An ionizable lipid molecule, its preparation method and application; lipid nanoparticles and their applications.

Abstract

This invention provides an ionizable lipid molecule, its preparation method, and its application, as well as lipid nanoparticles and their applications, belonging to the field of medicinal chemistry technology. This invention provides an ionizable lipid molecule, a novel type of ionizable lipid molecule, LNP prepared by combining it with non-cationic lipids, sterols, and polyethylene glycol lipids, which can improve the delivery efficiency of nucleic acid drugs loaded with it and has low toxicity. Data from the examples show that it has excellent ability to deliver nucleic acids into cells in both in vitro and in vivo delivery, and is significantly superior to the USFDA-approved positive control (SM-102) aminolipids.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, and in particular to an ionizable lipid molecule, its preparation method and application, and lipid nanoparticles and their applications. Background Technology

[0002] Nucleic acids have demonstrated strong therapeutic potential as drugs in a range of applications, including viral vaccines, protein replacement, therapy, cancer immunotherapy, cell therapy, reprogramming, and genome editing. However, due to inherent limitations such as easy degradation in vivo, low internalization efficiency, and poor endosome escape, the most effective nucleic acid delivery material approved by the US Food and Drug Administration (FDA), SM-102, only mediates 1% endosome escape efficiency. To ensure that nucleic acid molecules reach specific target cells and translate sufficient proteins, safe and effective nucleic acid delivery materials are needed, and nucleic acid delivery systems remain challenging.

[0003] Existing nucleic acid delivery systems mainly include viral vectors, polymeric nanoparticles, and lipid nanoparticles. While viral vector methods offer high gene delivery efficiency, they suffer from limitations in gene size and raise concerns about immunogenicity and safety. Furthermore, polymeric materials suffer from low incorporation escape efficiency, difficult synthesis, significant batch-to-batch variability, and are unsuitable for scale-up production. They also tend to generate antibodies that recognize polymeric materials, limiting their application as drug delivery systems. Liposome nanoparticles, containing liposomes and nucleic acid drugs, have a structure similar to biological membranes. They are biocompatible and non-toxic nanomaterials that can encapsulate both water-soluble and lipid-soluble drugs. They offer advantages such as easy scale-up production, low immunogenicity, reduced drug dosage, sustained release, targeted drug release, and protection of encapsulated nucleic acids from degradation and clearance in serum. Therefore, liposome nanoparticles are widely used in the field of nucleic acid drug delivery.

[0004] Lipid nanoparticles mainly consist of cationic lipids and cofactor lipids. The amino-based cationic lipids facilitate drug uptake and endosome escape by electrostatically complexing with nucleic acids, while the cofactor lipids prevent lipid oxidation, attach ligands to the surface of liposomes, or reduce lipid particle aggregation. Existing USFDA-approved lipid compound nucleic acid delivery systems suffer from low efficiency and high toxicity. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an ionizable lipid molecule, its preparation method and application, and lipid nanoparticles and their applications. The ionizable lipid molecule provided by this invention has high delivery efficiency and low toxicity.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides an ionizable lipid molecule having the structure shown in Formula I:

[0008] Formula I,

[0009] In Formula I, R1 and R2 are independently H, C1-C3 saturated or unsaturated aliphatic chains, halogenated or hydroxyl-substituted C1-C3 aliphatic chains;

[0010] R3, R4 and R5 are independently C1-C20 saturated or unsaturated aliphatic chains, halogenated or hydroxyl-substituted C1-C20 aliphatic chains.

[0011] G1, G2 and G3 are independently C1-C8 saturated or unsaturated aliphatic chains, halogenated or hydroxyl-substituted C1-C8 aliphatic chains;

[0012] L1 is -CH2-, -O-, -S-, -C(O)NH-, -HNC(O)-, -OC(O)O-, -OC(O)-, -C(O)O-, -HNC(O)O-, -OC(O)NH-, -HNC(O)NH-, -SC(O)O-, -OC(O)S-, -HNC(O)S- or -SC(O)NH-.

[0013] Preferably, R1 and R2 are independently H, C1-C2 aliphatic chains;

[0014] R3 and R4 are independently C6-C10 saturated or unsaturated aliphatic chains;

[0015] R5 is a C1-C20 saturated or unsaturated fatty acid chain;

[0016] G1 consists of C2-C4 fatty acid chains;

[0017] G2 consists of C5-C9 fatty acid chains;

[0018] G3 consists of C0-C8 fatty acid chains;

[0019] L1 can be -CH2-, -OC(O)-, or -C(O)O-.

[0020] Preferably, the ionizable lipid molecule has the structure shown in any one of Formulas 1 to 10:

[0021] Formula 1

[0022] Formula 2

[0023] Formula 3

[0024] Formula 4

[0025] Formula 5

[0026] Formula 6

[0027] Formula 7

[0028] Formula 8

[0029] Formula 9

[0030] Formula 10.

[0031] This invention also provides a method for preparing the ionizable lipid molecules described in the above technical solution, comprising the following steps:

[0032] Compound 1 and compound 2 were mixed and subjected to a substitution reaction to obtain a substituted product having the structure shown in compound 4.

[0033] The substituted product was mixed with compound 3 and subjected to an amidation reaction to obtain the ionizable lipid molecule;

[0034] The structures of compounds 1-4 are shown below:

[0035] Compound 1 Compound 2 Compound 3 Compound 4;

[0036] Of compounds 1-4:

[0037] R1 and R2 are independently H, C1-C3 saturated or unsaturated aliphatic chains, halogenated or hydroxyl-substituted C1-C3 aliphatic chains;

[0038] R3, R4 and R5 are independently C1-C20 saturated or unsaturated aliphatic chains, halogenated or hydroxyl-substituted C1-C20 aliphatic chains.

