Application of ionizable lipid compound in nucleic acid drug delivery system

Abstract

The invention provides an application of an ionizable lipid compound in a nucleic acid drug delivery system. According to the present invention, the types of the ionizable lipid compounds are enriched, the delivery carrier developed by using the ionizable lipid compounds has high nucleic acid molecule encapsulation efficiency, and the nucleic acid drug can be efficiently delivered and efficiently expressed in vivo so as to provide more choices for the delivery of the nucleic acid drug, such that the important significance is provided for the development and the application of the nucleic acid drug.

Description

Technical Field

[0001] The invention belongs to the technical field of nucleic acid drug delivery carriers, and particularly relates to application of an ionizable lipid compound in a nucleic acid drug delivery system. Background Art

[0002] As a new generation of biotechnology drugs, nucleic acids have broad application prospects in the medical field. However, nucleic acids have poor stability in vivo and in vitro and low delivery efficiency, which greatly limits their drugability. In recent years, lipid nanoparticles based on ionizable lipids have shown good clinical application potential and have been verified in vaccines. Lipid nanoparticles can show high delivery efficiency and good safety in vivo due to their unique structure and physicochemical properties, providing more possibilities for the clinical application of nucleic acid drugs in the future.

[0003] However, the development of ionizable lipid molecules is difficult, and there are still very few ionizable lipid molecules available for use. As the main component of the carrier for delivering nucleic acid molecules into the body, ionizable lipid molecules need to ensure high transfection efficiency and high expression rate of nucleic acid molecules after they reach the body. At the same time, they should also reduce toxicity in the body and avoid drug accumulation in the liver when necessary. Therefore, there is still a need to explore more lipid compounds suitable for the application of nucleic acid drugs, and further develop nucleic acid drug delivery carriers that truly take into account high transfection efficiency, high expression effect and low toxicity. Summary of the invention

[0004] The purpose of the present invention is to provide an ionizable lipid compound which has a simple preparation method, is easy to combine with nucleic acid and is easy to degrade.

[0005] Another object of the present invention is to provide a delivery vector that can efficiently deliver nucleic acid molecules in vivo.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] Ionizable lipid compounds represented by general formula (A), general formula (B), and general formula (C),

[0008]

[0009]

[0010] Wherein, in the general formula (A), D is O or N, G1 is a straight-chain alkylene group having 2 to 15 carbon atoms, R1 is a straight-chain alkyl group having 4 to 15 carbon atoms, a straight-chain alkenyl group having 12 to 20 carbon atoms and containing 1 to 3 carbon-carbon double bonds, or an alkenyl group having 8 to 15 carbon atoms and containing 1 to 3 carbon-carbon double bonds and 1 to 3 methyl branches,

[0011] In the general formula (B), G2 is a straight-chain alkylene group having 2 to 15 carbon atoms, R2 is a straight-chain alkyl group having 4 to 15 carbon atoms or an alkenyl group having 8 to 15 carbon atoms and containing 1 to 3 carbon-carbon double bonds and 1 to 3 methyl branches,

[0012] In the general formula (C), G3 is a straight-chain alkylene group having 2 to 15 carbon atoms, and R3 is an alkenyl group having 8 to 15 carbon atoms and containing 1 to 3 carbon-carbon double bonds and 1 to 3 methyl branches.

[0013] Preferably, the ionizable lipid compound is a compound represented by formula (I), formula (II), formula (III), formula (IV), formula (V), formula (VI), formula (VII), formula (VIII) and formula (IX):

[0014]

[0015]

[0016] a is an integer between 1 and 10;

[0017] b is an integer between 1 and 10;

[0018] c is an integer between 1 and 10;

[0019] d is an integer between 1 and 15;

[0020] g is an integer between 0 and 20;

[0021] p is an integer between 1 and 5;

[0022] e is an integer between 0 and 5;

[0023] h is an integer between 1 and 10;

[0024] i is an integer between 1 and 10;

[0025] f is an integer between 1 and 10;

[0026] j is an integer between 1 and 5;

[0027] m is an integer between 1 and 10.

[0028] According to some embodiments, the ionizable lipid compound is the following compound:

[0029]

[0030]

[0031] The present invention also provides a delivery vector, which comprises one or more of the above-mentioned ionizable lipid compounds.

[0032] Preferably, the delivery vector further comprises an auxiliary molecule.

[0033] More preferably, the molar ratio of the ionizable lipid compound to the auxiliary molecule is (0.1-1):(0.1-1), and more preferably (0.5-1):(0.5-1).

