 ) y -O-, provided that y is an integer between 0 and 4, L 2 、L 3 and L 5 are each independently connected to any three sites in Q, and the sites are carbon or nitrogen, a and d are each independently integers between 0 and 18, b and c are each independently integers between 1 and 18, e is an integer between 1 and 5, R 1 and R 10 are each independently selected from a methyl group or a cyclic hydrocarbon group, R 2 and R 3 are each independently H or C 1 -C 12 hydrocarbon group, or R 2 is H or C 1 -C 12 hydrocarbon group, and two adjacent R 3Two carbon atoms linked to form a carbon-carbon double bond, R 4 and R 5 are each independently H or a C 1 -C 12 hydrocarbon group, or R 4 is H or a C 1 -C 12 hydrocarbon group, and two carbon atoms linked to two adjacent R 5 form a carbon-carbon double bond, R 6 and R 7 are each independently H or a C 1 -C 12 hydrocarbon group, or R 6 is H or a C 1 -C 12 hydrocarbon group, and two carbon atoms linked to two adjacent R 7 form a carbon-carbon double bond, R 8 and R 9 are each independently H or a C 1 -C 12 hydrocarbon group, or R 8 is H or a C 1 -C 12 hydrocarbon group, and two carbon atoms linked to two adjacent R 9 form a carbon-carbon double bond, R 11 is H or a C 1 -C 6 hydrocarbon group selected individually, R 12 and R 13 are each independently a C 1 -C 6 hydrocarbon group.

[0014] Preferably, Q is selected from a benzene ring, a cyclopentyl group, or a cyclohexyl group, more preferably, Q is a benzene ring.

[0015] Preferably, L 2 、L 3 、L5 The linking pattern of Q is 1,2,5-trisubstituted, 1,3,5-trisubstituted, 1,2,4-trisubstituted, 1,4,5-trisubstituted or 1,3,6-trisubstituted. Preferably, y is 0, 1 or 2.

[0016] Preferably, a and d are each independently an integer from 6 to 10.

[0017] Preferably, b and c are each independently an integer from 1 to 10.

[0018] Preferably, e is selected from 2 or 3.

[0019] Preferably, R 1 and R 10 The cyclic hydrocarbon group in is selected from a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group or a cycloheptyl group. R 2 ~R 9 In the definition of C 1 -C 12 The hydrocarbon group is selected from a C 1 -C 12 alkyl group, a C 1 -C 12 alkenyl group or a C 1 -C 12 alkynyl group, more preferably a C 1 -C 6 alkyl group. R 11 is selected from H, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group or a hexyl group, more preferably a methyl group. R 12 and R 13 are each independently selected from a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group or a hexyl group, more preferably a methyl group. By way of example, Table 1 shows Representative Compounds One to Twenty-Five in the present application.

Table 1

[0020] Preferably, the ionizable lipid molecule is Compound 1. JPEG0007683017000008.jpg 46 131

[0021] Preferably, the ionizable lipid molecule is Compound 2. JPEG0007683017000009.jpg 30 140

[0022] Preferably, the ionizable lipid molecule is Compound 3. JPEG0007683017000010.jpg 30 140

[0023] This application provides a method for producing an ionizable lipid molecule. Taking Compound 1 as an example, in the method, using dichloromethane (DCM) as a solvent JPEG0007683017000011.jpg 15 133 is added to a mixed solution JPEG0007683017000012.jpg 10 129 4-dimethylaminopyridine (DMAP) and triethylamine (TEA) are added, and further 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) is added to the solution. After stirring and reacting, when the result of detection indicates that the reaction is completed, through extraction, washing, drying, concentration, and purification, a yellow oily JPEG0007683017000013.jpg 19 134 is obtained in Step 1, and using N,N-dimethylformamide (DMF) as a solvent A solution mixed with JPEG0007683017000014.jpg19129 and JPEG0007683017000015.jpg19148 was added with Cs 2 CO 3 and stirred for reaction. As a result of detection, when it is indicated that the reaction is completed, through extraction, washing, drying, concentration, and purification, a white solid form of JPEG0007683017000016.jpg21135 is obtained in Step 2, and A mixture of tetrahydrofuran (THF), ethanol, and JPEG0007683017000017.jpg17133 was added with NaBH at 0 °C or below 4 and stirred for reaction. As a result of detection, when it is indicated that the reaction is completed, through extraction, washing, drying, and concentration, a white solid form of JPEG0007683017000018.jpg19138 is obtained in Step 3, and JPEG0007683017000019.jpg19131, JPEG0007683017000020.jpg17136, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were stirred for reaction in a pyridine solution. As a result of detection, when it is indicated that the reaction is completed, through extraction, washing, drying, concentration, and purification, a colorless oily ionizable lipid molecule JPEG0007683017000021.jpg32141 is obtained in Step 4, including In the method for producing the ionizable lipid molecule, the mode in which it is indicated that the reaction is completed as a result of detection is TLC silicone board thin layer chromatography.

[0024] The present invention provides a composition comprising the ionizable lipid molecule, a pharmaceutically acceptable salt thereof, and a stereoisomer thereof, and a therapeutic agent.

[0025] Preferably, the therapeutic agent contains nucleic acid.

[0026] Preferably, the nucleic acid is selected from small interfering RNA (siRNA), microRNA (miRNA), and messenger RNA (mRNA).

[0027] Preferably, the mRNA is non-self-replicating mRNA or self-replicating mRNA.

[0028] Preferably, the composition further comprises one or more excipients selected from neutral lipids, steroid compounds, and lipids of polymer complexes. Preferably, the neutral lipid is one or more selected from 1,2-distearoyl-sn-glycerol-3-phosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), palmitoylphosphatidylcholine (POPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), and more preferably, DSPC and DOPE. The steroid compound is selected from cholesterol, sitosterol, fucosterol, campesterol, stigmasterol, and more preferably cholesterol. The lipid of the polymer complex is polyethylene glycol, preferably polyethylene glycolylated DMG, and more preferably DMG-PEG2000, where DMG represents 1,2-dimyristoyl-sn-glycerol.

[0029] Preferably, the molar ratio of the ionizable lipid molecule to the neutral lipid is 2:1 to 8:1, the molar ratio of the ionizable lipid molecule to cholesterol is 2:1 to 1:1, and the molar ratio of the ionizable lipid molecule to polyethylene glycolylated DMG is 100:1 to 25:1.

