Cationic lipid compounds, methods of making and using the same

Abstract

The application provides a cationic lipid compound and a preparation method and use thereof. The cationic lipid compound has a structure shown in formula I. The cationic lipid compound provided by the application has a simple chemical structure and is easy to synthesize, and can be used for preparing a drug carrier, in particular a nucleic acid drug carrier. The drug-loaded lipid nanoparticle containing the cationic lipid compound has a small particle size, a uniform particle size distribution, and good loading and delivery effects on nucleic acid drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a cationic lipid compound, its preparation method, and its uses. Background Technology

[0002] The biopharmaceutical field mainly comprises two types of drugs: therapeutic drugs and preventative drugs. Therapeutic drugs primarily include small-molecule chemical drugs and large-molecule protein drugs; while preventative drugs mainly include various vaccines, such as inactivated vaccines, live attenuated vaccines, split vaccines, and recombinant protein vaccines. The development of nucleic acid drugs (siRNA, mRNA, DNA, etc.) has greatly promoted the development of both therapeutic and preventative drugs, marking a significant milestone and propelling the biopharmaceutical field into the era of gene therapy. However, nucleic acid drugs also have drawbacks such as instability, high innate immunogenicity, low in vivo delivery efficiency, and susceptibility to clearance.

[0003] Lipid nanoparticles (LNPs) delivery systems refer to nanoparticles formed by the self-assembly of various lipid components to encapsulate and deliver nucleic acid drugs. They can effectively improve the stability of nucleic acid drugs, reduce their immunogenicity, and enhance their in vivo delivery efficiency. The first commercial application of LNPs was Onpattro, which was approved in the US and EU in 2018 for the treatment of amyloidosis. Since then, LNPs have received widespread attention as nucleic acid delivery carriers. In particular, since 2020, Moderna and BioNTech's novel coronavirus mRNA vaccines have both adopted LNP delivery systems. LNP delivery systems generally contain four lipid components [Pilkington, Emily H et al. “From influenza to COVID-19: Lipid nanoparticle mRNA vaccines at the frontiers of infectious diseases.” Acta biomaterialia vol. 131(2021):16-40. doi:10.1016 / j.actbio.2021.06.023]: 1) Ionizable cationic lipids, used to bind to negatively charged mRNA; 2) Cholesterol: mediates LNP endocytosis and stabilizes LNP structure; 3) Neutral phospholipids: helper lipids that can accelerate the release of mRNA during endocytosis; 4) PEG phospholipids: prolong metabolic time, improve LNP stability, and control particle size.

[0004] However, there are currently few types of lipid compounds that can be used for nucleic acid drug delivery, and there is an urgent need to develop more effective lipid compounds to promote the development of the nucleic acid drug industry. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a cationic lipid compound, its preparation method, and its uses. This cationic lipid compound has a simple chemical structure, is easy to synthesize, and can be used to prepare drug carriers. Drug-loaded lipid nanoparticles containing this cationic lipid compound have small particle sizes and uniform particle size distribution, exhibiting good loading and delivery effects for nucleic acid drugs.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a cationic lipid compound having the structure shown in Formula I:

[0008]

[0009] In Formula I, R1 is selected from one of C1-C10 alkylene and C2-C10 alkenylene;

[0010] R2 is CH and R3 is selected from One of them;

[0011] Alternatively, R2 is N and R3 is selected from *-L1-OH and One of them;

[0012] R4 is selected from C10-C20 alkyl, C10-C20 alkenyl, One of them;

[0013] Wherein, L1 is a single bond or a C1-C6 alkylene group, L2 is a single bond or a methylene group, L3 is a C1-C2 alkylene group, L4 is a C3-C12 alkylene group, and L5 is a C4-C15 alkyl group;

[0014] -* represents the linking bond of a group.

[0015] It should be noted that the alkenyl group described in this invention may contain one carbon-carbon double bond or multiple carbon-carbon double bonds.

[0016] In some embodiments of the present invention, R1 is selected from one of the following groups:

[0017]

[0018] Here, -* represents the linking bond of a group.

[0019] In some embodiments of the present invention, R2 is CH and R3 is selected from one of the following groups:

[0020]

[0021] Here, -* represents the linking bond of a group.

[0022] In some embodiments of the present invention, R2 is N and R3 is selected from one of the following groups:

[0023]

[0024] Here, -* represents the linking bond of a group.

[0025] In some embodiments of the present invention, R4 is selected from one of the following groups:

[0026]

[0027]

[0028] Here, -* represents the linking bond of a group.

[0029] In some embodiments of the present invention, the cationic lipid compound is selected from the following compounds 1 to 5:

[0030]

[0031]

[0032] In a second aspect, the present invention provides a method for preparing a cationic lipid compound as described in the first aspect, wherein when R2 is N, the preparation method includes the following steps:

[0033]

[0034] compound Imidazole was added to an organic solvent, and tert-butyldiphenylchlorosilane (TBDPSCl) was added dropwise to react and generate intermediate 1; intermediate 1 was dissolved in an organic solvent, and sodium hydride, sodium iodide and compound were added. The reaction proceeds to generate intermediate 2; intermediate 2 and lithium hydroxide are added to a mixture of tetrahydrofuran, methanol, and water to generate intermediate 3; intermediate 3 reacts with cholesterol under the catalysis of N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine (DMAP) to generate intermediate 4; intermediate 4 is dissolved in a mixture of dichloromethane and trifluoroacetic acid to generate intermediate 5; intermediate 5 reacts with compound Br-R4 in an organic solvent containing potassium carbonate and sodium iodide to generate intermediate 6; intermediate 6 and triethylamine hydrogen fluoride are added to an organic solvent, and the pH is adjusted to alkaline to generate the cationic lipid compound.