[0034] The auxiliary molecules can be auxiliary molecules commonly used in the art.

[0035] Preferably, the auxiliary molecules include one or more of artificially synthesized or naturally derived auxiliary lipids or lipid molecules, any species of animal origin, and any type of cells or vesicles (including exosomes) or their components, polypeptide molecules, polymer molecules, carbohydrate molecules or inorganic substances.

[0036] Further preferably, the auxiliary molecules include cholesterol, calcipotriol, stigmasterol, β-sitosterol, lupeol, betulin, ursolic acid, oleanolic acid, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, 1-stearoyl-2-oleoyl lecithin, dioleoylphosphatidylethanolamine, (1,2-dioleyloxypropyl)trimethylammonium chloride, didecyldimethylammonium bromide, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000, distearoylphosphatidylethanolamine-polyethylene glycol 2000, activated carbon, silicon dioxide and calcium phosphate.

[0037] According to some preferred embodiments, the delivery carrier includes the above-mentioned ionizable lipid compound, DSPC or DOPE, DMG-PEG2000 and cholesterol, and the molar ratio of the ionizable lipid compound, DSPC or DOPE, DMG-PEG2000 and cholesterol is 35-50:10-16:1.5-2.5:30-38.5.

[0038] Preferably, the ionizable lipid compound and / or the auxiliary molecule is modified with a targeting substance.

[0039] Still more preferably, the targeting substance includes one or more of folic acid, single-chain antibody or targeting polypeptide.

[0040] Preferably, the delivery vehicle is a nanolipid particle.

[0041] More preferably, the average size of the nanoparticle preparation is 50 nm to 200 nm.

[0042] Still more preferably, the average size of the nanoparticle preparation is 50 nm to 150 nm.

[0043] More preferably, the average size of the nanoparticle preparation is 50 nm to 100 nm.

[0044] The present invention also provides a nucleic acid pharmaceutical composition, which comprises the above-mentioned delivery vector and a nucleic acid molecule.

[0045] Preferably, the nucleic acid molecule is one or more of pDNA, siRNA, ASO or mRNA.

[0046] Preferably, the mass ratio of the nucleic acid molecule to the delivery vector is 1:(5-50); further preferably 1:(5-40), and further preferably 1:(5-30).

[0047] Preferably, the nucleic acid pharmaceutical composition further comprises a pharmaceutically acceptable additive, wherein the additive comprises one or more of an excipient, a stabilizer or a diluent.

[0048] Furthermore, the additives include but are not limited to sucrose, trehalose or other stabilizers.

[0049] Specifically, the additive is added in an amount of 1% to 20% of the total mass of the pharmaceutical composition.

[0050] Preferably, the nucleic acid pharmaceutical composition can be a lyophilized powder or an injection, and the injection is locally administered through microneedles, injection or perfusion into the muscle, subcutaneous tissue, endothelium, or tumor, or administered by intravenous injection.

[0051] Compared with the prior art, the present invention has the following advantages:

[0052] The present invention provides a new ionizable lipid compound, enriches the types of ionizable lipid compounds, and the delivery carrier formed by the compound has high encapsulation efficiency for nucleic acid molecules, and can efficiently deliver and express nucleic acid drugs in vivo, thereby providing more options for nucleic acid drug delivery, which is of great significance to the development and application of nucleic acid drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 is the mass spectrum of compound 1;

[0054] Figure 2 is the mass spectrum of compound 2;

[0055] Figure 3 is the mass spectrum of compound 3;

[0056] Figure 4is the mass spectrum of compound 4;

[0057] Figure 5 is the mass spectrum of compound 5;

[0058] Figure 6 is the mass spectrum of compound 6;

[0059] Figure 7 is the mass spectrum of compound 7;

[0060] Figure 8 is the mass spectrum of compound 8;

[0061] Fig. 9 is the mass spectrum of compound 9;

[0062] Fig.10 Shows the expression of luciferase in mice 4 h after liposome injection;

[0063] Fig.11 Shows the luciferase expression in mice 4 h after liposome injection;

[0064] Fig.12 The luciferase expression in various organs of mice 4 hours after liposome injection is shown. DETAILED DESCRIPTION

[0065] The present invention is further described below in conjunction with the examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0066] In order to reduce the production cost of nucleic acid drug delivery vectors and improve the in vivo delivery and expression effects of nucleic acid drugs, the inventors have conducted a lot of research and experimental verification and developed a new ionizable lipid compound, specifically an ionizable lipid compound represented by general formula (A), general formula (B), and general formula (C).