[0030] The present invention further provides the application of the ionizable lipid molecule, its pharmaceutically acceptable salt, and its stereoisomers in the manufacture of a carrier for delivering nucleic acids to cells.

[0031] Preferably, the nucleic acid is selected from small interfering RNA (siRNA), microRNA (miRNA), and messenger RNA (mRNA).

[0032] More preferably, the mRNA is non-self-replicating mRNA or self-replicating mRNA.

[0033] The present invention relates to the application of the ionizable lipid molecule, its pharmaceutically acceptable salt, and its stereoisomers in the production of lipid nanoparticles, which includes synthesizing an ionizable lipid molecule, phospholipid, cholesterol, and polyethylene glycol to obtain lipid nanoparticles LNP, and the LNP can improve the translation and expression level of the cargo-mRNA in cells.

[0034] Preferably, the polyethylene glycol is polyethylene glycolated DMG, and more preferably, it is DMG-PEG2000.

[0035] Preferably, the ionizable lipid molecule is selected from Compound 1 to Compound 3. Compound 1: JPEG0007683017000022.jpg47150 Compound 2: JPEG0007683017000023.jpg28148 Compound 3: JPEG0007683017000024.jpg29139

[0036] Preferably, the phospholipid is DSPC or DOPE.

[0037] Preferably, when the mixing ratio of the synthetic carrier LNP is calculated in mole percentage, the ionizable lipid molecule (for example, any one of Compound 1 to Compound 3): cholesterol: phospholipid: polyethylene glycol = 20-70%: 20-60%: 2-20%: 0.1-5%.

[0038] More preferably, when Compound 1 is used, the compounding ratio of the synthetic carrier LNP, calculated as a molar percentage, is ionizable lipid molecule:cholesterol:DSPC:DMG-PEG2000 = 31.5%:56%:10%:2.5%. When Compound 2 or Compound 3 is used, the compounding ratio of the synthetic carrier LNP, calculated as a molar percentage, is ionizable lipid molecule:cholesterol:DSPC:DMG-PEG2000 = 38.5%:50%:10%:1.5%.

[0039] Preferably, the ionizable lipid molecule, phospholipid, cholesterol, and polyethylene glycol are used to obtain LNP by a microfluidic control synthesis method.

[0040] Preferably, in the microfluidic control synthesis method, a step of preparing a mixed solution of an ionizable lipid molecule, phospholipid, cholesterol, and polyethylene glycol; a step of preparing an mRNA dilution solution; using a microfluidic control synthesis device and a pure microfluidic chip, injecting the mRNA dilution solution into one syringe and the mixed solution into the other syringe, setting the parameters, and repeating several times to use up all the mixed solution, collecting all the product LNP solutions, and adding a citrate buffer solution within 30 minutes to dilute the LNP into a solution with an ethanol ratio of less than 0.5%.

[0041] Preferably, the microfluidic control synthesis method further includes a step of obtaining LNP by purification by tangential flow filtration.

[0042] The present invention provides a method for producing a lipid nanoparticle composition containing enhanced green fluorescent protein mRNA (eGFP_mNRA). In this method, ionizable lipid, cholesterol, DSPC, and DMG-PEG2000 are dissolved in ethanol at a molar ratio of 38.5:50:10:1.5. Lipid nanoparticles (LNP) are produced at a weight ratio of total lipid to mRNA of 10:1 to 30:1 (preferably 10:1). Briefly, mRNA is diluted to 0.2 mg / mL in 10 - 50 mM (preferably 50 mM) citrate buffer (pH 6). Using a syringe pump, the ethanol solution of lipid and the aqueous solution of mRNA are mixed at a ratio of about 1:5 to 1:2 (volume / volume) (preferably 1:4), and the total flow rate is set to 15 mL / min or more. Then, ethanol is removed, and dialysis PBS is used instead of the external buffer. Finally, the lipid nanoparticles are filtered through a 0.22 μm sterile filter. The particle size of the lipid nanoparticles identified by quasi-elastic light scattering is 80 - 100 nm in diameter.

[0043] The present invention is based on the discovery of a novel ionizable lipid molecule, which exhibits advantages when used in lipid nanoparticles for delivering an active agent or therapeutic agent (e.g., nucleic acid) to mammalian cells in vivo. In particular, embodiments of the present invention provide a nucleic acid-lipid nanoparticle composition containing one or more of the novel ionizable lipid molecules described herein, with enhanced nucleic acid activity and in vivo tolerance of the composition, and a significantly increased therapeutic effect index compared to conventional nucleic acid-lipid nanoparticle compositions.

[0044] The above formula (I) may be used alone or in combination with other lipid components such as neutral lipids, charged lipids, steroid compounds (including, for example, all sterols) and / or their analogs and / or polymers complexed lipids to form lipid nanoparticles for delivering therapeutic agents. In some examples, the lipid nanoparticles are used for delivering nucleic acids such as siRNA, miRNA and / or mRNA. A method of using such lipid nanoparticles for the treatment of various diseases or conditions (such as solid tumors and / or diseases or conditions caused by protein deficiencies) is further provided. A method for administering a therapeutic agent to a patient is further provided, which includes manufacturing a composition of lipid nanoparticles containing the compound represented by the above formula (I) and a therapeutic agent, and delivering the composition to the patient.

[0045] In certain embodiments, the present invention provides novel ionizable lipids that can form compositions for improved delivery of mRNA and / or other oligonucleotides in vitro and in vivo. In some embodiments, those improved lipid nanoparticle compositions are useful for the expression of proteins encoded by mRNA. In some other embodiments, the lipid nanoparticles are also useful for the delivery of mRNA and plasmids for gene expression. In other embodiments, the lipid nanoparticle composition is useful for exerting a pharmacological effect by protein expression, and the pharmacological effect includes, for example, generating red blood cells that increase by delivering appropriate erythropoietin mRNA, or preventing infection by delivering mRNA for encoding an appropriate antibody.

[0046] The pharmaceutical composition of the present invention can be manufactured as a formulation in solid, semi-solid or liquid form, such as ointment, solution, suspension, injection, inhalant, rubber, microsphere and aerosol. Typical routes of administering such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation and intranasal routes, and include subcutaneous injection, intravenous, intramuscular, intradermal or infusion techniques.