[0035] Alternatively, when R2 is CH, the preparation method includes the following steps:

[0036]

[0037] compound Imidazole was added to an organic solvent, and tert-butyldiphenylchlorosilane (TBDPSCl) was added dropwise to react and generate intermediate 7. Intermediate 7 was dissolved in an organic solvent, and pyridinium chlorochromate was added to react and generate intermediate 8. Compound Br-L4-OBn was dissolved in an organic solvent, and molecular sieves, Mg, and iodine were added sequentially under nitrogen atmosphere to react and generate intermediate 9. Intermediate 8 was added to react and generate intermediate 10. Intermediate 10 reacted with compound R3-H to generate intermediate 11. Intermediate 11 was added to a mixture of tetrahydrofuran, methanol, and acetic acid, and palladium on carbon was added to react and generate intermediate 12 under hydrogen atmosphere. Intermediate 12 reacted with compound R3-H to generate intermediate 12. The intermediate 13 is generated by reacting 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) under catalysis. Intermediate 13 and triethylamine hydrogen fluoride are added to an organic solvent, and the pH is adjusted to alkaline to generate intermediate 14. Intermediate 14 is dissolved in acetone, and Jones' reagent is added dropwise to generate intermediate 15. Intermediate 15 is reacted with cholesterol under the catalysis of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) to generate the cationic lipid compound.

[0038] Thirdly, the present invention provides the use of the cationic lipid compound as described in the first aspect for preparing a drug carrier, preferably for preparing a nucleic acid drug carrier.

[0039] Fourthly, the present invention provides lipid nanoparticles comprising the cationic lipid compounds as described in the first aspect.

[0040] In some embodiments of the present invention, the lipid nanoparticles further include structural lipids, cholesterol, and polymer-modified lipids.

[0041] In some embodiments of the present invention, the structural lipid is a neutral phospholipid. The neutral phospholipid is preferably selected from one or more of distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), and distearylphosphatidylethanolamine (DSPE).

[0042] Neutral phospholipids are a commonly used type of structured lipid in the art, but in this invention, structured lipids are not limited to neutral phospholipids; other types of structured lipid compounds can also be used in this invention.

[0043] In some embodiments of the present invention, the polymer-modified lipid is a polyethylene glycol-modified lipid.

[0044] In some embodiments of the present invention, the lipid nanoparticles comprise the following components in molar percentages: 30-50% cationic lipid compound as described in the first aspect, 5-25% structural lipid, 28.5-48.5% cholesterol, and 0.5-3% polyethylene glycol modified lipid.

[0045] The molar percentage of the cationic lipid compound may be 30%, 32%, 35%, 38%, 40%, 42%, 45%, 48%, or 50%, etc.

[0046] The molar percentage of the structural lipids can be 5%, 8%, 10%, 12%, 15%, 18%, 20%, 22%, or 25%, etc.

[0047] The molar percentage of cholesterol can be 28.5%, 30%, 32%, 35%, 38%, 40%, 42%, 45%, 47%, or 48.5%, etc.

[0048] The molar percentage of the polyethylene glycol-modified lipid can be 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8%, or 3%, etc.

[0049] Fifthly, the present invention provides a drug-loaded lipid nanoparticle, comprising: the lipid nanoparticle as described in the fourth aspect and a nucleic acid drug loaded in the lipid nanoparticle.

[0050] Sixthly, the present invention provides a method for preparing drug-loaded lipid nanoparticles as described in the fifth aspect, the method comprising the following steps:

[0051] An oil phase containing the components of the lipid nanoparticles and an aqueous phase containing nucleic acid drugs were prepared separately. The oil phase and the aqueous phase were mixed using a microfluidic device to self-assemble the drug-loaded lipid nanoparticles.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] The cationic lipid compound provided by this invention has a simple chemical structure and is easy to synthesize, and can be used to prepare drug carriers, especially nucleic acid drug carriers. Drug-loaded lipid nanoparticles containing this cationic lipid compound have small particle size and uniform particle size distribution, exhibiting good encapsulation efficiency and transfection efficiency. They can effectively load nucleic acid drugs and deliver them to cells and animals for expression. Attached Figure Description

[0054] Figure 1 Compound 1 of the present invention1 H NMR spectrum;

[0055] Figure 2 Compound 2 of the present invention 1 H NMR spectrum;

[0056] Figure 3 Compound 3 of the present invention 1 H NMR spectrum;

[0057] Figure 4 Compound 4 of the present invention 1 H NMR spectrum;

[0058] Figure 5 Compound 5 of the present invention 1 H NMR spectrum;

[0059] Figure 6 The structural lipid used in Example 7 of this invention 1 H NMR spectrum;

[0060] Figure 7 The image shows the cell transfection results of the drug-loaded lipid nanoparticles prepared in Examples 6 and 7 of this invention. Detailed Implementation

[0061] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art should understand that the specific embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention.

[0062] Example 1

[0063] This embodiment provides compound 1, whose synthetic route is as follows:

[0064]

[0065] The specific preparation method is as follows:

[0066] Synthesis of Compound 1-1: N-(3-hydroxypropyl)carbamate tert-butyl ester (9 g, 51.362 mmol, 1 eq) was dissolved in dichloromethane (180 mL), and imidazole (6.99 g, 101.724 mmol, 2 eq) was added. The mixture was cooled to approximately 0 °C, and TBDPSCl (15.53 g, 56.498 mmol, 1.1 eq) was added dropwise. The reaction was carried out at room temperature for 16 hours. After the reaction was complete, excess TBDPSCl was quenched with water and extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, v / v 1:0–30:1), and the eluent was concentrated to give a yellow oily product (20 g, 94% yield). MS m / z [M+Na] + (ESI): 436.25.