[0039] G1, G2 and G3 are independently C0-C9 saturated or unsaturated aliphatic chains, halogenated or hydroxyl-substituted C1-C8 aliphatic chains;

[0040] L1 is -CH2-, -O-, -S-, -C(O)NH-, -HNC(O)-, -OC(O)O-, -OC(O)-, -C(O)O-, -HNC(O)O-, -OC(O)NH-, -HNC(O)NH-, -SC(O)O-, -OC(O)S-, -HNC(O)S- or -SC(O)NH-;

[0041] X is a halogen.

[0042] The present invention also provides the application of the ionizable lipid molecules described in the above technical solution in the preparation of nucleic acid delivery drugs.

[0043] The present invention also provides lipid nanoparticles (LNPs) comprising the following components:

[0044] The above technical solutions describe ionizable lipid molecules, non-cationic lipids, sterols, and polyethylene glycol lipids.

[0045] Preferably, the molar ratio of the ionizable lipid molecules, non-cationic lipids, sterols and polyethylene glycol lipids is (30~50):(5~20):(30~40):(0.1~3).

[0046] Preferably, the non-cationic lipids include non-cationic phospholipids.

[0047] Preferably, the polyethylene glycol lipid is PEG2000-DMG.

[0048] The present invention also provides the application of the lipid nanoparticles described above in the preparation of nucleic acid delivery drugs.

[0049] This invention provides an ionizable lipid molecule, and compared with the prior art, the advantages of this invention are as follows:

[0050] The ionizable lipid molecule of this invention is a novel type of ionizable lipid molecule. LNPs prepared with non-cationic lipids, sterols, and polyethylene glycol lipids can improve the delivery efficiency of nucleic acid drugs loaded with these molecules, and exhibit low toxicity. Data from the examples demonstrate excellent ability to deliver nucleic acids into cells in both in vitro and in vivo delivery, significantly outperforming the USFDA-approved positive control (SM-102) aminolipids. Attached Figure Description

[0051] Figure 1 This is a comparison chart of in vitro delivery efficiency;

[0052] Figure 2 This is a comparison chart of cytotoxicity. Detailed Implementation

[0053] This invention provides an ionizable lipid molecule having the structure shown in Formula I:

[0054] Formula I,

[0055] In Formula I, R1 and R2 are independently H, C1-C3 saturated or unsaturated aliphatic chains, halogenated or hydroxyl-substituted C1-C3 aliphatic chains;

[0056] R3, R4 and R5 are independently C1-C20 saturated or unsaturated aliphatic chains, halogenated or hydroxyl-substituted C1-C20 aliphatic chains.

[0057] G1, G2 and G3 are independently C1-C8 saturated or unsaturated aliphatic chains, halogenated or hydroxyl-substituted C1-C8 aliphatic chains;

[0058] L1 is -CH2-, -O-, -S-, -C(O)NH-, -HNC(O)-, -OC(O)O-, -OC(O)-, -C(O)O-, -HNC(O)O-, -OC(O)NH-, -HNC(O)NH-, -SC(O)O-, -OC(O)S-, -HNC(O)S- or -SC(O)NH-.

[0059] In this invention, R1 and R2 are preferably H and C1-C2 aliphatic chains independently;

[0060] R3 and R4 are preferably C6-C10 saturated or unsaturated aliphatic chains;

[0061] R5 is preferably composed of C1-C20 saturated or unsaturated fatty chains;

[0062] G1 is preferably composed of C2-C4 fatty acid chains;

[0063] G2 is preferably composed of C5-C9 fatty acid chains;

[0064] G3 is preferably composed of C0-C8 fatty acid chains;

[0065] L1 is preferably -CH2-, -OC(O)-, or -C(O)O-.

[0066] In this invention, the G3 preferably contains branched substituents, and the G3 is preferably a C1-C8 saturated or unsaturated branched aliphatic chain.

[0067] In this invention, the ionizable lipid molecule preferably has the structure shown in any one of Formulas 1 to 10:

[0068] Formula 1

[0069] Formula 2

[0070] Formula 3

[0071] Formula 4

[0072] Formula 5

[0073] Formula 6

[0074] Formula 7

[0075] Formula 8

[0076] Formula 9

[0077] Formula 10

[0078] This invention also provides a method for preparing the ionizable lipid molecules described in the above technical solution, comprising the following steps:

[0079] Compound 1 and compound 2 were mixed and subjected to a substitution reaction to obtain a substituted product having the structure shown in compound 4.

[0080] The substituted product was mixed with compound 3 and subjected to an amidation reaction to obtain the ionizable lipid molecule;

[0081] The structures of compounds 1-4 are shown below:

[0082] Compound 1 Compound 2 Compound 3 Compound 4;

[0083] Of compounds 1-4:

[0084] R1 and R2 are independently H, C1-C3 saturated or unsaturated aliphatic chains, halogenated or hydroxyl-substituted C1-C3 aliphatic chains;

[0085] R3, R4 and R5 are independently C1-C20 saturated or unsaturated aliphatic chains, halogenated or hydroxyl-substituted C1-C20 aliphatic chains.