[0047] The liquid pharmaceutical composition of the present invention may be in the form of a solution, suspension, or other similar form, such as water for injection, saline solution, preferably physiological saline, Ringer's solution, a sterile diluent such as isotonic sodium chloride, for example, synthetic mono- or bidirectional mannitol, polyethylene glycol, glycerin, propylene glycol or other solvents that can be used as a solvent or suspension medium, non-volatile oils, such as antioxidants like ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffering agents such as acetate, tris(hydroxymethyl)aminomethane, citrate or phosphate, formulation agents for adjusting tonicity such as sodium chloride or glucose, and reagents used as cryoprotectants such as sucrose or trehalose, and may contain one or more of such adjuvants. The formulation may be enclosed in an ampoule made of glass or plastic, a disposable syringe or a multi-dose bottle. Physiological saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

[0048] The pharmaceutical composition of the present invention is used in the case of topical administration, and the carrier may appropriately contain a solution base, an emulsion base, an ointment base or a gel base. For example, the base may contain one or more of petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, emulsifiers and stabilizers. A thickening agent may be present in the pharmaceutical composition for topical administration. In the case of transdermal administration, the composition may include a transdermal patch or an iontophoresis device.

[0049] The composition of the present invention may be administered simultaneously with, before, or after the administration of one or more other therapeutic agents. Such combination therapies include administering a single dosage formulation of the composition according to the present invention and one or more other active agents, and administering the composition according to the present invention and the active agents in their respective single dosage formulations. For example, the composition according to the present invention and other active agents may be administered to a patient together as a single injection dosage composition (e.g., an injection solution), or each reagent may be administered as a different injection dosage formulation. When using different dosage formulations, the composition according to the present invention and one or more other active agents may be administered substantially simultaneously or at staggered times with respect to each other.

[0050] The beneficial effects of the present invention are as follows.

[0051] In the present invention, by improving the structure of ionizable lipid molecules, novel ionizable lipid molecules are obtained, and the ionizable lipid molecules can effectively deliver nucleic acids into the cytoplasm of cells.

[0052] In the present invention, an LNP obtained by synthesizing an ionizable lipid molecule, a phospholipid, cholesterol, and polyethylene glycol has better mRNA carrier performance, and can significantly improve the intracellular translation and expression level of the carried mRNA.

Brief Description of the Drawings

[0053]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0054] The present invention will be described in detail below with reference to the accompanying drawings and specific examples. The examples are only for illustrative purposes and should not be construed as limiting the scope of protection of the claims in this application.

[0055] Examples The following alphabet abbreviations represent the following reagents, respectively. DCM: Dichloromethane, DMAP: 4-Dimethylaminopyridine, TEA: Triethylamine, EDCI: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, TLC: Silicone board thin layer chromatography, THF: Tetrahydrofuran, DMF: N,N-Dimethylformamide, DSPC: 1,2-Distearoyl-sn-glycerophosphorylcholine, DOPE: 1,2-Dioleoyl-sn-glycerol-3-phosphate ethanolamine, TFF: Tangential flow filtration.

[0056] Example 1 Synthesis of Compound 1 (Code: XH-04) JPEG0007683017000025.jpg40139 (Compound 1, Code: XH-04) The manufacturing method of Compound 1 specifically includes the following steps as shown in Figure 1.

[0057] In Step 1, Compound 1 in DCM dichloromethane (100 mL) JPEG0007683017000026.jpg13136 (20.0 g, 89.6 mmol, 1.00 equivalent) solution, Compound 1A JPEG0007683017000027.jpg8136 (14.2 g, 98.6 mmol, 1.10 eq), DMAP 4-dimethylaminopyridine (1.10 g, 8.96 mmol, 0.10 eq), and TEA triethylamine (36.3 g, 358.6 mmol, 49.9 mL, 4.00 eq) are added. To the solution, EDCI 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (22.3 g, 116.5 mmol, 1.30 eq) is added. The reaction is stirred at 25 °C for 12 h. TLC silicone board thin layer chromatography (petroleum ether:ethyl acetate = 10:1, Rf = 0.62) indicates that the reaction is complete. The reaction solution is poured into H 2 O (100 mL). The solution is extracted with dichloromethane (200 mL × 2). The organic layer is rinsed with brine (100 mL). The organic layer is dried over sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product is purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate = 1:0 - 0:1) to obtain the yellow oily compound 2 JPEG0007683017000028.jpg17147 (22.0 g, 63.0 mmol, 70.3%). 1 1H NMR: (400 MHz, CDCl3) δ 4.03 (t, J = 6.8 Hz, 2H), 3.38 (t, J = 6.0 Hz, 2H), 2.27 (t, J = 7.6 Hz, 2H), 1.71 - 1.86 (m, 2H), 1.56 - 1.61 (m, 4H), 1.40 - 1.43 (m, 2H), 1.22 - 1.33 (m, 16H), 0.84 - 0.88 (m, 3H).

[0058] The reaction formula of Step 1 is as follows. JPEG0007683017000029.jpg35151

[0059] In Step 2, compound 2 JPEG0007683017000030.jpg18144 (20.0 g, 57.25 mmol, 1.00 eq) and compound 2A A solution of 22149 (3.95 g, 28.6 mmol, 0.50 equiv) in DMF (dimethylformamide, 120 mL) was added to Cs 2 CO 3 (37.3 g, 114.5 mmol, 2.00 equiv). The suspension was stirred at 25 °C for 6 h. TLC (thin layer chromatography) on a silica gel plate (petroleum ether:ethyl acetate = 8:1, Rf = 0.40) indicated that the reaction was complete. The reaction mixture was poured into H 2 O (200 mL). The solution was extracted with dichloromethane (200 mL × 3). The organic layer was rinsed with brine (100 mL). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate = 1:0 - 10:1) to obtain compound 3 in the form of a white solid: dinonyl-8,8'-((2-formyl-1,4-phenylene)bis(oxy))octanedioate (15.0 g, 22.2 mmol, 77.6% yield). 1 1H NMR: (400 MHz, CDCl3) δ 10.47 (s, 1H), 7.31 (d, J = 3.2 Hz, 1H), 7.10 (dd, J1 = 8.8 Hz, J2 = 3.2 Hz, 1H), 6.92 (d, J = 9.2 Hz, 1H), 4.01 - 4.08 (m, 6H), 3.94 (t, J = 6.4 Hz, 2H), 2.31 (t, J = 7.2 Hz, 4H), 1.74 - 1.84 (m, 4H), 1.59 - 1.68 (m, 8H), 1.27 - 1.43 (m, 36H), 0.87 - 0.90 (m, 6H).