[0067] Synthesis of compounds 1-2: Compound 1-1 (20 g, 48.352 mmol, 1 eq) was dissolved in N,N-dimethylformamide (200 mL), cooled to 0 °C, and sodium hydride (2.9 g, 72.528 mmol, 1.5 eq) was added in portions, followed by sodium iodide (724.8 mg, 4.835 mmol, 0.1 eq) and ethyl 8-bromooctanoate (18.22 g, 75.528 mmol, 1.5 eq). The mixture was stirred at 40 °C for 1 hour. After the reaction was complete, the mixture was quenched with saturated ammonium chloride aqueous solution. The mixture was then extracted with dichloromethane, and the combined organic phases were washed successively with water and saturated sodium chloride aqueous solution. The mixture was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, v / v ratio 1:0–1:9). The eluent was concentrated to give a yellow oily product (17 g, yield 54%). MS m / z [M+Na] + (ESI): 606.45.

[0068] Synthesis of compounds 1-3: Compounds 1-2 (17 g, 29.115 mmol, 1 eq) were dissolved in a mixture of tetrahydrofuran / methanol / water (2:1:1, 340 mL, v / v), and then lithium hydroxide (1.05 g, 43.672 mmol, 1.5 eq) was added. The reaction was allowed to proceed overnight at room temperature. After the reaction was complete, the pH of the system was adjusted to 4 with 1 mol / L hydrochloric acid, and the mixture was extracted three times with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give a crude product as a colorless oil (16 g, 99% yield). MS m / z [M+Na] + (ESI): 578.30.

[0069] Synthesis of compounds 1-4: Compounds 1-3 (16 g, 28.786 mmol, 1 eq) were dissolved in dichloromethane (320 mL), and cholesterol (13.36 g, 34.543 mmol, 1.2 eq), N,N'-dicyclohexylcarbodiimide (8.91 g, 43.179 mmol, 1.5 eq), and 4-dimethylaminopyridine (1.06 g, 8.636 mmol, 0.3 eq) were added sequentially. The reaction was carried out overnight at room temperature. After the reaction was completed, the solids were filtered off, and the filtrate was concentrated. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, v / v ratio 1:0-40:1) to give a yellow oily product (13 g, yield 49%).

[0070] Synthesis of compounds 1-5: Compounds 1-4 (13 g, 14.062 mmol, 1 eq) were dissolved in a mixture of dichloromethane and trifluoroacetic acid (v / v = 5:1, 260 mL) and reacted at room temperature for 10 min. After the reaction was complete, the mixture was extracted twice with dichloromethane. The combined organic phases were washed successively with water and saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, v / v = 1:0–20:1) to give a white solid (8.5 g, yield 73%). MS m / z [M+H] + (ESI): 824.60.

[0071] Synthesis of compounds 1-6: Oleyl alcohol (3 g, 11.174 mmol, 1 eq) was dissolved in dichloromethane (60 mL), carbon tetrabromide (4.45 g, 13.409 mmol, 1.2 eq) was added, and triphenylphosphine (3.52 g, 13.409 mmol, 1.2 eq) was slowly added at 0 °C. The reaction was carried out at room temperature for 60 minutes. After the reaction was complete, n-heptane was added until a solid was formed, and the mixture was concentrated and evaporated to dryness. The solid was filtered off, and the filtrate was concentrated to give a colorless oily product (2.8 g, yield 76%). MS m / z [M+H] + (ESI): 330.19.

[0072] Synthesis of compounds 1-7: Compounds 1-5 (1.5 g, 1.820 mmol, 1 eq) were dissolved in N,N-dimethylformamide (30 mL), and anhydrous potassium carbonate (0.3 g, 2.184 mmol, 1.2 eq) was added. The reaction was carried out at 100 °C for 60 min. After the reaction was completed, compounds 1-6 (0.72 g, 2.184 mmol, 1.2 eq) and sodium iodide (0.03 g, 0.182 mmol, 0.1 eq) were added, and the reaction was carried out at room temperature for 6 h. After the reaction was completed, the solid was filtered off with diatomaceous earth. The filtrate was washed successively with water and sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, v / v ratio 1:0-8:1). The eluent was concentrated to give a yellow oily substance (1.0 g, yield 51%). MS m / z [M+H] + (ESI): 1074.80.

[0073] Synthesis of Compound 1: Compounds 1-7 (1.0 g, 0.930 mmol, 1 eq) were dissolved in dichloromethane (20 mL), and a triethylamine hydrogen fluoride solution (1.50 mg, 9.300 mmol, 10 eq) was added. The reaction was carried out at room temperature for 2 hours. The pH was adjusted to 8 with triethylamine, and the product was concentrated and evaporated to dryness. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, v / v 1:0-60:1). The eluent was concentrated to give a pale yellow semi-solid (0.472 g, yield 61%). MS m / z [M+H]+ (ESI): 836.85.

[0074] Compound 1 1 H NMR spectrum as shown Figure 1 As shown, the NMR data are 1 H NMR (300MHz, CD3OD-d4): 5.28-5.23(m,3H), δ4.75(s,1H), 3.61-3.58(m,2H), 3.21-3.13(m,2H), 3.13-3.00(m,4H), 2.22-2.19(m,4H) ,1.94-1.61(m,11H),1.54-1.50(m,13H),1.46-1.19(m,32H),1.19-1.01(m,7H),1.01-0.96(m,6H),0.96-0.77(m,12H),0.62(s,3H).

[0075] Example 2

[0076] This embodiment provides compound 2, whose synthetic route is as follows:

[0077]

[0078] The specific preparation method is as follows:

[0079] Synthesis of Compound 2-1: 6-Bromo-1-hexanol (3 g, 16.667 mmol, 1 eq), 2-ethyldecanoic acid (4.3 g, 16.797 mmol, 1 eq), EDCI (3.9 g, 20.313 mmol, 1.2 eq), and DMAP (0.41 g, 3.361 mmol, 0.2 eq) were dissolved in dichloromethane (60 mL). DIEA (N,N-diisopropylethylamine, 8.6 g, 66.667 mmol, 4 eq) was added, and the reaction was carried out at room temperature for 16 hours. The reaction solution was diluted with dichloromethane, washed once with saturated sodium chloride aqueous solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane:methanol v / v = 1:0-50:1). The eluent was concentrated to give the product as a white solid (4 g, yield 59%).