[0067]

[0068] Wherein, in the general formula (A), D is O or N, G1 is a straight-chain alkylene group having 2 to 15 carbon atoms, R1 is a straight-chain alkyl group having 4 to 15 carbon atoms, a straight-chain alkenyl group having 12 to 20 carbon atoms and containing 1 to 3 carbon-carbon double bonds, or an alkenyl group having 8 to 15 carbon atoms and containing 1 to 3 carbon-carbon double bonds and 1 to 3 methyl branches,

[0069] In the general formula (B), G2 is a straight-chain alkylene group having 2 to 15 carbon atoms, R2 is a straight-chain alkyl group having 4 to 15 carbon atoms or an alkenyl group having 8 to 15 carbon atoms and containing 1 to 3 carbon-carbon double bonds and 1 to 3 methyl branches,

[0070] In the general formula (C), G3 is a straight-chain alkylene group having 2 to 15 carbon atoms, and R3 is an alkenyl group having 8 to 15 carbon atoms and containing 1 to 3 carbon-carbon double bonds and 1 to 3 methyl branches.

[0071] The nucleic acid carriers composed of the above-mentioned ionizable lipid compounds and other auxiliary molecules have high encapsulation efficiency for nucleic acid molecules, and can efficiently deliver and express nucleic acid drugs in vivo. Some of them have achieved non-liver targeting and efficient delivery to spleen or lung organs. Due to the extremely low expression in the liver, the toxic side effects of the nucleic acid delivery system on the liver are greatly reduced. Some show obvious lung organ targeting, which will provide new nucleic acid delivery carriers for the treatment of lung diseases.

[0072] Among them, the non-liver-targeted ionizable lipid compounds include compounds represented by formula (I), formula (II), formula (IV), formula (VI), formula (VII), formula (VIII) and formula (IX)

[0073]

[0074]

[0075] a is an integer between 1 and 10; b is an integer between 1 and 10; g is an integer between 0 and 20; h is an integer between 1 and 10; i is an integer between 1 and 10; f is an integer between 1 and 10; j is an integer between 1 and 5; and m is an integer between 1 and 10.

[0076] The vector prepared from the above-mentioned ionizable lipid compound can reduce the toxic side effects caused by the accumulation of nucleic acid molecules in the liver organ, and can form a nucleic acid drug delivery vector that truly takes into account high transfection efficiency, high expression effect and low toxicity.

[0077] The technical scheme and technical effects of the present invention are further described below in conjunction with specific embodiments.

[0078] In the following examples, unless otherwise specified, conventional methods are used; the experimental materials used, unless otherwise specified, are purchased from conventional biochemical reagent manufacturers.

[0079] Example 1

[0080] Synthesis route of compound 1:

[0081]

[0082] Synthesis of compound 1-1:

[0083] In a 250 mL reaction bottle, 8-bromooctanoic acid (11.44 g, 51.25 mmol), 3,7-dimethyloct-6-octen-1-ol (7 g, 51.25 mmol), and 150 mL of dichloromethane were added in sequence. After stirring to dissolve, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (9.8 g, 51.25 mmol) and 4-dimethylaminopyridine (12.5 g, 122.5 mmol) were added. The mixture was reacted at room temperature for 48 h. After washing with saturated NaHCO3 solution 3 times and saturated NaCl solution 3 times, it was dried over anhydrous Na2SO4, the solvent was spin-dried, and purified using a silica gel chromatography column (petroleum ether: ethyl acetate = 100 / 1) to obtain compound 1-1 (5 g, yield: 40%).

[0084] Synthesis of compound 1:

[0085] Compound 1-1 (0.4 g, 2 mmol) and 1,4-bis(3-aminopropyl)piperazine (3.32 g, 10 mmol) were added to a reaction flask and dissolved in THF / CH3CN (1:1, 20 mL), and then DIPEA (1.67 g, 10 mmol) was added. The reactants were stirred at 63°C for 72 h. The reaction was completed by TLC detection. The crude product was washed three times with ethyl acetate and saturated NaHCO3 and saturated NaCl solutions. The combined organic layers were dried over anhydrous Na2SO4, and the crude product was purified by silica gel chromatography (dichloromethane / methanol = 80 / 1) to obtain yellow oily compound 1 (369 mg, yield: 14%). The mass spectrum of compound 1 is shown in Figure 1 .