[0060] The reaction scheme for step 2 is as follows. JPEG0007683017000032.jpg31148

[0061] In step 3, compound 3 A solution of 19148 (12.0 g, 17.78 mmol, 1.00 equivalent) in tetrahydrofuran THF (72 mL) and ethanol EtOH (36 mL) is treated with NaBH 4 (739.9 mg, 19.6 mmol, 1.00 equivalent) at 0 °C. After addition, the reaction solution is stirred at 25 °C for 2 h. TLC silica board thin layer chromatography (petroleum ether:ethyl acetate = 5:1, Rf = 0.20) indicates that the reaction is complete. The reaction mixture is poured into H 2 O (100 mL). The solution is extracted with dichloromethane (200 mL × 2). The organic layer is rinsed with brine (200 mL). The organic layer is dried over sodium sulfate and concentrated under reduced pressure to give compound 4 JPEG0007683017000034.jpg19146 (11.8 g, crude product). 1 H NMR: (400 MHz, CDCl3) δ 6.87 (d, J = 2.4 Hz, 1H), 6.71 - 6.76 (m, 2H), 4.63 (s, 2H), 4.04 (t, J = 6.8 Hz, 4H), 3.93 (t, J = 6.4 Hz, 2H), 3.88 (t, J = 6.4 Hz, 2H), 3.69 - 3.74 (m, 1H), 2.28 (t, J = 7.2 Hz, 4H), 1.69 - 1.78 (m, 4H), 1.56 - 1.65 (m, 8H), 1.21 - 1.45 (m, 36H), 0.84 - 0.88 (m, 6H).

[0062] The reaction formula for step 3 is as follows. JPEG0007683017000035.jpg22150

[0063] In step 4, compound 4 JPEG0007683017000036.jpg19144 (11.0 g, 16.3 mmol, 1.00 equivalent), compound 4A A solution of JPEG0007683017000037.jpg17146 (3.13 g, 18.69 mmol, 1.15 eq., HCl) and EDCI 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (7.16 g, 37.4 mmol, 2.30 eq.) in pyridine (55 mL) is stirred at 40 °C for 20 h. TLC silicone board thin layer chromatography (dichloromethane:methanol = 10:1, Rf = 0.39) indicates that the reaction is complete. The reaction solution is poured into saturated ammonium chloride solution (60 mL). The solution is extracted with ethyl acetate (120 mL × 3). The organic layer is dried over sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product is purified by silica gel column chromatography (petroleum ether:ethyl acetate = 100:1 - 0:1) to obtain a colorless oily compound I, XH-04 (4.00 g, 5.06 mmol, 31.2% yield). 1 1H NMR: (400 MHz, CDCl3) δ 6.89 (s, 1H), 6.77 (d, J = 2.4 Hz, 2H), 5.14 (s, 2H), 4.06 (t, J = 6.8 Hz, 4H), 3.90 (q, J = 6.4 Hz, 4H), 2.30 - 2.42 (m, 4H), 2.26 - 2.29 (m, 10H), 1.82 - 1.89 (m, 2H), 1.72 - 1.78 (m, 4H), 1.27 - 1.45 (m, 36H), 0.86 - 0.90 (m, 6H).

[0064] The reaction formula of Step 4 is as follows. JPEG0007683017000038.jpg37154

[0065] Example 2 Synthesis of Compound II Compound II: JPEG0007683017000039.jpg To a dichloromethane solution (210 mL) of 27139 nonyl alcohol (19.4 g) and bromooctanoic acid (30 g), triethylamine (40.8 g), dimethylaminopyridine (1.64 g) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (25.7 g) were added. After stirring at room temperature for 12 hours, a hydrochloric acid (20 mL) solution was added to terminate the reaction. The mixture was extracted three times with ethyl acetate, and the filtrate was washed with brine. It was dried over anhydrous magnesium sulfate, filtered to remove the solvent, purified by a chromatography column, and 14 g of ethyl nonyl-8-bromocaprylate was obtained.

[0066] To a dichloromethane solution (350 mL) of heptaamine-1,9,17-triol (63.2 g) and bromooctanoic acid (50 g), triethylamine (68 g), dimethylaminopyridine (2.74 g) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (94.5 g) were added. After stirring at room temperature for 12 hours, a hydrochloric acid solution was added to terminate the reaction. Then, the mixture was extracted three times with ethyl acetate, and the filtrate was washed with brine. It was dried over anhydrous magnesium sulfate, filtered to remove the solvent, and purified by a chromatography column to obtain a colorless oil. To a solution of 5-bromo-2-hydroxybenzaldehyde (25 g) in dimethylformamide (175 mL), the colorless oil (47.7 g) and potassium carbonate (34.3 g) were added. After stirring at 80 °C for 12 hours, deionized water (300 mL) was added to terminate the reaction. The mixture was extracted three times with ethyl acetate, and the filtrate was washed with brine. The organic part was dried over anhydrous magnesium sulfate, filtered to remove the solvent, purified by a chromatography column, and 57 g of JPEG0007683017000040.jpg 21134 was obtained. 11H NMR: (400 MHz, CDCl3) δ 10.41 (s, 1H), 7.92 (d, J = 2.4 Hz, 1H), 7.61 - 7.59 (m, 1H), 6.87 (d, J = 8.8 Hz, 1H), 4.88 - 4.84 (m, 1H), 4.05 (t, J = 6.4 Hz, 2H), 2.31 - 2.27 (m, 2H), 1.86 - 1.81 (m, 2H), 1.71 - 1.64 (m, 2H), 1.51 - 1.37 (m, 36H), 0.87 - 0.85 (m, 6H).

[0067] Dissolve JPEG0007683017000041.jpg22129 (20 g) in a mixed solution of tetrahydrofuran (40 mL) and methanol (100 mL), pre - cool it to 0 °C, and treat it with sodium borohydride (520 mg) for 3 hours. Add ammonium chloride solution (20 mL) to terminate the reaction. Then, extract it three times with ethyl acetate, wash the filtrate with brine, dry it over anhydrous magnesium sulfate, filter it, and purify it by a chromatography column to obtain a brown oil. Dissolve the brown oil in dimethylformamide (21 mL), add p - methylphenylsulfonylimidazole (3.5 g) and tert - butyldiphenylchlorosilane (11.3 g), treat it for 12 hours, and purify it by a chromatography column to obtain 24 g of a yellow oil.