[0080] Synthesis of Compound 2-2: Compound 1-5 (1 g, 1.214 mmol, 1 eq) and anhydrous potassium carbonate (0.25 g, 1.812 mmol, 1.5 eq) were dissolved in DMF (20 mL). Compound 2-1 (0.76 g, 1.881 mmol, 1.5 eq) and sodium iodide (0.02 g, 0.138 mmol, 0.1 eq) were added, and the reaction was carried out at 100 °C for 12 hours. The reaction solution was diluted with dichloromethane and extracted once with saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate v / v = 1:0-70:1). The eluent was concentrated to give a grayish-white oil (1.2 g, yield 85%). MS m / z [M+H] + (ESI): 1162.85.

[0081] Synthesis of Compound 2: Compound 2-2 (1 g, 0.861 mmol, 1 eq) was dissolved in dichloromethane (200 mL), and 3HF·TEA (1.4 g, 8.696 mmol, 10 eq) was added. The reaction mixture was reacted at room temperature for 16 hours. The pH of the reaction solution was adjusted to 8 with triethylamine, and the crude product was directly concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane and methanol, v / v ratio 1:0-50:1). The eluent was concentrated to give a yellow semi-solid (505.5 mg, yield 63%, purity 98.7%). MS m / z [M+H] + (ESI): 924.75.

[0082] Compound 2 1 H NMR spectrum as shown Figure 2 As shown, the NMR data are 1H NMR (300MHz, Chloroform-d): δ5.30-5.28(m,1H),4.49-4.39(m,1H),4.00(t,J =6Hz,2H),3.60(t,J=6Hz,2H),3.18-3.13(m,2H),3.06-3.00(m,4H),2.29-2.18 (m,5H),1.98-1.71(m,7H),1.69-1.39(m,17H),1.38-1.23(m,17H),1.24-1.14( m,21H),1.11-0.99(m,6H),0.96-0.93(m,4H),0.89-0.79(m,16H),0.63(s,3H).

[0083] Example 3

[0084] This embodiment provides compound 3, whose synthetic route is as follows:

[0085]

[0086] The specific preparation method is as follows:

[0087] Synthesis of compound 3-1: Tetradecanoic acid (3 g, 13.158 mmol, 1 eq), EDCI (2.8 g, 14.583 mmol, 1.1 eq), and DMAP (0.18 g, 1.475 mmol, 0.1 eq) were dissolved in dichloromethane (60 mL), and 3-bromo-1-propanol (1.7 g, 12.319 mmol, 0.91 eq) was added. The reaction mixture was reacted at room temperature for 16 hours. The reaction solution was diluted with dichloromethane, washed once with saturated sodium chloride aqueous solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel column chromatography (petroleum ether and ethyl acetate, v / v ratio 1:0-50:1). The eluent was concentrated to give the product as a white solid (3 g, yield 66%).

[0088] Synthesis of compound 3-2: Compound 1-5 (1 g, 1.214 mmol, 1 eq) was dissolved in anhydrous potassium carbonate (0.25 g, 1.812 mmol, 1.5 eq) in DMF (20 mL), and reacted at 100 °C for 1 h. Compound 3-2 (0.63 g, 1.822 mmol, 1.5 eq) and sodium iodide (0.02 g, 0.138 mmol, 0.1 eq) were added, and the reaction was carried out at 100 °C for 12 h. The pH of the reaction solution was adjusted to 7. The reaction solution was diluted with dichloromethane and extracted once with saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether and ethyl acetate, v / v ratio 1:0-5:1). The eluent was concentrated to give a grayish-white oil (1.2 g, yield 92%). MS m / z [M+H]+ (ESI): 1092.75.

[0089] Synthesis of Compound 3: Compound 3-2 (1 g, 0.916 mmol, 1 eq) was dissolved in dichloromethane (20 mL), and triethylamine hydrogen fluoride (1.5 g, 9.158 mmol, 10 eq) was added. The reaction mixture was reacted at room temperature for 16 hours. The pH of the reaction solution was adjusted to 8 with triethylamine, and the crude product was directly concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane and methanol, v / v 1:0-50:1). The eluent was concentrated to give the product as a yellow semi-solid (572.8 mg, yield 70%, purity 99.345%). MS m / z [M+H] + (ESI): 854.65.

[0090] Compound 3 1 H NMR spectrum as shown Figure 3 As shown, the NMR data are 1 H NMR (400MHz, Chloroform-d): δ5.39-5.38(m,1H),4.87-4.49(m,1H),4.16(t,J=6Hz ,2H),3.69(t,J=5.6Hz,2H),3.18-3.10(m,4H),3.05-3.01(m,2H),2.36-2.28(m,6H) ,2.06-1.96(m,4H),1.93-1.81(m,5H),1.69-1.45(m,13H),1.39-1.26(m,30H),1.22 -1.09(m,7H),1.07-0.98(m,6H),0.96-0.92(m,3H),0.95-0.81(m,9H),0.72(s,3H).

[0091] Example 4

[0092] This embodiment provides compound 4, whose synthetic route is as follows:

[0093]

[0094] The specific preparation method is as follows:

[0095] Synthesis of compound 4-1: 1,9-Nonanediol (20 g, 124.8 mmol, 1 eq) and imidazole (8.5 g, 124.8 mmol, 1 eq) were dissolved in tetrahydrofuran (1250 mL), and TBDPSCl (34.3 g, 124.8 mmol, 1 eq) was added dropwise. The reaction was carried out at room temperature for 3 hours. After the reaction was complete, the solid was filtered off, and the filtrate was concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, v / v 1:0–8:1). The eluent was concentrated to give a colorless oily product (13 g, yield 26%). MS m / z [M+Na]+ (ESI): 421.20.