[0086] Example 2

[0087] Synthesis route of compound 2:

[0088]

[0089] Synthesis of compound 2-1:

[0090] In a 500mL reaction bottle, 8-bromooctanoic acid (22.88g, 103mmol), 3,7-dimethyl-2,6-octadien-1-ol (15.78g, 103mmol), and 300mL of dichloromethane were added in sequence. After stirring and dissolving, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (19.6g, 103mmol) and 4-dimethylaminopyridine (25g, 245mmol) were added. The mixture was reacted at room temperature for 48h. The product was washed three times with ethyl acetate and saturated NaHCO3 and saturated NaCl solution, dried over anhydrous Na2SO4, and the solvent was spin-dried. It was purified by silica gel chromatography (petroleum ether: ethyl acetate = 100-200: 1) to obtain compound 2-1 (9g, yield: 38%).

[0091] Synthesis of compound 2:

[0092] 1,4-bis(3-aminopropyl)piperazine (0.8 g, 4 mmol) and compound 2-1 (7.16 g, 20 mmol) were added to a reaction flask and dissolved in THF / CH3CN (1:1, 40 mL), followed by the addition of DIPEA (3.33 g, 20 mmol). The reactants were stirred at 63°C for 72 h. The reaction was completed by TLC detection. The crude product was washed three times with ethyl acetate, saturated NaHCO3 and saturated NaCl. The combined organic layers were dried over anhydrous Na2SO4. The crude product was purified by silica gel chromatography (dichloromethane / methanol = 70 / 1) to obtain a yellow oily compound 2 (789 mg, yield: 15%). The mass spectrum of compound 2 is shown in Figure 2 .

[0093] Example 3

[0094] Synthesis route of compound 3:

[0095]

[0096] Synthesis of compound 3-1:

[0097] 8-Bromooctanol (11.2 g, 53.7 mmol) and undecanoic acid (10 g, 53.7 mmol) were dissolved in dichloromethane (100 mL), and EDC (10.3 g, 53.7 mmol) and DMAP (1.3 g, 10.74 mmol) were added. The mixture was stirred at room temperature for 48 h. The reaction was detected by TLC, and the crude product was washed 3 times with ethyl acetate and saturated NaHCO3 and saturated NaCl. The combined organic layers were dried over anhydrous Na2SO4, and the crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate = 100 / 1) to obtain a light yellow oily compound 3-1 (15.15 g, yield: 75%).

[0098] Synthesis of compound 3:

[0099] 1,4-bis(3-aminopropyl)piperazine (0.8 g, 4 mmol) and compound 3-1 (7.56 g, 20 mmol) were added to a reaction flask and dissolved in THF / CH3CN (1:1, 40 mL), followed by the addition of DIPEA (3.33 g, 20 mmol). The reactants were stirred at 63°C for 72 h. The reaction was completed by TLC detection. The crude product was washed three times with ethyl acetate, saturated NaHCO3 and saturated NaCl. The combined organic layers were dried over anhydrous Na2SO4. The crude product was purified by silica gel chromatography (dichloromethane / methanol = 70 / 1) to obtain a yellow oily compound 3 (831 mg, yield: 15%). The mass spectrum of compound 3 is shown in Figure 3 .

[0100] Example 4

[0101] Synthesis route of compound 4:

[0102]

[0103] Synthesis of compound 4-1:

[0104] n-Decylamine (9.4 g, 60 mmol) and triethylamine (7.8 g, 78 mmol) were added to a round-bottom flask containing 240 mL of dichloromethane, and 8 mL of acryloyl chloride was slowly added under ice bath conditions, and the reaction was carried out at 0 ° C for 24 h. After the reaction was completed, the dichloromethane was suspended to dryness, resuspended with ethyl acetate, filtered to remove the white solid, extracted three times with ethyl acetate and saturated NaCl, dried over anhydrous Na2SO4, and the organic phase was suspended to dryness to obtain a light yellow liquid. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate = 5 / 1) to obtain a colorless oily product 4-1 (4.0 g, yield: 32%).