[0068] Dissolve the yellow oil (10 g) in dioxane (70 mL), and 3.71 g of JPEG0007683017000042.jpg16144 and [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium (890 mg) and potassium acetate (2.39 g) were treated for 12 hours under a nitrogen atmosphere and purified by a chromatography column, and then 13 g of a yellow oil was obtained. The yellow oil (13 g) was dissolved in tetrahydrofuran (130 mL), hydrogen peroxide (3.39 g) and sodium hydroxide (1 M, 14.9 mL) were added, and the mixture was treated for 1 hour. Sodium thiosulfate was added to terminate the reaction. The mixture was extracted twice with ethyl acetate, the filtrate was washed with brine, dried over anhydrous magnesium sulfate, filtered, and purified by a chromatography column, and then a brown oily intermediate product 3 JPEG0007683017000043.jpg22140 was obtained. 1 H NMR: (400 MHz, CDCl3) δ 7.76 - 7.67 (m, 4H), 7.46 - 7.34 (m, 7H), 7.18 (s, 1H), 6.67 (s, 1H), 4.81 - 4.72 (m, 1H), 4.69 (s, 1H), 3.81 - 3.78 (m, 2H), 2.25 (t, J = 7.4 Hz, 2H), 1.66 - 1.55 (m, 5H), 1.42 - 1.43 (m, 4H), 1.30 - 1.22 (m, 34H), 1.12 (s, 9H), 0.88 (t, J = 6.8 Hz, 6H).

[0069] A solution of the brown oily intermediate 3 (9.5 g) in dimethylformamide (63 mL) is treated with nonyl-8-bromo octanoate (5.03 g) and potassium carbonate (3.46 g), and stirred at 90 °C for 12 hours. Extract three times with a solvent, wash the filtrate with brine, dry over anhydrous magnesium sulfate, filter, and purify by a chromatography column to obtain 6.3 g of a yellow oil. Dissolve the yellow oil in tetrahydrofuran (30 mL), add tetrabutylammonium fluoride (1 M, 12.2 mL), and stir and react for 5 hours. Extract twice with ethyl acetate, wash the filtrate with brine, dry over anhydrous magnesium sulfate, filter, and purify by a chromatography column to obtain 6 g of a yellow oil. Dissolve the yellow oil in pyridine (6 mL), add 4-aminobutyric acid (1.91 g) and carbodiimide (2.91 g), and stir and react at 50 °C for 12 hours. Concentrate under reduced pressure and purify by a chromatography column to obtain 3 g of the final product compound two (43.7% yield). 1 H NMR: (400 MHz, CDCl3) δ 6.86 (s, 1H), 6.79 (s, 2H), 5.15 (s, 2H), 4.86 (t, J = 6.2 Hz, 1H), 4.06 (t, J = 6.6 Hz, 2H), 3.91 (td, J = 6.4, 9.0 Hz, 4H), 3.09 - 2.91 (m, 2H), 2.74 (s, 6H), 2.52 (t, J = 6.6 Hz, 2H), 2.32 - 2.27 (m, 4H), 2.23 - 2.10 (m, 2H), 1.79 - 1.74 (m, 4H), 1.65 - 1.58 (m, 6H), 1.55 - 1.41 (m, 9H), 1.41 - 1.34 (m, 9H), 1.33 - 1.23 (m, 34H), 0.93 - 0.83 (m, 9H).

[0070] Example 3 Synthesis of Compound Three Compound Three: For the synthesis of Compound 3, refer to the synthesis method of Compound 2. The nuclear magnetic resonance spectrum of Compound 3 is as follows. 1 H NMR: (400 MHz, CDCl3) δ 6.86 (s, 1H), 6.79 (s, 2H), 5.15 (s, 2H), 4.86 (t, J = 6.2 Hz, 1H), 4.06 (t, J = 6.6 Hz, 2H), 3.91 (td, J = 6.4, 9.0 Hz, 4H), 3.09 - 2.91 (m, 2H), 2.74 (s, 6H), 2.52 (t, J = 6.6 Hz, 2H), 2.32 - 2.27 (m, 4H), 2.23 - 2.10 (m, 2H), 1.79 - 1.74 (m, 4H), 1.65 - 1.58 (m, 6H), 1.55 - 1.41 (m, 9H), 1.41 - 1.34 (m, 9H), 1.33 - 1.23 (m, 34H), 0.93 - 0.83 (m, 9H).

[0071] Example 4 Synthesis of Compounds 4 - 25 With reference to the synthesis method of Compound 1, Compounds 4 - 25 were prepared and verified by NMR and mass spectrometry. Due to limitations in the paper width of the specification, the details of the synthesis process and characterization data are not listed.

[0072] Example 5 Application of Compound 1 in the Production of Lipid Nanoparticles LNP In the application of ionizable lipid molecules in the production of lipid nanoparticles, the ionizable lipid molecules JPEG0007683017000045.jpg40150, lipids, cholesterol, and polyethylene glycol are synthesized by a microfluidic control synthesis method to obtain LNP, and the LNP can improve the translation and expression level of the carried mRNA in cells. Preferably, the lipid is DSPC (1,2-distearoyl-sn-glycerophosphocholine) or DOPE (1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine), and polyethylene glycol, and more preferably DMG-PEG2000. It should be noted here that the microfluidic control synthesis method is only a suitable method, and LNP may also be synthesized by the thin film hydration followed by extrusion method. None of these methods are exhaustive, and any method for obtaining LNP by the synthesis with XH-04 in the present invention falls within the protection scope of the present invention.

[0073] When the mixing ratio of the synthesized novel carrier LNP is calculated in mole percentage, it is XH-04:cholesterol:lipid:polyethylene glycol = 20 - 70%:20 - 60%:2 - 20%:0.1 - 5%. Preferably, when the mixing ratio of the synthesized novel carrier LNP is calculated in mole percentage, it is XH-04:cholesterol:DSPC:DMG-PEG2000 = 31.5:56:10:2.5 (mol%). It should be noted here that the optimal mixing ratio has been verified by the following experiments. For those skilled in the art, the mixing ratio of mole percentage within this range is a commonly seen flexible range, and LNP can be obtained by any of them. It should be known that any LNP obtained with such a mixing ratio falls within the protection scope of the present invention. Here, the description of the experimental process and data is omitted. Note that it is not limited to the combination pattern of XH-04, cholesterol, DSPC, and DMG-PEG2000. Instead of DSPC, DOPE may be used, and instead of DMG-PEG2000, any PEG (polyethylene glycol) may be used.