[0096] Synthesis of compound 4-2: Compound 4-1 (5 g, 12.542 mmol, 1 eq) was dissolved in dichloromethane (100 mL), and pyridinium chlorochromate (4.06 g, 18.813 mmol, 1.5 eq) was added. The reaction was carried out at room temperature for 3 hours. After the reaction was complete, the solid was filtered off. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, v / v ratio 1:0–50:1). The eluent was concentrated to give a colorless oily product (4 g, yield 80%). MS m / z [M+Na]+ (ESI): 419.10

[0097] Synthesis of compound 4-3: Benzyl-6-bromohexane (2 g, 7.375 mmol, 1 eq) was dissolved in tetrahydrofuran (30 mL). A certain amount of the above solution was added sequentially under nitrogen atmosphere with molecular sieve (4A), Mg (537.7 mg, 22.125 mol, 3 eq), and a catalytic amount of elemental iodine. The mixture was heated to 50 °C and stirred until the solution changed from yellow to colorless. The remaining benzyl-6-bromohexane tetrahydrofuran solution was slowly added dropwise to the reaction system, and the reaction was continued for 2 h to obtain compound 4-3.

[0098] Synthesis of compound 4-4: Compound 4-2 (2.34 g, 8.587 mmol, 0.8 eq) was dissolved in tetrahydrofuran (50 mL), and molecular sieve (4A) was added under nitrogen atmosphere. A tetrahydrofuran solution of compound 4-3 was added dropwise to the reaction system, and the reaction was carried out at room temperature for 2 hours. The reaction solution was added to a saturated aqueous ammonium chloride solution and extracted twice with dichloromethane. The combined organic phases were washed with saturated sodium chloride, then dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, v / v 1:0–20:1) to give a colorless oil (3.5 g, yield 40%). MS m / z [M+Na]+ (ESI): 611.45.

[0099] Synthesis of compounds 4-5: Compound 4-4 (3.5 g, 5.943 mmol, 1 eq) was dissolved in dichloromethane (70 mL), and N,N-dimethylglycine hydrochloride (1.24 g, 8.915 mmol, 1.5 eq) and N,N-diisopropylethylamine (1.15 g, 8.915 mmol, 1.5 eq) were added sequentially. The reaction mixture was allowed to react overnight at room temperature. After the reaction was complete, the reaction solution was diluted with dichloromethane, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, v / v ratio 1:0-3:1) to give a colorless oily product (3 g, yield 75%). MS m / z [M+H] + (ESI): 674.40.

[0100] Synthesis of compounds 4-6: Compounds 4-5 (2.5 g, 3.624 mmol, 1 eq) were dissolved in a mixture of tetrahydrofuran / methanol / acetic acid (v / v 20:20:1, 100 mL), and anhydrous palladium on carbon (10%, 2.5 g, 100% wt) was added under nitrogen atmosphere. The reaction system was then purged with hydrogen (50 atm) and allowed to react overnight at room temperature. The pH of the system was adjusted to ~8 with triethylamine, and then filtered through diatomaceous earth. The filtrate was concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol, v / v 1:0–10:1). The eluent was concentrated to give a colorless oily product (1.1 g, yield 41%). MS m / z [M+H] + (ESI): 584.70.

[0101] Synthesis of compounds 4-7: Compounds 4-6 (1.1 g, 1.507 mmol, 1 eq) were dissolved in dichloromethane (22 mL), followed by the addition of 2-hexyldecanoic acid (579.7 mg, 2.260 mmol, 1.5 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (433.3 mg, 2.260 mmol, 1.5 eq), and 4-dimethylaminopyridine (52.2 mg, 0.452 mmol, 0.3 eq). The reaction mixture was allowed to react overnight at room temperature. The reaction solution was diluted with dichloromethane, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. MS m / z [M+H]+ (ESI): 822.75.

[0102] Synthesis of compounds 4-8: Compounds 4-8 were dissolved in dichloromethane (30 mL), and triethylamine hydrogen fluoride (2.43 g, 15.070 mmol, 10 eq) was added. The reaction was carried out at room temperature for 16 hours. The pH of the system was adjusted to 8 with triethylamine and the solution was concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, v / v 1:0–30:1). The eluent was concentrated to give a colorless oily product (700 mg, 80% yield). MS m / z [M+H] + (ESI): 584.55.

[0103] Synthesis of compounds 4-9: Compounds 4-8 (700 mg, 1.199 mmol, 1 eq) were dissolved in acetone (25 mL), and Jones' reagent (2.672 mol / L, 1.44 mL, 3.2 eq) was added dropwise. The mixture was stirred at 0 °C for 1 hour. Then, 20 mL of water was added, and the mixture was extracted twice with ethyl acetate. The combined organic phases were washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, v / v 1:0–20:1). The eluent was concentrated to give the product as a white solid (450 mg, 63% yield). MS m / z [M+H]+ (ESI): 599.00.

[0104] Synthesis of Compound 4: Compounds 4-9 (600 mg, 1.003 mmol, 1 eq) were dissolved in dichloromethane (12 mL), and cholesterol (582 mg, 1.504 mmol, 1.5 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (288.5 mg, 1.504 mmol, 1.5 eq), and 4-dimethylaminopyridine (36.8 mg, 0.301 mmol, 0.3 eq) were added sequentially. The reaction mixture was allowed to react overnight at room temperature. The reaction solution was diluted with dichloromethane, washed with saturated sodium chloride, and then dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate, v / v 1:0.5 / 1), and the eluent was concentrated to give a milky white oil (578.5 mg, yield 59%). MS m / z [M+H]+ (ESI): 966.70.