[0105] Synthesis route of compound 4:

[0106] 1,4-bis(3-aminopropyl)piperazine (0.8 g, 4 mmol) and compound 4-1 (4.22 g, 20 mmol) were added to the reaction flask, and the reactants were stirred at 80°C for 48 h. The reaction was completed by TLC detection, and the crude product was washed three times with ethyl acetate and saturated NaHCO3 and saturated NaCl. The combined organic layers were dried over anhydrous Na2SO4, and the crude product was purified by silica gel chromatography (dichloromethane / methanol = 70 / 1) to obtain yellow oily compound 4 (626 mg, yield: 15%). The mass spectrum of compound 4 is shown in Figure 4 .

[0107] Example 5

[0108] Synthesis route of compound 5:

[0109]

[0110] Synthesis of compound 5-1:

[0111] Octadecenylamine (8.01 g, 30 mmol) and triethylamine (3.9 g, 39 mmol) were added to a round-bottomed flask containing 240 mL of dichloromethane, and 4 mL of acryloyl chloride was slowly added under ice bath conditions, and the reaction was continued at 0 ° C for 24 h. After the reaction was completed, the dichloromethane was suspended and resuspended with ethyl acetate, and the white solid was removed by suction filtration, and then extracted three times with ethyl acetate and saturated NaCl, and dried over anhydrous Na2SO4, and the organic phase was suspended to obtain a light yellow liquid. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate = 5 / 1) to obtain a colorless oily product 5-1 (3.0 g, yield: 32%).

[0112] Synthesis of compound 5:

[0113] 1,4-bis(3-aminopropyl)piperazine (0.4 g, 2 mmol) and compound 5-1 (3.85 g, 12 mmol) were added to the reaction flask, and the reactants were stirred at 80°C for 48 h. The reaction was completed by TLC detection, and the crude product was washed three times with ethyl acetate and saturated NaHCO3 and saturated NaCl. The combined organic layers were dried over anhydrous Na2SO4, and the crude product was purified by silica gel chromatography (dichloromethane / methanol = 60 / 1) to obtain yellow oily compound 5 (446 mg, yield: 15%). The mass spectrum of compound 5 is shown in Figure 5 .

[0114] Example 6

[0115] Synthesis route of compound 6:

[0116]

[0117] Synthesis of compound 6-1:

[0118] 8-Bromooctanoic acid (11.97 g, 53.7 mmol) and undecanol (9.23 g, 53.7 mmol) were dissolved in dichloromethane (200 mL), and EDC (10.3 g, 53.7 mmol) and DMAP (1.3 g, 10.74 mmol) were added. The mixture was stirred at room temperature for 48 h. TLC detected that the reaction was complete, and the crude product was washed 3 times with ethyl acetate and saturated NaHCO3 and saturated NaCl. The combined organic layers were dried over anhydrous Na2SO4, and the crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate = 100 / 1) to obtain a light yellow oily compound 6-1 (14.15 g, 72%).

[0119] Synthesis of compound 6:

[0120] 1,4-bis(3-aminopropyl)piperazine (0.8 g, 4 mmol) and compound 6-1 (9 g, 24 mmol) were added to a reaction flask and dissolved in THF / CH3CN (1:1, 30 mL), followed by the addition of DIPEA (4 g, 24 mmol). The reactants were stirred at 63°C for 72 h. The reaction was completed by TLC detection. The crude product was washed three times with ethyl acetate and saturated NaHCO3 and saturated NaCl. The combined organic layers were dried over anhydrous Na2SO4. The crude product was purified by silica gel chromatography (dichloromethane / methanol = 70 / 1) to obtain a yellow oily compound 6 (887 mg, yield: 16%). The mass spectrum of compound 6 is shown in Figure 6 .

[0121] Example 7

[0122] Synthesis route of compound 7:

[0123]

[0124] Synthesis of compound 7-1:

[0125] 8-Bromooctanol (12.28 g, 58.72 mmol) and 3,7-dimethyl-6-octenoic acid (10 g, 58.73 mmol) were dissolved in dichloromethane (140 mL), and EDC (11.22 g, 58.73 mmol) and DMAP (1.4 g, 11.75 mmol) were added. The mixture was stirred at room temperature for 48 h. TLC detected that the reaction was complete, and the crude product was washed three times with ethyl acetate and saturated NaHCO3 and saturated NaCl. The combined organic layers were dried over anhydrous Na2SO4, and the crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate = 100 / 1) to obtain a light yellow oily compound 7-1 (15.9 g, yield: 75%).