[0074] The specific steps in performance detection are as follows.

[0075] (1) Purify the LNP by tangential flow filtration (TFF). It should be noted that the method for purifying the LNP includes, in addition to the purification method by tangential flow filtration, a dialysis + concentration method. None of these methods are comprehensive, and as long as the LNP is purified according to the formulation ratio of the present invention, it falls within the protection scope of the present invention.

[0076] (2) Use spike protein_mRNA as the labeled mRNA to verify the LNP carrier delivery performance in vitro.

[0077] The following verifies the technical effects of the present invention through experiments.

[0078] First, prepare the LNP. The specific process is as follows.

[0079] 1. Prepare the working fluid. (1) Prepare the mother liquor of each component (Cholesterol: 10 mg / mL, DSPC 1,2-distearoyl-sn-glycerol-3-phosphocholine: 10 mg / mL, DMG-PEG2000 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000: 4 mg / mL), and the ionizable lipid molecule XH-04 (Compound I, 10 mg).

[0080] (2) Dissolve 10 mg of XH-04 thoroughly in 1 mL of absolute ethanol. Also, put XH-04, DMG-PEG2000, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, and Cholesterol in a water bath at 37°C for 40 minutes, and put DSPC 1,2-distearoyl-sn-glycerol-3-phosphocholine in a water bath at 55°C for 40 minutes.

[0081] (3) Add the mother liquors of each preheated component according to the volumes shown in Table 2 below, mix well, and then continue to add absolute ethanol (7.32 mL) for dilution. Maintain a water bath at 37 °C.

Table 2

[0082] 2. Synthesize LNP by the microfluidic control synthesis method. Manufacture LNP using the NanoAssemblr microfluidic device and a genuine microfluidic chip. After cleaning and preprocessing the microfluidic control chip channel, pour 8 mL of ultrapure water into the left syringe and 2 mL of the mixed solution of each lipid component into the right syringe. In the device control software, set the parameters with a total liquid flow rate of 16 mL / min, a flow rate ratio of the left and right sides of 4:1, an initial waste volume of 0.550 mL, and a final waste volume of 0.150 mL. Repeat several times. After using up all the lipid component mixed solutions, collect all the product LNP solutions and add ultrapure water within 30 minutes to dilute until the ethanol ratio in the solution is less than 0.5%.

[0083] 3. Purify LNP by tangential flow filtration (TFF). Use a hollow fiber ultrafiltration membrane column to perform liquid exchange, remove the ethanol component in the LNP solution, disperse LNP in ultrapure water, and store it at 2 - 8 °C. The cryo-electron microscopy image of the obtained XH-04 LNP is shown in Figure 2.

[0084] Manufacture LNP encapsulating spike protein_mRNA and conduct a comparative experiment with conventional LNP products. The specific process is as follows.

[0085] The ionizable lipid molecule Dlin-MC3-DMA (CAS number 1224606-06-7) is the main component of conventional LNP products. In this example, using Dlin-MC3-DMA and other essential components, an MC3-LNP product was synthesized and manufactured to encapsulate spike protein_mRNA for reference. At the same time, instead of the Dlin-MC3-DMA molecule, the ionizable lipid molecule XH-04 provided by the present invention was used, together with components such as Cholesterol cholesterol, DSPC 1,2-distearoyl-SN-glycerol-3-phosphocholine, DMG-PEG2000 1,2-dimyristoyl-RAC-glycerol-3-methoxypolyethylene glycol 2000, etc., to synthesize an XH 04-LNP product to encapsulate spike protein_mRNA. The mRNA delivery performance of XH04 LNP was verified in vitro.

[0086] 1. Prepare the working solution. (1) Accurately prepare the stock solutions of each component (Cholesterol cholesterol: 10 mg / mL, DSPC 1,2-distearoyl-SN-glycerol-3-phosphocholine: 10 mg / mL, DMG-PEG2000 1,2-dimyristoyl-RAC-glycerol-3-methoxypolyethylene glycol 2000: 4 mg / mL), the ionizable lipid molecule XH-04 or (10 mg) Dlin-MC3-DMA.

[0087] (2) Dissolve 10 mg of XH-04 (or Dlin-MC3-DMA) thoroughly in 1 mL of absolute ethanol. Also, put XH-04, DMG-PEG2000 1,2-dimyristoyl-RAC-glycerol-3-methoxypolyethylene glycol 2000, Cholesterol cholesterol in a water bath at 37°C for 40 minutes of water bath, and put DSPC 1,2-distearoyl-SN-glycerol-3-phosphocholine in a water bath at 55°C for 40 minutes of water bath.

[0088] (3) According to the volume shown in the following table, add the mother liquor of each preheated component and mix well. Then, continue to add anhydrous ethanol (7.32 mL) for dilution. Maintain a water bath state at 37 °C. JPEG0007683017000047.jpg49148

[0089] (4) Take 507 μg of spike protein_mRNA, dilute it to 39.37 mL with citrate buffer (pH 6.0), and place it in a 50 mL centrifuge tube (place it on ice).

[0090] 2. Synthesize LNP by the microfluidic control synthesis method. Use the NanoAssemblr microfluidic control synthesis equipment and a pure microfluidic chip to manufacture LNP. After cleaning and preprocessing the microfluidic control chip channel, pour 8 mL of the mRNA dilution into the left syringe and 2 mL of the mixed solution of each lipid component into the right syringe. In the equipment control software, set the parameters with the total liquid flow rate of 16 mL / min, the flow rate ratio of the left and right sides of 4:1, the initial waste volume of 0.550 mL, and the final waste volume of 0.150 mL. Repeat several times. After using up all the lipid component mixed solutions, collect all the product LNP solutions and add citrate buffer within 30 minutes to dilute until the ethanol ratio in the solution is less than 0.5%.