[0105] Compound 4 1 H NMR spectrum as shown Figure 4 As shown, the NMR data are 1H NMR (400MHz, Chloroform-d): δ5.37(d,J=5.1Hz,1H),5.00-4.87(m,1H),4.66 -4.54(m,1H),4.05(t,J=6.6Hz,2H),3.17(s,2H),2.38(s,6H),2.34-2.23(m, 5H),2.05-1.92(m,2H),1.90-1.77(m,3H),1.68-1.39(m,21H),1.38-1.19(m, 38H),1.18-1.06(m,6H),1.04-0.94(m,5H),0.92-0.83(m,15H),0.68(s,3H).

[0106] Example 5

[0107] This embodiment provides compound 5, whose synthetic route is as follows:

[0108]

[0109] The specific preparation method is as follows:

[0110] Synthesis of Compound 5-1: Linoleol (1 g, 3.753 mmol, 1 eq) was dissolved in dichloromethane (20 mL), carbon tetrabromide (1.49 g, 4.504 mmol, 1.2 eq) was added, and triphenylphosphine (1.18 g, 4.504 mmol, 1.2 eq) was added. The reaction was carried out at room temperature for 1 hour. Heptane was added until a solid was formed, and the mixture was concentrated and evaporated to dryness. Heptane was added again, the solid was filtered off, and the filtrate was concentrated to give a colorless oily product (1 g, yield 81%).

[0111] Synthesis of compound 5-2: Compound 1-5 (1.0 g, 1.213 mmol, 1 eq) was dissolved in N,N-dimethylformamide (20 mL), and anhydrous potassium carbonate (0.25 g, 1.820 mmol, 1.5 eq) was added. The reaction was carried out at 100 °C for 30 min. Then, compound 5-1 (0.60 g, 1.820 mmol, 1.5 eq) was added, and the reaction was carried out at 100 °C for 16 h. After cooling to room temperature, the reaction solution was diluted with dichloromethane and filtered off the solid with diatomaceous earth. The filtrate was washed successively with water and sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, v / v ratio 1:0-8:1). The eluent was concentrated to give a yellow oily substance (0.8 g, yield 61%). MS m / z [M+H]+ (ESI): 1072.80.

[0112] Synthesis of Compound 5: Compound 5-2 (0.8 g, 0.746 mmol, 1 eq) was dissolved in dichloromethane (16 mL), and a triethylamine hydrogen fluoride solution (1.2 g, 7.460 mmol, 10 eq) was added. The reaction was carried out at room temperature for 2 hours. The pH was adjusted to 8 with triethylamine, and the solution was concentrated and evaporated to dryness. The crude product was purified by silica gel column chromatography (dichloromethane / methanol, v / v 1:0–30:1). The eluent was concentrated to give a pale yellow semi-solid (0.531 g, 85% yield). MS m / z [M+H]+ (ESI): 834.70.

[0113] Compound 5 1 H NMR spectrum as shown Figure 5 As shown, the NMR data are 1 H NMR (300MHz, CD3OD-d4): δ5.31-5.18(m,5H),4.49-4.48(m,1H),3.60(t,J=6H z,2H),3.22-3.19(m,2H),3.05-2.99(m,4H),2.68(t,J=6Hz,2H),2.23-2.19(m ,4H),1.99-1.94(m,5H),1.86-1.70(m,5H),1.63-1.39(m,13H),1.30-1.19(m ,27H),1.14-1.01(m,7H),0.98-0.90(m,5H),0.86-0.77(m,13H),0.63(s,3H).

[0114] Example 6

[0115] This embodiment provides a series of drug-loaded lipid nanoparticles, the preparation method of which is as follows:

[0116] (1) The cationic lipid compound, DSPC (distearate phosphatidylcholine), CHOL (cholesterol) and ALC-0159 (polyethylene glycol modified lipid, purchased from Xiamen Sinobond Biotechnology Co., Ltd.) were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 to prepare an oil phase with a total lipid concentration of 14.4 mmol / L.

[0117] Among them, the cationic lipid compounds are compounds 1 to 5 and ALC-0315 (purchased from Xiamen Sinobond Biotechnology Co., Ltd.);

[0118] (2) Dissolve mRNA or Luciferase-mRNA (luciferase-labeled mRNA) in citrate buffer (pH = 4.5, 50 mM) to prepare an aqueous phase with an mRNA concentration of 0.133 mg / mL (0.4 mmol / L);

[0119] (3) The above oil phase and aqueous phase were introduced into a microfluidic nanofabrication system, and the oil-water volume ratio was controlled to be 1:3, so that the N / P ratio of lipid to mRNA (the molar ratio of nitrogen in lipid to phosphorus in mRNA) was 6:1, the total flow rate was 12 mL / min, the waste was discharged at the beginning and 0.1 mL at the end, and the prothrombus was prepared. 3 mL of prothrombus was loaded into a 10 kD dialysis card, the magnetic stirring speed was set to 120 rpm, and the mixture was dialyzed in 500 mL PBS dialysis solution for 4 h to obtain drug-loaded lipid nanoparticles (named mRNA-LNP or Luciferase-mRNA-LNP).

[0120] Example 7

[0121] This embodiment provides a series of drug-loaded lipid nanoparticles, the preparation method of which is as follows:

[0122] (1) The cationic lipid compound, structural lipid, CHOL (cholesterol) and ALC-0159 (polyethylene glycol modified lipid, purchased from Xiamen Sinobond Biotechnology Co., Ltd.) were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 to prepare an oil phase with a total lipid concentration of 14.4 mmol / L.