[0086] G1, G2 and G3 are independently C0-C9 saturated or unsaturated aliphatic chains, halogenated or hydroxyl-substituted C1-C8 aliphatic chains;

[0087] L1 is -CH2-, -O-, -S-, -C(O)NH-, -HNC(O)-, -OC(O)O-, -OC(O)-, -C(O)O-, -HNC(O)O-, -OC(O)NH-, -HNC(O)NH-, -SC(O)O-, -OC(O)S-, -HNC(O)S- or -SC(O)NH-;

[0088] X is a halogen.

[0089] In this invention, the halogen is preferably -F, -Cl or -Br.

[0090] In this invention, unless otherwise specified, all raw materials used are commercially available products in the field or products prepared by conventional methods in the field.

[0091] In this invention, the molar equivalent ratio of compound 1 to compound 2 is preferably 10:1.

[0092] In this invention, the temperature of the substitution reaction is preferably 60°C, and the time is preferably 24 hours.

[0093] In this invention, the substitution reaction is preferably carried out in an organic solvent, which preferably includes anhydrous ethanol.

[0094] After the substitution reaction is completed, the organic solvent is preferably removed by rotary evaporation. The crude product is dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, concentrated by vacuum rotary evaporation, and then purified by silica gel column elution to obtain the substituted product.

[0095] In this invention, the eluent used for silica gel column elution and purification is preferably a mixture of dichloromethane, methanol and ammonia.

[0096] In this invention, the molar equivalent ratio of compound 4 to compound 3 is preferably 1:2.

[0097] In this invention, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl) and N,N-diisopropylethylamine (DIEPA) are preferably added during the amidation reaction.

[0098] In this invention, the molar equivalent ratio of compound 4 to EDC HCl is preferably 1:2.

[0099] In this invention, the molar equivalent ratio of compound 4 to DIEPA is preferably 1:4.

[0100] In this invention, the temperature of the amidation reaction is preferably room temperature, and the time is preferably 12 hours.

[0101] In this invention, the amidation reaction is preferably carried out in an organic solvent, which preferably includes dichloromethane.

[0102] After the amidation reaction is completed, the organic solvent is preferably removed by rotary evaporation. The crude product is dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, concentrated by vacuum rotary evaporation, and then purified by silica gel column chromatography to obtain the ionizable lipid molecule.

[0103] In this invention, the eluent used for silica gel column purification is preferably a mixture of dichloromethane, methanol, and ammonia.

[0104] The present invention also provides the application of the ionizable lipid molecules described in the above technical solution in the preparation of nucleic acid delivery drugs.

[0105] In this invention, the ionizable lipid molecules are preferably first prepared into lipid nanoparticles, and then the nucleic acid delivery drug is prepared.

[0106] The present invention also provides lipid nanoparticles comprising the following components:

[0107] The above technical solutions describe ionizable lipid molecules, non-cationic lipids, sterols, and polyethylene glycol lipids.

[0108] In this invention, the preferred molar ratio of the ionizable lipid molecules, non-cationic lipids, sterols and polyethylene glycol lipids is (30~50):(5~20):(30~40):(0.1~3), specifically it can be 50:10:38.5:1.5.

[0109] In this invention, the non-cationic lipids preferably include non-cationic phospholipids, which preferably include 1,2-distearate-sn-glycerol-3-phosphocholine and / or 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine.

[0110] In this invention, the polyethylene glycol lipid is preferably PEG2000-DMG.

[0111] The present invention also provides a method for preparing the lipid nanoparticles, comprising the following steps:

[0112] An ionizable lipid molecule, non-cationic lipid, sterol, polyethylene glycol lipid and anhydrous ethanol were mixed to obtain a mixture.

[0113] The mixture was then mixed with a citric acid-sodium citrate solution to obtain a crude solution.

[0114] The crude solution was dialyzed sequentially to obtain the lipid nanoparticles.

[0115] In this invention, the concentration of the citric acid-sodium citrate solution is preferably 10 mM, and the pH value is preferably 4.5.

[0116] In this invention, the volume ratio of the mixture to the citric acid-sodium citrate solution is preferably 1:1 to 1:10, and more specifically, it can be 1:3.

[0117] The present invention preferably uses a pipette to mix the mixture with the citric acid-sodium citrate solution, or mixes the mixture with the citric acid-sodium citrate solution in a microfluidic chip.

[0118] In this invention, the dialysis is performed using a dialysis bag (MWCO 14 kDa).

[0119] In this invention, the dialysate used for dialysis is preferably a sucrose-PBS solution (10%).

[0120] In this invention, the preferred temperature for dialysis is 4°C, and the preferred number of dialysis sessions is 3.

[0121] In this invention, filtration is preferably performed after dialysis, and the filtration is preferably performed using a 0.22 μm microporous membrane.

[0122] The present invention also provides the application of the lipid nanoparticles described above in the preparation of nucleic acid delivery drugs.

[0123] In this invention, the nucleic acid delivery drug preferably comprises lipid nanoparticles and luciferase mRNA (FlucmRNA).

[0124] In this invention, the preferred mass ratio of ionizable lipid molecules to luciferase mRNA in the lipid nanoparticles is 10:1.

[0125] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0126] Example 1

[0127] Preparation of ionizable lipid molecules

[0128] Synthesis of Intermediate 1

[0129]

[0130] 25 g of 8-bromooctyl-1-ol was dissolved in 300 mL of dichloromethane, and 1 molar equivalent of pyridinium chlorochromate (PCC) was added. The mixture was stirred at room temperature for 6 h. The reaction mixture was filtered and concentrated by rotary evaporation under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether in ethyl acetate) to give intermediate 1 (12.7 g), which was a colorless oil. 1 H NMR (400 MHz, CDCl3): δ 9.77 (t, J = 1.8 Hz, 1H), 3.40 (t, J = 6.8 Hz, 2H), 2.43 (m, 2H), 1.86 (m, 2H), 1.63 (m, 2H), 1.45 (m, 2H), 1.34 (m, 4H).

