CN119638646A

CN119638646A - Application of an ionizable lipid compound in a nucleic acid drug delivery system

Info

Publication number: CN119638646A
Application number: CN202311205960.7A
Authority: CN
Inventors: 崔艳芳; 吉帅洁
Original assignee: Central China Normal University
Current assignee: Central China Normal University
Priority date: 2023-09-18
Filing date: 2023-09-18
Publication date: 2025-03-18
Also published as: WO2025061211A1

Abstract

The present invention provides the use of an ionizable lipid compound in a nucleic acid drug delivery system. The invention enriches the types of the ionizable lipid compounds, and the delivery carrier developed by the invention has high encapsulation efficiency on nucleic acid molecules, and can efficiently deliver and express the nucleic acid drugs in vivo, thereby providing more choices for the delivery of the nucleic acid drugs and having important significance for the development and application of the nucleic acid drugs.

Description

Application of ionizable lipid compound in nucleic acid drug delivery system

Technical Field

The invention belongs to the technical field of nucleic acid drug delivery vectors, and particularly relates to application of an ionizable lipid compound in a nucleic acid drug delivery system.

Background

The nucleic acid as a new generation biotechnology medicine has wide application prospect in the medical field. However, nucleic acids have poor in vivo and in vitro stability, low delivery efficiency, and greatly limit their drug formation. In recent years, lipid nanoparticles based on ionizable lipids have demonstrated good clinical application potential and have been validated in vaccines. The lipid nanoparticle can show higher delivery efficiency and better safety in vivo by virtue of the unique structural and physicochemical properties of the lipid nanoparticle, and provides more possibility for clinical application of future nucleic acid medicaments.

However, the development difficulty of the ionizable lipid molecules is high, the number of the ionizable lipid molecules available at present is still small, and as a main component of a vector for delivering nucleic acid molecules into the body, the ionizable lipid molecules are required to ensure high transfection efficiency, high expression rate of the nucleic acid molecules after reaching the body, and reduce toxicity in the body, and avoid accumulation of drugs in the liver if necessary. Thus, there is still a need to explore more lipid compounds suitable for nucleic acid drug applications and to further develop nucleic acid drug delivery vehicles truly compromising high transfection efficiency, high expression effect and low toxicity.

Disclosure of Invention

The invention aims to provide an ionizable lipid compound which is simple in preparation method, easy to combine with nucleic acid and easy to degrade.

It is another object of the present invention to provide a delivery vehicle that is capable of delivering nucleic acid molecules in vivo with high efficiency.

In order to solve the technical problems, the invention adopts the following technical scheme:

An ionizable lipid compound represented by the general formula (A), the general formula (B) and the general formula (C),

Wherein in the general formula (A), D is O or N, G ₁ is a linear alkylene group having 2 to 15 carbon atoms, R ₁ is a linear alkyl group having 4 to 15 carbon atoms, a linear alkenyl group having 12 to 20 carbon atoms and having 1 to 3 carbon-carbon double bonds, or an alkenyl group having 8 to 15 carbon atoms and having 1 to 3 carbon-carbon double bonds and 1 to 3 methyl branches,

In the general formula (B), G ₂ is a linear alkylene group with 2-15 carbon atoms, R ₂ is a linear alkyl group with 4-15 carbon atoms or an alkenyl group with 8-15 carbon atoms and containing 1-3 carbon-carbon double bonds and 1-3 methyl branches,

In the general formula (C), G ₃ is a linear alkylene group having 2 to 15 carbon atoms, and R ₃ 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.

Preferably, the ionizable lipid compound is a compound 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 of 1 to 10;

b is an integer of 1 to 10;

c is an integer between 1 and 10;

d is an integer of 1-15;

g is an integer between 0 and 20;

p is an integer of 1 to 5;

e is an integer between 0 and 5;

h is an integer of 1 to 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 of 1 to 10.

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

The invention also provides a delivery vehicle comprising one or more of the above-described ionizable lipid compounds.

Preferably, the delivery vehicle further comprises an auxiliary molecule.