[0123] Among them, the cationic lipid compounds are compounds 1 to 5 and ALC-0315 (purchased from Xiamen Sinobond Biotechnology Co., Ltd.);

[0124] (2) Dissolve mRNA or Luciferase-mRNA (luciferase-labeled mRNA) in citrate buffer (pH = 4.5, 50 mM) to prepare an aqueous phase with an mRNA concentration of 0.133 mg / mL (0.4 mmol / L);

[0125] (3) The above oil phase and aqueous phase were introduced into a microfluidic nanofabrication system with an oil-water volume ratio of 1:3, so that the N / P ratio of lipid to mRNA (the molar ratio of nitrogen in lipid to phosphorus in mRNA) was 6:1. The total flow rate was 12 mL / min, with 0.2 mL of waste discharged before and 0.1 mL of waste discharged after, to prepare the promulgated emulsion. 3 mL of the promulgated emulsion was loaded into a 10 kD dialysis card, and the magnetic stirring speed was set to 120 rpm. Dialysis was performed in 500 mL of PBS dialysis solution for 4 h to obtain drug-loaded lipid nanoparticles (named mRNA-LNP or Luciferase-mRNA-LNP).

[0126] In this embodiment, the structural lipid is:

[0127]

[0128] The synthetic route and preparation method are as follows:

[0129]

[0130] Synthesis of Intermediate 1: Cholic acid (30 g, 73.529 mmol, 1 eq) was dissolved in N,N-dimethylformamide (600 mL), followed by the addition of dimethylaminoethanol (13.088 g, 147.059 mmol, 2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (21.066 g, 110.294 mmol, 1.5 eq), and 4-dimethylaminopyridine (2.691 g, 22.059 mmol, 0.3 eq). The mixture was stirred overnight at room temperature. The reaction solution was collected and purified by reverse-phase column chromatography. The eluent was extracted twice with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate and concentrated to give the product as a white solid (30 g, yield 85%, purity 99%).

[0131] Synthesis of Intermediate 2: Intermediate 1 (3 g, 6.254 mmol, 1 eq) was dissolved in dichloromethane (30 mL), and 4-dimethylaminopyridine (840.5 mg, 6.879 mmol, 1.1 eq) was added. A solution of octadecanoyl chloride (2.084 g, 6.879 mmol, 1.1 eq) in dichloromethane (30 mL) was slowly added dropwise to the reaction mixture. The reaction was carried out at room temperature for 2 hours, and the reaction mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane:methanol v / v = 20:1) to give a white solid (1.6 g, yield 34%, purity 99%).

[0132] Synthesis of structural lipids: Intermediate 2 (1.4 g, 0.899 mmol, 1 eq) was dissolved in dichloromethane (23 mL), and iodomethane (534 mg, 3.752 mmol, 2 eq) was added. The reaction was carried out at room temperature for 16 hours, and the reaction solution was concentrated. The crude product was purified by silica gel column chromatography (dichloromethane:methanol volume ratio = 10:1) to obtain a yellow solid (1.1243 g, yield 66%, purity 98%).

[0133] The structural lipid 1 H NMR spectrum as shown Figure 6 As shown, the NMR data are 1H NMR (400MHz, DMSO-d6): δ4.44 (s, 3H), 4.12 (dd, J = 19.8Hz, 3.4Hz, 2H), 3.79 (s, 1H),3.72-3.56(m,3H),3.12(s,9H),2.46-2.32(m,2H),2.32-2.08(m,4H),2.0 4-1.91(m,1H),1.88-1.59(m,6H),1.59-1.43(m,5H),1.43-1.32(m,7H),1.23( s,28H),1.20-1.12(m,1H),1.02-0.92(m,5H),0.89-0.75(m,6H),0.59(s,3H).

[0134] Physical property testing:

[0135] The particle size, PDI (dispersion index), and encapsulation efficiency of the mRNA-LNPs provided in Examples 6 and 7 were tested using the following methods:

[0136] Particle size and PDI: Mix 100 μL of mRNA-LNP sample with 900 μL of PBS and add it to the sample cell. Place the sample cell into the sample chamber of the Malvern Zetasizer Ultra nanoparticle size potentiometry instrument, select liposomes as the sample type, set the equilibration time to 30 seconds, and measure the particle size and PDI.

[0137] Encapsulation efficiency: The encapsulation efficiency of mRNA was determined using the Ribogreen fluorescence assay. Two mRNA-LNP samples were taken. One sample was diluted 50-fold with 1×TE buffer and then bound to Ribogreen fluorescent dye. The content of unencapsulated mRNA (F) was measured using a microplate reader (excitation wavelength 480 nm and emission wavelength 520 nm). free Another sample was demulsified with 2% Triton X-100 solution (polyethylene glycol octylphenyl ether), then diluted to 1 / 50 of the original mRNA concentration, bound to Ribogreen fluorescent dye, and the total mRNA concentration was determined using a microplate reader (excitation wavelength 480 nm and emission wavelength 520 nm). total According to the formula EE% = (F total -F free ) / F total Calculate the encapsulation efficiency of mRNA-LNP by multiplying by 100%.

[0138] The results of the above tests are shown in Table 1 below:

[0139] Table 1

[0140]

[0141] As shown in Table 1, the mRNA-LNPs prepared using compounds 1-5 and the commercial lipid ALC-0315 have particle sizes ranging from 50 to 145 nm and PDI ranging from 0.02 to 0.31, exhibiting small and uniform particle sizes. Furthermore, the encapsulation efficiency of the mRNA-LNPs prepared using compounds 1-5 is 93-98%, significantly higher than that of the commercial lipid ALC-0315, indicating that the cationic lipid compounds provided in this invention have better encapsulation effects.

[0142] Cell transfection efficiency test:

[0143] 293T cells were added to a 96-well plate at a cell density of 2×10⁻⁶. 4 Each well contains 100 μL of enzyme-linked immunosorbent assay (ELISA) substrate and is incubated overnight at 37°C with 5% CO2. Luciferase-mRNA-LNP prepared in Examples 6 and 7 are added to each well, maintaining an mRNA concentration of 50 ng / well. After 18 h of incubation, 100 μL of Luciferase fluorescent substrate is added to each well, and the reaction is allowed to proceed for 5 min. The fluorescence value of each well is detected using a fluorescence microplate reader. The results are shown below. Figure 7 As shown.