[0131] Synthesis of intermediate 2

[0132]

[0133] Intermediate 1 (12.7 g) was dissolved in 300 mL of dichloromethane, and 0.15 molar equivalents of p-toluenesulfonic acid hydrate, 3 molar equivalents of sodium sulfate, and 3 molar equivalents of n-octanol were added. The reaction mixture was reacted at room temperature for 24 h. The reaction mixture was filtered and concentrated by rotary evaporation under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether-ethyl acetate) to give intermediate 2 (6.9 g), which was a colorless oil. 1 HNMR (400 MHz, CDCl3): δ 4.46 (t,J = 5.6, 1H), 3.56 (m, 2H), 3.41 (m, 4H), 1.84 (m, 2H), 1.59 (m, 6H), 1.33-1.28 (m, 34H) , 0.89 (t,J = 6.6 Hz, 6H).

[0134] Synthesis of intermediate 3

[0135]

[0136] Intermediate 2 (900 mg) was dissolved in 5 mL of anhydrous ethanol, and 10 molar equivalents of N,N-dimethyl-1,3-diaminopropane were added. The mixture was reacted at 60 °C for 24 h. The ethanol was removed by rotary evaporation. The crude product was dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The reaction mixture was filtered and concentrated by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane-methanol-ammonia) to give intermediate 3 (300 mg), which was a colorless oil. 1 H NMR (400 MHz, CDCl3): δ 5.53(1H), 4.47 (1H), 3.78 (4H), 2.69 (2H), 2.62 (2H), 2.42 (2H), 2.27 (6H), 1.78(4H), 1.68 (2H), 1.58 (2H), 1.48 (2H), 1.45 (4H), 1.38 (2H), 1.30 (2H), 1.29(4H), 1.27 (4H), 1.26 (12H), 0.89 (6H).

[0137] Intermediate 4 Synthesis

[0138]

[0139] 25 g of 6-bromooctyl-1-ol was dissolved in 300 mL of dichloromethane, and 1 molar equivalent of pyridinium chlorochromate (PCC) was added. The mixture was stirred at room temperature for 6 h. The reaction mixture was filtered and concentrated by rotary evaporation under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether in ethyl acetate) to give intermediate 1 (11.6 g), which was a colorless oil. 1 H NMR (400 MHz, CDCl3): 1 H NMR (400MHz, CDCl3): δ 9.76 (1H), 3.42 (2H), 2.40 (2H), 1.87 (2H), 1.62 (2H), 1.48(2H).

[0140] Synthesis of intermediate 5

[0141]

[0142] Intermediate 4 (11.6 g) was dissolved in 300 mL of dichloromethane, and 0.15 molar equivalents of p-toluenesulfonic acid hydrate, 3 molar equivalents of sodium sulfate, and 3 molar equivalents of decanol were added. The reaction mixture was reacted at room temperature for 24 h. The reaction mixture was filtered and concentrated by rotary evaporation under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether-ethyl acetate) to give intermediate 5 (7.9 g), which was a colorless oil. 1 HNMR (400 MHz, CDCl3): δ 4.47 (1H), 3.78 (4H), 3.42 (2H), 1.87 (2H), 1.78 (4H), 1.58 (2H), 1.48 (2H), 1.45 (4H), 1.38 (2H), 1.29 (4H), 1.27 (4H), 1.26 (16H), 0.89 (6H).

[0143] Intermediate 6 Synthesis

[0144]

[0145] Intermediate 5 (800 mg) was dissolved in 5 mL of anhydrous ethanol, and 10 molar equivalents of N,N-dimethyl-1,3-diaminopropane were added. The mixture was reacted at 60 °C for 24 h. The ethanol was removed by rotary evaporation. The crude product was dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The reaction mixture was filtered and concentrated by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane-methanol-ammonia) to give intermediate 6 (277 mg), which was a colorless oil. 1H NMR (400 MHz, CDCl3): δ 5.53(1H), 4.47 (1H), 3.78 (4H), 2.69 (2H), 2.62 (2H), 2.42 (2H), 2.27 (6H), 1.78(4H), 1.68 (2H), 1.58 (2H), 1.48 (2H), 1.45 (4H), 1.38 (2H), 1.31 (2H), 1.30(2H), 1.29 (4H), 1.27 (2H), 1.26 (16H), 0.89 (6H).

[0146] Synthesis of intermediate 7

[0147]

[0148] Intermediate 2 (900 mg) was dissolved in 5 mL of anhydrous ethanol, and 10 molar equivalents of N,N-diethyl-1,3-diaminopropane were added. The mixture was reacted at 60 °C for 24 h. The ethanol was removed by rotary evaporation. The crude product was dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The reaction mixture was filtered and concentrated by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane-methanol-ammonia) to give intermediate 7 (263 mg), which was a colorless oil. 1 H NMR (400 MHz, CDCl3): δ5.53 (1H), 4.47 (1H), 3.78 (4H), 2.69 (2H), 2.62 (2H), 2.55 (4H), 2.52 (2H),1.78 (4H), 1.68 (2H), 1.58 (2H), 1.48 (2H), 1.45 (4H), 1.38 (2H), 1.31 (2H), 1.30 (2H), 1.29 (4H), 1.27 (2H), 1.26 (16H), 1.19 (6H), 0.89 (6H).