[0126] Synthesis of compound 7:

[0127] 1,4-bis(3-aminopropyl)piperazine (0.8 g, 4 mmol) and compound 7-1 (8.64 g, 24 mmol) were added to a reaction flask and dissolved in THF / CH3CN (1:1, 40 mL), followed by the addition of DIPEA (4 g, 24 mmol). The reactants were stirred at 63°C for 72 h. The reaction was completed by TLC detection, and the crude product was washed with ethyl acetate, saturated NaHCO3 and brine. The combined organic layers were dried over anhydrous Na2SO4, and the crude product was purified by silica gel chromatography (dichloromethane / methanol = 60 / 1) to obtain a yellow oily compound 7 (825 mg, yield: 16%). The mass spectrum of compound 7 is shown in Figure 7 .

[0128] Example 8

[0129] Synthesis route of compound 8:

[0130]

[0131] Synthesis of compound 8-1:

[0132] Linolenic alcohol (8.01 g, 30 mmol) and triethylamine (3.99 g, 39 mmol) were added to the reaction bottle in an ice-water bath, and dichloromethane (180 mL) was added. Acryloyl chloride (3.3 g, 39 mmol) was dissolved in dichloromethane (66 mL) and slowly added dropwise to the reaction bottle. The reaction lasted for 10 minutes. The reaction was kept below 10°C. Finally, the ice bath was removed and the reaction solution was reacted at room temperature for 2 hours. Washed with saturated NaCl to obtain a crude product, which was purified by chromatography (silica gel column, eluent was petroleum ether containing 0.5% EA (volume percentage)), and the pure product was evaporated to obtain a light yellow oily compound 8-1 (5.19 g, yield: 50%)

[0133] Synthesis of compound 8:

[0134] 1,4-bis(3-aminopropyl)piperazine (0.4 g, 2 mmol) and compound 8-1 (3.84 g, 12 mmol) were added to the reaction flask, and the reactants were stirred at 80°C for 48 h. The reaction was completed by TLC detection, and the crude product was washed three times with ethyl acetate and saturated NaHCO3 and saturated NaCl. The combined organic layers were dried over anhydrous Na2SO4, and the crude product was purified by silica gel chromatography (dichloromethane / methanol = 60 / 1) to obtain yellow oily compound 8 (446 mg, yield: 18%). The mass spectrum of compound 8 is shown in Figure 8 .

[0135] Example 9

[0136] Synthesis route of compound 9:

[0137]

[0138] Synthesis of compound 9-1:

[0139] 8-Bromooctanoic acid (11.2 g, 50 mmol) and (E)-3,7-dimethyl-2,6-octadiene-1-ol (11 g, 50 mmol) were dissolved in dichloromethane (140 mL), and EDC (9.55 g, 50 mmol) and DMAP (1.22 g, 10 mmol) were added. The mixture was stirred at room temperature for 48 h. TLC detected that the reaction was complete, and the crude product was washed 3 times with ethyl acetate and saturated NaHCO3 and saturated NaCl. The combined organic layers were dried over anhydrous Na2SO4, and the crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate = 100 / 1) to obtain a light yellow oily compound 9-1 (14.0 g, yield: 78%).

[0140] Synthesis of compound 9:

[0141] 2-Hydroxy-N,N-bis(2-aminoethyl)ethylamine (0.4 g, 2.7 mmol) and compound 9-1 (4.9 g, 13.5 mmol) were added to a reaction flask and dissolved in THF / CH3CN (1:1, 30 mL), and then DIPEA (1.74 g, 13.5 mmol) was added. The reactants were stirred at 63°C for 72 h. The reaction was completed by TLC detection. The crude product was washed three times with ethyl acetate and saturated NaHCO3 and saturated NaCl. The combined organic layers were dried over anhydrous Na2SO4, and the crude product was purified by silica gel chromatography (dichloromethane / methanol = 70 / 1) to obtain a yellow oily compound 9 (510 mg, yield: 15%). The mass spectrum of compound 9 is shown in Fig. 9 .

[0142] Embodiment 10:

[0143] Preparation of liposomes, determination of particle size, potential and encapsulation efficiency.

[0144] (1) According to the molar ratio of ionizable compound: DSPC: DMG-PEG2000: cholesterol of 50:10:1.5:38.5, anhydrous ethanol was used as solvent to prepare a liposome solution, and the sum of the concentrations of each component was controlled to be 50 mM. After dissolving and mixing, the solution was stored at -20°C for future use.

[0145] (2) mRNA was dissolved in 25 mM sodium acetate buffer at a pH of about 5.2 to prepare a nucleic acid preparation with a final concentration of about 0.17 mg / mL.