[0144] from Figure 7 It can be seen that the transfection efficiency of Luciferase-mRNA-LNP prepared using compounds 1, 2, and 5 is similar to that of the commercial lipid ALC-0315, while the transfection efficiency of Luciferase-mRNA-LNP prepared using compounds 3 and 4 is significantly higher than that of the commercial lipid ALC-0315. When structural lipids are used to replace DSPC, the transfection efficiency of Luciferase-mRNA-LNP is significantly improved in all cases.

[0145] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A cationic lipid compound, characterized in that, The cationic lipid compound has the structure shown in Formula I: In Formula I, R1 is selected from one of C1-C10 alkylene and C2-C10 alkenylene; R2 is CH and R3 is selected from One of them; Alternatively, R2 is N and R3 is selected from... One of them; R4 is selected from C10-C20 alkenyl groups. and One of them; Wherein, L1 is a single bond or a C1-C6 alkylene group, L2 is a single bond or a methylene group, L3 is a C1-C2 alkylene group, L4 is a C3-C12 alkylene group, and L5 is a C4-C15 alkyl group; -* represents the linking bond of a group.

2. The cationic lipid compound according to claim 1, characterized in that, The R1 is selected from one of the following groups: Here, -* represents the linking bond of a group.

3. The cationic lipid compound according to claim 1, characterized in that, R2 is CH and R3 is selected from one of the following groups: Alternatively, R2 is N and R3 is selected from one of the following groups: Here, -* represents the linking bond of a group.

4. The cationic lipid compound according to any one of claims 1-3, characterized in that, The R4 is selected from one of the following groups: Here, -* represents the linking bond of a group.

5. The cationic lipid compound according to any one of claims 1-3, characterized in that, The cationic lipid compound is selected from the following compounds 1 to 5:

6. A method for preparing a cationic lipid compound according to any one of claims 1-5, characterized in that, R2 is N, and the preparation method includes the following steps: compound Imidazole was added to an organic solvent, and tert-butyldiphenylchlorosilane was added dropwise to react and generate intermediate 1; intermediate 1 was dissolved in an organic solvent, and sodium hydride, sodium iodide and compound were added. The reaction proceeds to generate intermediate 2; intermediate 2 and lithium hydroxide are added to a mixture of tetrahydrofuran, methanol, and water to generate intermediate 3; intermediate 3 reacts with cholesterol under the catalysis of N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine to generate intermediate 4; intermediate 4 is dissolved in a mixture of dichloromethane and trifluoroacetic acid to generate intermediate 5; intermediate 5 reacts with compound Br-R4 in an organic solvent containing potassium carbonate and sodium iodide to generate intermediate 6; intermediate 6 and triethylamine hydrogen fluoride are added to an organic solvent, and the pH is adjusted to alkaline to generate the cationic lipid compound. Alternatively, R2 is CH, and the preparation method includes the following steps: compound Imidazole was added to an organic solvent, and tert-butyldiphenylchlorosilane was added dropwise to react and generate intermediate 7. Intermediate 7 was dissolved in an organic solvent, and pyridine chlorochromate was added to react and generate intermediate 8. Compound Br-L4-OBn was dissolved in an organic solvent, and molecular sieves, Mg, and iodine were added sequentially under nitrogen atmosphere to react and generate intermediate 9. Intermediate 8 was added to react and generate intermediate 10. Intermediate 10 reacted with compound R3-H to generate intermediate 11. Intermediate 11 was added to a mixture of tetrahydrofuran, methanol, and acetic acid, and palladium on carbon was added to react and generate intermediate 12 under hydrogen atmosphere. Intermediate 12 reacted with compound R3-H to generate intermediate 12. The intermediate 13 is generated by reacting 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine under the catalysis of these two compounds. Intermediate 13 and triethylamine hydrogen fluoride are added to an organic solvent, and the pH is adjusted to alkaline to generate intermediate 14. Intermediate 14 is dissolved in acetone, and Jones' reagent is added dropwise to generate intermediate 15. Intermediate 15 is then reacted with cholesterol under the catalysis of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine to generate the cationic lipid compound.

7. Use of a cationic lipid compound as described in any one of claims 1-5 for the preparation of a drug carrier.

8. The use according to claim 7, characterized in that, The drug carrier is a nucleic acid drug carrier.

9. A lipid nanoparticle, characterized in that, Includes the cationic lipid compounds as described in any one of claims 1-5.

10. The lipid nanoparticles according to claim 9, characterized in that, The lipid nanoparticles also include structural lipids, cholesterol, and polymer-modified lipids.

11. The lipid nanoparticles according to claim 10, characterized in that, The structural lipids are neutral phospholipids.

12. The lipid nanoparticles according to claim 11, characterized in that, The neutral phospholipid is selected from one or more of distearylphosphatidylcholine, dioleoylphosphatidylcholine, and distearylphosphatidylethanolamine.

13. The lipid nanoparticles according to claim 10, characterized in that, The polymer-modified lipid is a polyethylene glycol-modified lipid.

14. The lipid nanoparticles according to claim 10, characterized in that, The lipid nanoparticles comprise the following components in molar percentages: 30-50% cationic lipid compound as described in any one of claims 1-5, 5-25% structural lipid, 28.5-48.5% cholesterol, and 0.5-3% polyethylene glycol modified lipid.

15. A drug-loaded lipid nanoparticle, characterized in that, include: The lipid nanoparticles as described in any one of claims 9-14 and the nucleic acid drug loaded in the lipid nanoparticles.

16. A method for preparing drug-loaded lipid nanoparticles as described in claim 15, characterized in that, The preparation method includes the following steps: An oil phase containing the components of the lipid nanoparticles and an aqueous phase containing nucleic acid drugs were prepared separately. The oil phase and the aqueous phase were mixed using a microfluidic device to self-assemble the drug-loaded lipid nanoparticles.

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