[0149] Synthesis of ionizable lipid molecules (Formula 1)

[0150] Intermediate 3 (300 mg) was dissolved in 10 mL of dichloromethane, and 2 molar equivalents of linoleic acid, 2 molar equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), and 4 molar equivalents of N,N-diisopropylethylamine (DIEPA) were added. The reaction was carried out for 12 h, and dichloromethane was removed by rotary evaporation. The crude product was dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The mixture was filtered and concentrated by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane-methanol-ammonia) to give an ionizable lipid molecule (Formula 1) (153 mg). 1 H NMR (400 MHz, CDCl3): δ 5.26 (dd, J= 15.4, 8.5 Hz, 4H), 4.38 (s, 1H), 3.52 – 3.43 (m, 2H), 3.38 – 3.25 (m, 3H), 3.25 – 3.09 (m, 3H), 2.70 (t, J = 6.1 Hz, 2H), 2.61 – 2.31 (m, 6H), 2.22 (dd,J = 18.2, 10.8 Hz, 5H), 2.04 – 1.91 (m, 5H), 1.89 – 1.67 (m, 7H), 1.23 (d, J= 11.6 Hz, 43H), 0.81 (d, J = (4.1 Hz, 9H). MS: 733.7190.

[0151] Synthesis of ionizable lipid molecules (Equation 2)

[0152] Intermediate 3 (300 mg) was dissolved in 10 mL of dichloromethane, and 2 mol equivalents of nonanoic acid, 2 mol equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), and 4 mol equivalents of N,N-diisopropylethylamine (DIEPA) were added. The reaction mixture was reacted for 12 h, and the dichloromethane was removed by rotary evaporation. The crude product was dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The reaction mixture was filtered and concentrated by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane-methanol-ammonia) to give an ionizable lipid molecule (Formula 2) (122 mg). 1H NMR (400 MHz, CDCl3): δ 4.38 (d, J= 4.3 Hz, 1H), 3.54 – 3.44 (m, 2H), 3.38 – 3.10 (m, 6H), 2.55 – 2.13 (m,12H), 1.79 (dd, J = 41.5, 34.6 Hz, 6H), 1.63 (dd, J = 14.2, 6.9 Hz, 1H), 1.34– 1.00 (m, 40H), 0.81 (t, J = 5.7 Hz, 9H). MS:611.6089.

[0153] Synthesis of ionizable lipid molecules (Equation 3)

[0154] Intermediate 3 (300 mg) was dissolved in 10 mL of dichloromethane, and 2 molar equivalents of 2-hexyldecanoic acid, 2 molar equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl) and 4 molar equivalents of N,N-diisopropylethylamine (DIEPA) were added. The reaction mixture was reacted for 12 h, and the dichloromethane was removed by rotary evaporation. The crude product was dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The reaction mixture was filtered and concentrated by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane-methanol-ammonia) to give an ionizable lipid molecule (Formula 3) (131 mg). 1 H NMR (400 MHz, CDCl3): δ3.69 (s, 2H), 3.49 (d, J = 6.9 Hz, 1H), 3.37 – 3.14 (m, 5H), 2.36 (d, J =25.0 Hz, 5H), 2.20 (d, J = 17.7 Hz, 8H), 1.87 – 1.71 (m, 6H), 1.45 – 1.07 (m,52H), 0.80 (d, J = 6.3 Hz, 12H). MS:709.7186.

[0155] intermediate 8 synthesis

[0156]

[0157] Octanoic acid (5 g) was dissolved in 100 mL of anhydrous ethanol. 0.2 mol equivalents of n-decanol, 1 mol equivalent of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), and 1 mol equivalent of N,N-diisopropylethylamine (DIEPA) were added. The reaction mixture was reacted for 12 h. Ethanol was removed by rotary evaporation. The crude product was dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The reaction mixture was filtered and concentrated by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography (n-hexane-ethyl acetate) to give intermediate 8 (1.8 g). 1 H NMR (400 MHz, CDCl3): δ 5.37 (1H), 4.14 (2H), 2.33(2H), 2.32 (2H), 1.78 (2H), 1.62 (4H), 1.45 (2H), 1.29 (2H), 1.27 (2H), 1.26(12H), 0.89 (3H).

[0158] Synthesis of intermediate 9

[0159]

[0160] Intermediate 1 (10.5 g) was dissolved in 300 mL of dichloromethane, and 0.15 molar equivalents of p-toluenesulfonic acid hydrate, 3 molar equivalents of sodium sulfate, and 3 molar equivalents of n-hexanol were added. The reaction mixture was reacted at room temperature for 24 h. The reaction mixture was filtered and concentrated by rotary evaporation under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether-ethyl acetate) to give intermediate 9 (7.2 g), which was a colorless oil. 1 HNMR (400 MHz, CDCl3): δ 4.47 (1H), 3.78 (4H), 3.42 (2H), 1.87 (2H), 1.78(4H), 1.58 (2H), 1.48 (2H), 1.45 (4H), 1.38 (2H), 1.31 (2H), 1.29 (4H), 1.27(2H), 1.26 (4H), 0.89 (6H).

[0161] Synthesis of intermediate 10

[0162]

[0163] Intermediate 9 (900 mg) was dissolved in 5 mL of anhydrous ethanol, and 10 molar equivalents of N,N-diethyl-1,3-diaminopropane were added. The mixture was reacted at 60 °C for 24 h. The ethanol was removed by rotary evaporation. The crude product was dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The reaction mixture was filtered and concentrated by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane-methanol-ammonia) to give intermediate 10 (279 mg), which was a colorless oil. 1 H NMR (400 MHz, CDCl3): 5.53(1H), 4.47 (1H), 3.78 (4H), 2.69 (2H), 2.62 (2H), 2.55 (4H), 2.52 (2H), 1.78(4H), 1.68 (2H), 1.58 (2H), 1.48 (2H), 1.45 (4H), 1.38 (2H), 1.30 (2H), 1.29(4H), 1.27 (4H), 1.26 (4H), 1.19 (6H), 0.89 (6H).