[0146] (3) The liposome solution and the nucleic acid preparation are mixed at a volume ratio of about 15:1 and a total rate of 12 mL / min through the Nano Assemblr microfluidic system or by vortexing to form uniform and stable liposome nanoparticles. After the liposomes are formed, the liposome environment is quickly changed from pH 4.0 to 7.2-7.4. The specific operation is to dilute it 20 times with a PBS buffer with a pH of 7.2 or a sodium acetate buffer with a pH of 7.4, and then concentrate it using a 50KD ultrafiltration tube. The speed of the centrifuge should not exceed the maximum speed limit of the ultrafiltration tube. After 4-5 changes of liquid, the pH of the liposome solution environment is about 7.2-7.4. It is concentrated to a final concentration of about 200 mM and placed at 4°C for use.

[0147] The particle size and PDI of liposomes were detected by Zetasizer Nano ZS (Malvern, Worcestershire, UK). The particle size was measured by diluting the liposome solution 50 times with 1×PBS, and the Zeta potential was measured by diluting the liposomes into 15mM pure water. The encapsulation efficiency was determined using the Quant-It RiboGreenRNA quantitative detection kit on the Modulus microporous multifunctional detector. The results of the particle size, PDI, encapsulation efficiency and potential are shown in Table 1.

[0148] Table 1. Physical parameters of representative LNP-mRNA nanolipid particles

[0149] Lipid Nanoparticles (LNP Formulations) Particle size (nm) Encapsulation rate (%) Potential (mV) LNP-1 (containing compound 1) 79.1 96 -2.3 LNP-2 (containing compound 2) 83.3 97 3.4 LNP-3 (containing compound 3) 89.4 92 4.5 LNP-4 (containing compound 4) 94.4 95 -5.1 LNP-5 (containing compound 5) 95.6 96 -6.2 LNP-6 (containing compound 6) 89.1 90 2.1 LNP-7 (containing compound 7) 84.1 91 7.9 LNP-8 (containing compound 8) 79.2 92 -3.4 LNP-9 (containing compound 9) 80.3 93 -7.8

[0150] Table 1 shows that the average particle size of the assembled LNPs is between 80 and 100 nm, the encapsulation efficiency is above 90%, the potentials of LNP-2, LNP-3, LNP-6 and LNP-7 are positively charged, and the potentials of LNP-1, LNP-4, LNP-5, LNP-8 and LNP-9 are negatively charged.

[0151] Example 11: Liposome transfection experiment in animals:

[0152] Nanolipid particles were prepared using mRNA expressing Luciferase fluorescent protein according to the preparation method of Example 4, wherein the amount of mRNA was 20 μg, the total amount of ionizable liposome compound, DSPC, DMG-PEG2000 and cholesterol was 200 μg, and 200 μL of neutral PBS buffer was used to quickly convert the liposome environment.

[0153] The prepared nanolipid particles were rapidly injected intramuscularly into the medial hind limb muscles of 6-8 week old female Babl / c mice, and 30 μg of mRNA was injected into the left and right hind limbs respectively. At different time periods after injection, the expression of luciferase in the injected mice was observed by small animal imaging.

[0154] The prepared nanolipid particles were rapidly injected (IV) into 6-8 week old female Babl / c mice via the tail vein, with an mRNA injection of 30 μg. At different time periods after injection, the expression of luciferase in the injected mice was observed by a small animal imaging device. After 4 hours, the heart, liver, spleen, lung, and kidney of the mice were subjected to fluorescence imaging.

[0155] The fluorescence expression intensity of each liposome 4 hours after intravenous injection is as follows Fig.10 and Fig.11 As shown in the figure, the fluorescence intensity in the circle is integrated to obtain the fluorescence intensity value, which is used to quantitatively characterize the expression of luciferase and draw a bar graph. The fluorescence intensity in the figure is closely related to the delivery efficiency of the liposome. The stronger the fluorescence intensity, the higher the delivery efficiency of the liposome. Among them, LNP-5 has the highest fluorescence expression intensity, reaching 1.3×10 11 , followed by LNP-3, whose fluorescence expression intensity was 2.5×10 10 The fluorescence intensity of other liposomes was 10 8 ~10 9 about.

[0156] After intravenous injection of each LNP, the fluorescence expression in various organs of mice is shown in Fig.12 The results showed that LNP-1, LNP-2 and LNP-6 showed obvious lung organ targeting; LNP-3 showed significant liver organ targeting; LNP-5 and LNP-8 were mainly liver targeting, but there was fluorescence expression in the spleen and lungs; LNP-4, LNP-7 and LNP-9 showed significant spleen targeting.