[0091] 3. Purify LNP by ultrafiltration through TFF. Use a hollow fiber ultrafiltration membrane column to perform liquid exchange, remove components such as ethanol and citrate in the LNP solution, and disperse LNP in the formulation buffer. Measure the concentration of mRNA in the solution and adjust it to 10 μg / mL. Aliquot the LNP encapsulating mRNA and store it at -80 °C.

[0092] 4. Verify the carrier delivery performance of LNP in vitro. An LNP formulation encapsulating mRNA is cultured with BHK cells for a total of 72 hours, and the expression level of the spike protein inside the cells is detected using a flow cytometer.

[0093] (1) Cell resuscitation and subculture. Resuscitate BHK-21 cells and subculture them in a culture flask until the desired number of cells is reached.

[0094] (2) Culture in plates. Digest and count the cells in the culture flask, seed 10,000 cells per well onto a 96-well plate, and culture overnight so that the cells adhere to the wall.

[0095] (3) Drug injection. Aspirate and dry the medium in the 96-well plate, add PBS and wash 3 times, then aspirate and dry the liquid in the medium. Add 200 μL of the LNP formulation and 1800 μL of the medium to each well and culture in an incubator for 72 hours.

[0096] (4) After collecting the cells, incubate with the corresponding primary antibody and fluorescent secondary antibody for labeling, and compare the positive cell rate using a flow cytometer. As shown in Figure 3. As can be seen from this, the intracellular expression effect of mRNA using the XH 04-LNP carrier (57.62%) is much higher than that of the conventional product MC3-LNP (5.77%).

[0097] In the present invention, an ionizable lipid molecule XH-04 (Compound I) with a novel structure is proposed. The LNP obtained by blending with a lipid raw material has better mRNA carrier performance, and can significantly improve the intracellular translation and expression level of its carrier - mRNA.

[0098] Example 6: Preparation of a lipid nanoparticle composition containing enhanced green fluorescent protein mRNA Ionizable lipid molecular compound two or compound three (10 mg), cholesterol, DSPC and DMG-PEG2000 are dissolved in ethanol at a molar ratio of 38.5:50:10:1.5. Lipid nanoparticles (LNPs) are produced at a weight ratio of total lipid to mRNA of 10:1. That is, the mRNA is diluted to 0.2 mg / mL with 50 mM citrate buffer (pH 6). Using a syringe pump, the ethanol solution of the lipid and the aqueous mRNA solution are mixed at a ratio of 1:4 (volume / volume), and the total flow rate is set to 15 mL / min. Then, the ethanol is removed and dialysis PBS is used instead of the external buffer. Finally, the lipid nanoparticles are filtered through a 0.22 μm sterile filter. The particle size of the lipid nanoparticles identified by the quasi-elastic light scattering method is 80-100 nm in diameter.

[0099] Example 7: Evaluation of the loading performance of the lipid nanoparticle composition The BHK cells are from ATCC and are maintained in Dulbecco's modified Eagle medium (HyClone) supplemented with 10% fetal bovine serum (HyClone) under the conditions of 37 °C and 5% CO 2 Incubate the cells with the lipid nanoparticles containing enhanced green fluorescent protein mRNA, either 0.1 μg or 0.5 μg of the lipid nanoparticle composition prepared in Example 6 or the commercially available MC3 nanoparticle composition (Dlin-MC3-DMA ionizable lipid molecule, CAS number 1224606-06-7) to deliver the nucleic acid therapeutic agent to the BHK cells. After incubating for a total of 24 hours, observe the expression of green fluorescent protein in the cells using a fluorescence microscope and calculate the average fluorescence intensity. The results are shown in Figure 4.

[0100] As can be seen by referring to Fig. 4, after performing cell transfection using the lipid nanoparticle composition produced by 0.1 μg or 0.5 μg of Compound II in the present invention, the expression intensity of the green fluorescent protein in the cells exceeded that of the commercially available MC3 nanoparticle composition. Also, after performing cell transfection using the lipid nanoparticle composition produced by 0.1 μg or 0.5 μg of Compound III in the present invention, the expression intensity of the green fluorescent protein in the cells approached the fluorescence expression of the commercially available MC3 nanoparticle composition. Thereby, it is shown that the ionizable lipid molecule in the present invention can effectively deliver nucleic acid therapeutic substances to BHK cells.

[0101] Example 8: Evaluation of the in vitro protein expression ability using a lipid nanoparticle composition containing an mRNA nucleic acid therapeutic agent The BHK cells are from ATCC and are maintained in Dulbecco's modified Eagle's medium (HyClone) supplemented with 10% fetal bovine serum (HyClone) under the conditions of 37 °C and 5% CO 2 . By incubating with the lipid nanoparticle composition produced by 0.1 μg or 0.5 μg of the ionizable lipid compound in the present invention or the commercially available MC3 nanoparticle composition, the mRNA nucleic acid encoding the green fluorescent protein contained therein is delivered to BHK cells to express the corresponding protein. After incubating for a total of 24 hours, the cells are digested with trypsin and collected, and the signal intensity of the green fluorescent protein in the cells is collected using a flow cytometer. Using the cell group added with the commercially available MC3 nanoparticle composition that does not contain the mRNA encoding the enhanced green fluorescent protein as a control, the relative proportion analysis of protein expression is performed. The results are shown in Table 3.

[0102] Table 3: Fluorescent protein expression rates in BHK cells at different concentrations of the composition of lipid nanoparticles produced by commercially available or ionizable lipids in the present invention and enhanced green fluorescent protein mRNA

Table 3

[0103] Based on the fluorescence intensity after incubating a commercially available MC3 nanoparticle composition that does not contain mRNA encoding enhanced green fluorescent protein with cells, the fluorescence protein expression rate is 0. As can be seen from Table 3, by employing 0.5 μg of the lipid nanoparticle composition of the present invention, an effect equivalent to the expression effect of green fluorescent protein by the commercially available MC3 nanoparticle composition can be obtained. Thereby, it is shown that the ionizable lipid molecule in the present invention can effectively deliver a nucleic acid therapeutic substance to BHK cells and has an excellent fluorescence protein expression rate.

[0104] The above has been described by showing the basic principle, main features and advantages of the present invention. As can be understood by those skilled in the art, the above embodiments are not intended to limit the present invention in any way, and any technical solution obtained by equivalent replacement or equivalent conversion shall fall within the protection scope of the present invention.