[0164] Synthesis of ionizable lipid molecules (Equation 4)

[0165] Intermediate 3 (300 mg) was dissolved in 10 mL of dichloromethane, and 2 molar equivalents of intermediate 8, 2 molar equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), and 4 molar equivalents of N,N-diisopropylethylamine (DIEPA) were added. The reaction mixture was reacted for 12 h, and the dichloromethane was removed by rotary evaporation. The crude product was dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The reaction mixture was filtered and concentrated by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane-methanol-ammonia) to give an ionizable lipid molecule (Formula 4) (115 mg). 1 H NMR (400 MHz, CDCl3): δ4.38 (d, J = 4.5 Hz, 1H), 3.98 (t, J = 6.6 Hz, 3H), 3.68 (t, J = 6.9 Hz, 1H), 3.53 – 3.44 (m, 2H), 3.37 – 3.10 (m, 6H), 2.58 – 2.49 (m, 2H), 2.48 – 2.30(m, 4H), 2.29 – 2.15 (m, 9H), 1.87 – 1.62 (m, 8H), 1.35 – 1.04 (m, 49H), 0.81(t, J = 5.9 Hz, 9H). MS:767.7231.

[0166] Synthesis of ionizable lipid molecules (Equation 5)

[0167] Intermediate 7 (263 mg) was dissolved in 10 mL of dichloromethane, and 2 molar equivalents of linoleic acid, 2 molar equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), and 4 molar equivalents of N,N-diisopropylethylamine (DIEPA) were added. The reaction mixture was reacted for 12 h, and the dichloromethane was removed by rotary evaporation. The crude product was dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The reaction mixture was filtered and concentrated by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane-methanol-ammonia) to give an ionizable lipid molecule (Formula 5) (102 mg). 1 H NMR (400 MHz, CDCl3): δ 5.41 –5.28 (m, 6H), 4.45 (t, J = 4.8 Hz, 1H), 3.74 (t, J = 7.3 Hz, 1H), 3.55 (dd, J= 14.6, 7.6 Hz, 1H), 3.43 – 3.17 (m, 5H), 2.76 (dd, J = 13.5, 7.1 Hz, 3H), 2.56 (dt, J = 14.6, 7.4 Hz, 3H), 2.39 – 1.96 (m, 20H), 1.31 (dd, J = 18.3,13.0 Hz, 43H), 1.14 (dt, J = 26.9, 7.1 Hz, 4H), 0.94 – 0.83 (m, 9H). MS:761.7495.

[0168] Synthesis of ionizable lipid molecules (Equation 6)

[0169] Intermediate 7 (225 mg) was dissolved in 10 mL of dichloromethane, and 2 molar equivalents of intermediate 8, 2 molar equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), and 4 molar equivalents of N,N-diisopropylethylamine (DIEPA) were added. The reaction mixture was reacted for 12 h, and the dichloromethane was removed by rotary evaporation. The crude product was dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The reaction mixture was filtered and concentrated by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane-methanol-ammonia) to give an ionizable lipid molecule (Formula 6) (92 mg). 1H NMR (400 MHz, CDCl3): δ4.44 (dd, J = 10.1, 5.5 Hz, 1H), 4.05 (td, J = 6.7, 1.9 Hz, 3H), 3.76 – 3.69(m, 1H), 3.59 – 3.51 (m, 2H), 3.43 – 0.88 (t, J = 6.8 Hz, 9H). MS:795.7752.

[0170] Synthesis of ionizable lipid molecules (Formula 7)

[0171] Intermediate 10 (279 mg) was dissolved in 10 mL of dichloromethane, and 2 molar equivalents of linoleic acid, 2 molar equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), and 4 molar equivalents of N,N-diisopropylethylamine (DIEPA) were added. The reaction mixture was reacted for 12 h, and the dichloromethane was removed by rotary evaporation. The crude product was dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The reaction mixture was filtered and concentrated by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane-methanol-ammonia) to give an ionizable lipid molecule (Formula 7) (112 mg). 1 H NMR (400 MHz, CDCl3): δ5.41 – 5.28 (m, 5H), 4.45 (dd, J = 9.8, 5.7 Hz, 1H), 3.77 – 3.70 (m, 1H), 3.59 – 3.51 (m, 2H), 3.44 – 3.14 (m, 6H), 2.77 (t, J = 6.3 Hz, 3H), 2.65 –2.38 (m, 6H), 2.33 – 2.18 (m, 6H), 2.05 (q, J = 6.8 Hz, 5H), 1.83 – 1.71 (m,2H), 1.41 – 1.22 (m, 37H), 1.17 (t, J = 7.3 Hz, 2H), 1.04 (dt, J = 14.3, 6.8Hz, 5H), 0.89 (t, J = 6.8 Hz, 9H).MS:705.6876.