[0157] The above detailed description of the present invention is intended to enable persons familiar with the art to understand the contents of the present invention and implement them. It does not limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. An ionizable lipid compound represented by general formula (A), general formula (B) or general formula (C), in, In the general formula (A), D is O or N, G1 is a straight-chain alkylene group having 2 to 15 carbon atoms, R1 is a straight-chain alkyl group having 4 to 15 carbon atoms, a straight-chain alkenyl group having 12 to 20 carbon atoms and containing 1 to 3 carbon-carbon double bonds, or an alkenyl group having 8 to 15 carbon atoms and containing 1 to 3 carbon-carbon double bonds and 1 to 3 methyl branches, In the general formula (B), G2 is a straight-chain alkylene group having 2 to 15 carbon atoms, R2 is a straight-chain alkyl group having 4 to 15 carbon atoms or an alkenyl group having 8 to 15 carbon atoms and containing 1 to 3 carbon-carbon double bonds and 1 to 3 methyl branches, In the general formula (C), G3 is a straight-chain alkylene group having 2 to 15 carbon atoms, and R3 is an alkenyl group having 8 to 15 carbon atoms and containing 1 to 3 carbon-carbon double bonds and 1 to 3 methyl branches.

2. The ionizable lipid compound according to claim 1, characterized in that The ionizable lipid compound is one or more of the compounds represented by general formula (I), general formula (II), general formula (III), general formula (IV), general formula (V), general formula (VI), general formula (VII), general formula (VIII) and general formula (IX): a is an integer between 1 and 10; b is an integer between 1 and 10; c is an integer between 1 and 10; d is an integer between 1 and 15; g is an integer between 0 and 20; p is an integer between 1 and 5; e is an integer between 0 and 5; h is an integer between 1 and 10; i is an integer between 1 and 10; f is an integer between 1 and 10; j is an integer between 1 and 5; m is an integer between 1 and 10.

3. The ionizable lipid compound according to claim 1, characterized in that The ionizable lipid compound is one or more of the following compounds:

4. A delivery vector, characterized in that The delivery vehicle comprises one or more of the ionizable lipid compounds described in any one of claims 1 to 3.

5. The delivery vector according to claim 4, characterized in that The delivery carrier also includes auxiliary molecules, and the molar ratio of the ionizable lipid compound to the auxiliary molecules is (0.1-1): (0.1-1); the auxiliary molecules include one or more of artificially synthesized or naturally derived auxiliary lipid / lipid molecules, any species of animal origin and any type of cells or vesicles or their components, polypeptide molecules, polymer molecules, carbohydrate molecules or inorganic substances.

6. The delivery vector according to claim 5, characterized in that The auxiliary molecules include one or more of cholesterol, calcipotriol, stigmasterol, lupeol, β-sitosterol, betulin, ursolic acid, oleanolic acid, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, 1-stearoyl-2-oleoyl lecithin, dioleoylphosphatidylethanolamine, (1,2-dioleyloxypropyl)trimethylammonium chloride, didecyldimethylammonium bromide, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000, distearoylphosphatidylethanolamine-polyethylene glycol 2000, activated carbon, silicon dioxide and calcium phosphate.

7. The delivery vector according to claim 4, characterized in that The delivery carrier is a nano lipid particle, and the particle size of the nano lipid particle is 50nm to 200nm.

8. A nucleic acid pharmaceutical composition, characterized in that: The nucleic acid pharmaceutical composition comprises the delivery vector and a nucleic acid molecule according to any one of claims 4 to 7.

9. The nucleic acid pharmaceutical composition according to claim 8, characterized in that: The nucleic acid molecule includes one or more of plasmid DNA, siRNA, ASO or mRNA; and / or, the mass ratio of the nucleic acid molecule to the delivery vector is 1:(5-50); And / or, the nucleic acid pharmaceutical composition further comprises a pharmaceutically acceptable additive, wherein the additive comprises one or more of an excipient, a stabilizer or a diluent.

10. The nucleic acid pharmaceutical composition according to claim 8, characterized in that: The nucleic acid pharmaceutical composition is a lyophilized powder or an injection, which is administered locally through microneedles, injection or perfusion into the muscle, subcutaneous tissue, endothelium or tumor, or administered by intravenous injection.

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