Claims

1. The ionizable lipid molecule is selected from the following compounds: Compound 1: Compound 2: Compound 3: Ionizable lipid molecule, its pharmaceutically acceptable salt and its stereoisomer.

2. The ionizable lipid molecule is Compound 1. Ionizable lipid molecule, its pharmaceutically acceptable salt and its stereoisomer according to Claim 1.

3. The ionizable lipid molecule is Compound 2. Ionizable lipid molecule, its pharmaceutically acceptable salt and its stereoisomer according to Claim 1.

4. The ionizable lipid molecule is Compound 3. Ionizable lipid molecule, its pharmaceutically acceptable salt and its stereoisomer according to Claim 1.

5. A method for producing an ionizable lipid molecule, its pharmaceutically acceptable salt and its stereoisomer according to Claim 1, wherein the ionizable lipid molecule is Compound 1 below, in the method, using dichloromethane (DCM) as a solvent, 4-dimethylaminopyridine (DMAP) and triethylamine (TEA) are added to the mixed solution, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) is further added to the solution, followed by stirring and reacting. As a result of detection, when it is indicated that the reaction is complete, through extraction, washing, drying, concentration, and purification, a yellow oily is obtained in Step 1, using N,N-dimethylformamide (DMF) as a solvent, to obtain Cs is added to the mixed solution ２ CO ３ is added, stirred and reacted. As a result of detection, when it is indicated that the reaction is completed, through extraction, washing, drying, concentration, and purification, in Step 2, tetrahydrofuran (THF), ethanol and Add NaBH at 0°C or below to the mixed solution, stir and react. As a result of detection, when it is indicated that the reaction is complete, after extraction, washing, drying, and concentration, ４ a white solid form is obtained is obtained in Step 3, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) are stirred and reacted in a pyridine solution. As a result of detection, when it is indicated that the reaction is complete, through extraction, washing, drying, concentration, and purification, a colorless oily ionized lipid molecule is obtained in Step 4, including A method for producing an ionizable lipid molecule, its pharmaceutically acceptable salt and its stereoisomer.

6. The mode in which it is indicated that the reaction is complete as a result of detection is TLC silicone board thin layer chromatography, and the production method according to Claim 5.

7. A composition comprising an ionizable lipid molecule, its pharmaceutically acceptable salt and its stereoisomer according to any one of Claims 1 to 4, and a therapeutic agent.

8. The therapeutic agent contains nucleic acid, and the composition according to Claim 7.

9. The nucleic acid is the composition according to claim 8, selected from small interfering RNA (siRNA), microRNA (miRNA), and messenger RNA (mRNA).

10. The mRNA is non-self-replicating mRNA or self-replicating mRNA, and the composition according to claim 9.

11. The composition further comprises one or more excipients selected from neutral lipids, steroid compounds, and lipids of polymer complexes. Preferably, the neutral lipid is one or more selected from 1,2-distearoyl-sn-glycerol-3-phosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), palmitoylphosphatidylcholine (POPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), and more preferably, DSPC and DOPE. The steroid compound is selected from cholesterol, sitosterol, fucosterol, campesterol, stigmasterol, and more preferably cholesterol. The lipid of the polymer complex is polyethylene glycol, preferably polyethylene glycolylated DMG, and more preferably DMG-PEG2000. The composition according to any one of claims 7 to 10.

12. The molar ratio of the ionizable lipid molecule to the neutral lipid is 2:1 to 8:

1. The molar ratio of the ionizable lipid molecule to cholesterol is 2:1 to 1:

1. The molar ratio of the ionizable lipid molecule to polyethylene glycolylated DMG is 100:1 to 25:1, and the composition according to claim 11.

13. A carrier for delivering a nucleic acid to a cell, comprising the ionizable lipid molecule according to any one of claims 1 to 4, a pharmaceutically acceptable salt thereof, and a stereoisomer thereof.

14. The nucleic acid is the carrier according to claim 13, selected from small interfering RNA (siRNA), microRNA (miRNA), and messenger RNA (mRNA).

15. The mRNA is non-self-replicating mRNA or self-replicating mRNA, and the carrier according to claim 14.

16. The ionizable lipid molecule according to any one of claims 1 to 4, a pharmaceutically acceptable salt thereof, and a stereoisomer thereof, and Comprising synthesizing a lipid nanoparticle LNP from phospholipid, cholesterol, and polyethylene glycol, The method for producing a lipid nanoparticle, wherein the LNP can improve the translation expression level of the cargo-mRNA intracellularly. **Claim 17** The phospholipid is DSPC or DOPE, The polyethylene glycol is DMG-PEG2000, according to the method of claim 16. **Claim 18** When the mixing ratio of the synthetic carrier LNP is calculated in mole percentage, the ionizable lipid molecule: cholesterol: phospholipid: polyethylene glycol = 20-70%: 20-60%: 2-20%: 0.1-5%, according to the method of claim 16 or 17. **Claim 19** When Compound 1 is used, When the mixing ratio of the synthetic carrier LNP is calculated in mole percentage, the ionizable lipid molecule: cholesterol: DSPC: DMG-PEG2000 = 31.5%: 56%: 10%: 2.5%, or When Compound 2 or Compound 3 is used, When the mixing ratio of the synthetic carrier LNP is calculated in mole percentage, the ionizable lipid molecule: cholesterol: DSPC: DMG-PEG2000 = 38.5%: 50%: 10%: 1.5%, according to the method of claim 16 or 17. **Claim 20** The ionizable lipid molecule, phospholipid, cholesterol, and polyethylene glycol are used to obtain LNP by a microfluidic control synthesis method, according to the method of claim 16 or 17. **Claim 21** In the microfluidic control synthesis method, A step of preparing a mixed solution of an ionizable lipid molecule, phospholipid, cholesterol, and polyethylene glycol, A step of preparing an mRNA dilution solution, Using a microfluidic control synthesis device and a pure microfluidic chip, injecting the mRNA dilution solution into one syringe and the mixed solution into the other syringe, setting the parameters, and repeating several times to use up all the mixed solution, collecting all the product LNP solutions, and adding a citrate buffer within 30 minutes to dilute the LNP into a solution with an ethanol ratio of less than 0.5%, including the step of manufacturing LNP, Characterized by the method of claim 20. **Claim 22** The microfluidic control synthesis method further includes a step of obtaining LNP by purification by tangential flow filtration, characterized by the method of claim 20 or 21.

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