[0172] Synthesis of ionizable lipid molecules (Equation 8)

[0173] Intermediate 6 (277 mg) was dissolved in 10 mL of dichloromethane, and 2 molar equivalents of linoleic acid, 2 molar equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), and 4 molar equivalents of N,N-diisopropylethylamine (DIEPA) were added. The reaction mixture was reacted for 12 h, and the dichloromethane was removed by rotary evaporation. The crude product was dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The reaction mixture was filtered and concentrated by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane-methanol-ammonia) to give an ionizable lipid molecule (Formula 8) (109 mg). 1 H NMR (400 MHz, CDCl3): 5.35 (pd,J = 10.7, 6.9 Hz, 6H), 4.44 (q, J = 5.4 Hz, 1H), 3.77 – 3.69 (m, 1H), 3.59 –3.50 (m, 2H), 3.45 – 3.18 (m, 6H), 2.77 (t, J = 6.3 Hz, 3H), 2.58 (t, J = 7.4Hz, 1H), 2.34 – 2.19 (m, 13H), 2.11 – 1.86 (m, 9H), 1.44 – 1.21 (m, 46H), 1.17 (t, J = 7.3 Hz, 2H), 0.88 (q, J = (6.7 Hz, 9H). MS: 761.7521.

[0174] Synthesis of intermediate 11

[0175]

[0176] Oxalic acid (5 g) was dissolved in 100 mL of anhydrous ethanol. 0.2 mol equivalents of n-heptanol, 1 mol equivalent of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), and 1 mol equivalent of N,N-diisopropylethylamine (DIEPA) were added. The reaction mixture was reacted for 12 h. Ethanol was removed by rotary evaporation. The crude product was dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The reaction mixture was filtered and concentrated by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography (n-hexane-ethyl acetate) to give intermediate 11 (1.67 g). 1H NMR (400 MHz, CDCl3): δ 5.37 (1H), 4.14 (2H), 2.33 (2H), 2.32 (2H), 1.78 (2H), 1.62 (4H), 1.45 (2H), 1.29 (2H), 1.27 (2H), 1.26 (6H), 0.89 (3H).

[0177] Synthesis of intermediate 12

[0178]

[0179] Octanoic acid (5 g) was dissolved in 100 mL of anhydrous ethanol. 0.2 mol equivalents of cis-3-nonen-1-ol, 1 mol equivalent of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), and 1 mol equivalent of N,N-diisopropylethylamine (DIEPA) were added. The reaction mixture was reacted for 12 h. Ethanol was removed by rotary evaporation. The crude product was dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The reaction mixture was filtered and concentrated by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography (n-hexane-ethyl acetate) to give intermediate 12 (1.5 g). 1 H NMR (400 MHz, CDCl3): δ 5.50 (1H), 5.38(1H), 5.37 (1H), 4.16 (2H), 2.41 (2H), 2.32 (2H), 1.90 (4H), 1.62 (4H), 1.37(2H), 1.29 (2H), 1.27 (2H), 1.26 (4H), 0.89 (3H).

[0180] Synthesis of ionizable lipid molecules (Formula 9)

[0181] Intermediate 3 (300 mg) was dissolved in 10 mL of dichloromethane, and 2 molar equivalents of intermediate 11, 2 molar equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), and 4 molar equivalents of N,N-diisopropylethylamine (DIEPA) were added. The reaction mixture was reacted for 12 h, and the dichloromethane was removed by rotary evaporation. The crude product was dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The reaction mixture was filtered and concentrated by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane-methanol-ammonia) to give an ionizable lipid molecule (Formula 9) (66 mg). 1H NMR (400 MHz, CDCl3): δ4.48 (d, J = 4.5 Hz, 1H), 4.08 (t, J = 6.6 Hz, 3H), 3.78 (t, J = 6.9 Hz, 1H), 3.59 – 3.45 (m, 2H), 3.44 – 3.23 (m, 6H), 2.62 – 2.55 (m, 2H), 2.54 – 2.34(m, 4H), 2.30 – 2.17 (m, 9H), 1.91 – 1.66 (m, 8H), 1.37 – 1.08 (m, 40H), 0.83(t, J = 5.9 Hz, 9H). MS:725.3963.

[0182] Synthesis of ionizable lipid molecules (Formula 10)

[0183] Intermediate 3 (300 mg) was dissolved in 10 mL of dichloromethane, and 2 molar equivalents of intermediate 12, 2 molar equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), and 4 molar equivalents of N,N-diisopropylethylamine (DIEPA) were added. The reaction mixture was reacted for 12 h, and the dichloromethane was removed by rotary evaporation. The crude product was dissolved in ethyl acetate, washed three times with saturated sodium chloride solution, and then dried over anhydrous sodium sulfate. The reaction mixture was filtered and concentrated by vacuum rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane-methanol-ammonia) to give an ionizable lipid molecule (Formula 10) (57 mg). 1 H NMR (400 MHz, CDCl3): δ5.43-5.36 (m, 2H), 4.43 (d, J = 4.5 Hz, 1H), 4.03 (t, J = 6.6 Hz, 3H), 3.77(t, J = 6.9 Hz, 1H), 3.56 – 3.43 (m, 2H), 3.42 – 3.22 (m, 6H), 2.57 – 2.54(m, 2H), 2.52 – 2.31 (m, 4H), 2.28 – 2.16 (m, 9H), 1.92 – 1.67 (m, 8H), 1.32– 1.05 (m, 42H), 0.85 (t, J = 5.9 Hz, 9H). MS:751.5267.

[0184] Preparation of lipid nanoparticles encapsulating luciferase mRNA (Fluc-mRNA)

[0185] The prepared ionizable lipid molecules, DSPC, cholesterol, and PEG2000-DMG were mixed and dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 to obtain a mixture. Using two micro-injection pumps, the volume ratio of the mixture to a citrate-sodium citrate solution (10 mM, pH=4.5) containing luciferase mRNA (Fluc-mRNA) was controlled at 1:3, and the mass ratio of ionizable lipid molecules to luciferase mRNA was controlled at 10:1. A crude solution of lipid nanoparticles was prepared in a microfluidic chip. The solution was then dialyzed three times with sucrose-PBS solution (10%) at 4°C using a dialysis bag (MWCO 14 kDa). The solution was then filtered through a 0.22 μm microporous membrane to obtain lipid nanoparticles, which were then prepared as LNP1~LNP10. LNP1 is the lipid nanoparticle containing Fluc-mRNA prepared by the ionizable lipid molecules of Formula 1. The other nanoparticles were prepared in the same way.

[0186] In vitro delivery efficiency assessment

[0187] HCT-116 cells were seeded in 96-well plates at a density of 1 × 10⁶ cells per well. 4 HCT-116 cells were cultured in a cell culture incubator for 24 hours. Lipid nanoparticles containing Fluc-mRNA, prepared from different ionizable lipid molecules, were diluted in DMEM medium. After 24 hours of incubation, the culture medium was removed from the HCT-116 cells, and the cells were washed once with PBS before luciferase expression efficiency was measured. The USFDA-approved ionizable lipid SM-102 was used as a positive control.

[0188] Figure 1 The in vitro delivery efficiency comparison chart shows that, compared with LNP-SM102 synthesized from SM102, the in vitro mRNA delivery efficiency of LNP1 synthesized from ionizable lipid molecules of Formula 1 is increased by 10.6 times, the in vitro mRNA delivery efficiency of LNP4 synthesized from ionizable lipid molecules of Formula 4 is increased by 2.4 times, the in vitro mRNA delivery efficiency of LNP5 synthesized from ionizable lipid molecules of Formula 5 is increased by 6.4 times, and the in vitro mRNA delivery efficiency of LNP8 synthesized from ionizable lipid molecules of Formula 8 is increased by 3.4 times.

[0189] Cytotoxicity testing methods

[0190] HCT-116 cells were seeded in 96-well plates at a density of 2 × 10⁶ cells per well. 4 Cells were cultured for 24 hours in a cell culture incubator. Lipid nanoparticles containing Fluc-mRNA (1.2 μg / mL), prepared from different ionizable lipid molecules, were diluted in DMEM medium. After 24 hours of incubation, 10 μL of CCK-8 was added, and the cells were cultured for another 4 hours. Absorbance at 450 and 650 nm was measured. PBS was used as a positive control.

[0191] The formula for calculating cell viability is shown in formula (1):

[0192] Cell viability = experimental group (OD) 450nm -OD 650nm ) / Control group (OD 450nm -OD 650nm )×100% formula (1).

[0193] Figure 2 The cytotoxicity comparison diagram shows that, compared with LNP-SM102 synthesized from SM102, LNP1 synthesized from ionizable lipid molecules of Formula 1, LNP4 synthesized from ionizable lipid molecules of Formula 4, and LNP5 synthesized from ionizable lipid molecules of Formula 5 did not show significant cytotoxicity while improving delivery efficiency.

[0194] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An ionizable lipid molecule having the structure shown in any one of Formulas 1 to 10: Formula 1 Formula 2 Formula 3 Formula 4 Formula 5 Formula 6 Formula 7 Formula 8 Formula 9 Formula 10.

2. The method for preparing ionizable lipid molecules according to claim 1, characterized in that, Includes the following steps: Compound 1 and compound 2 were mixed and subjected to a substitution reaction to obtain a substituted product having the structure shown in compound 4. The substituted product was mixed with compound 3 and subjected to an amidation reaction to obtain the ionizable lipid molecule; The structures of compounds 1-4 are shown below: Compound 1 Compound 2 Compound 3 Compound 4; Of compounds 1-4: R1 and R2 are independently H, C1-C3 saturated or unsaturated aliphatic chains, halogenated or hydroxyl-substituted C1-C3 aliphatic chains; R3, R4 and R5 are independently C1-C20 saturated or unsaturated aliphatic chains, halogenated or hydroxyl-substituted C1-C20 aliphatic chains. G1, G2 and G3 are independently C0-C9 saturated or unsaturated aliphatic chains, halogenated or hydroxyl-substituted C1-C8 aliphatic chains; L1 is -CH2-, -O-, -S-, -C(O)NH-, -HNC(O)-, -OC(O)O-, -OC(O)-, -C(O)O-, -HNC(O)O-, -OC(O)NH-, -HNC(O)NH-, -SC(O)O-, -OC(O)S-, -HNC(O)S- or -SC(O)NH-; X is a halogen.

3. The use of the ionizable lipid molecule according to claim 1 in the preparation of nucleic acid delivery drugs.

4. A lipid nanoparticle, characterized in that, Includes the following components: The ionizable lipid molecules, non-cationic lipids, sterols, and polyethylene glycol lipids as described in claim 1.

5. The lipid nanoparticles according to claim 4, characterized in that, The molar ratio of the ionizable lipid molecules, non-cationic lipids, sterols and polyethylene glycol lipids is (30~50):(5~20):(30~40):(0.1~3).

6. The lipid nanoparticles according to claim 4 or 5, characterized in that, The non-cationic lipids include non-cationic phospholipids.

7. The lipid nanoparticles according to claim 4 or 5, characterized in that, The polyethylene glycol lipid is PEG2000-DMG.

8. The use of the lipid nanoparticles according to any one of claims 4 to 7 in the preparation of nucleic acid delivery drugs.

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