Further preferably, the charging molar ratio of the ionizable lipid compound to the auxiliary molecule is (0.1-1): (0.1-1), and further preferably (0.5-1): (0.5-1).

The auxiliary molecule may be an auxiliary molecule commonly used in the art.

Preferably, the accessory molecules include one or more of an accessory lipid or lipid molecule of synthetic or natural origin, an animal source of any species, and any kind of cell or vesicle (including exosomes) or component parts thereof, a polypeptide molecule, a polymer molecule, a carbohydrate molecule, or an inorganic substance.

Further preferred, the auxiliary molecule comprises one or more of cholesterol, calcipotriol, stigmasterol, beta-sitosterol, lupeol, betulin, ursolic acid, oleanolic acid, dioleoyl phosphatidylcholine, distearoyl phosphatidylcholine, 1-stearoyl-2-oleoyl lecithin, dioleoyl phosphatidylethanolamine, (1, 2-dioleoxypropyl) trimethylammonium chloride, didecyl dimethylammonium bromide, 1, 2-dimyristoyl-sn-glycero-3-ethyl phosphorylcholine, dipalmitoyl phosphatidylethanolamine-methoxypolyethylene glycol 5000, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, activated carbon, silica, and calcium phosphate.

According to some preferred embodiments, the delivery vehicle comprises the above mentioned ionizable lipid compound, DSPC or DOPE, DMG-PEG2000 and cholesterol in a molar ratio of 35-50:10-16:1.5-2.5:30-38.5.

Preferably, the ionizable lipid compound and/or the helper molecule is modified with a targeting agent.

Still further preferably, the targeting agent comprises one or more of folic acid, a single chain antibody, or a targeting polypeptide.

Preferably, the delivery vehicle is a nanolipid particle.

Further preferably, the average size of the nanoparticle preparation is 50nm to 200nm.

Still further preferably, the nanoparticle formulation has an average size of 50nm to 150nm.

Still more preferably, the nanoparticle formulation has an average size of 50nm to 100nm.

The invention also provides a nucleic acid pharmaceutical composition comprising the delivery vehicle and a nucleic acid molecule.

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

Preferably, the mass ratio of the nucleic acid molecule to the delivery vehicle is 1 (5-50), more preferably 1 (5-40), still more preferably 1 (5-30).

Preferably, the nucleic acid pharmaceutical composition further comprises pharmaceutically acceptable additives, including one or more of excipients, stabilizers or diluents.

Further, the additives include, but are not limited to, sucrose, trehalose, or other stabilizers.

Specifically, the additive is added in an amount of 1% -20% of the total mass of the pharmaceutical composition.

Preferably, the nucleic acid pharmaceutical composition can be lyophilized powder or an injection, and the injection is locally administered by muscle, subcutaneous, endothelial, intratumoral administration by micro needle, injection or infusion, or by intravenous injection.

Compared with the prior art, the invention has the following advantages:

The invention provides a new ionizable lipid compound, enriches the types of the ionizable lipid compound, forms a delivery carrier with high encapsulation efficiency on nucleic acid molecules, and can efficiently deliver and express nucleic acid drugs in vivo, thereby providing more choices for nucleic acid drug delivery and having important significance for development and application of the nucleic acid drugs.

Drawings

FIG. 1 is a mass spectrum of Compound 1;

FIG. 2 is a mass spectrum of Compound 2;

FIG. 3 is a mass spectrum of Compound 3;

FIG. 4 is a mass spectrum of Compound 4;

FIG. 5 is a mass spectrum of Compound 5;

FIG. 6 is a mass spectrum of Compound 6;

FIG. 7 is a mass spectrum of Compound 7;

FIG. 8 is a mass spectrum of Compound 8;

FIG. 9 is a mass spectrum of Compound 9;

FIG. 10 shows luciferase expression in mice 4h after liposome injection;

FIG. 11 shows luciferase expression levels in mice 4h after liposome injection;

FIG. 12 shows luciferase expression in various organs of mice 4h after liposome injection.

Detailed Description

The invention is further described below with reference to examples. The present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions which are not noted are conventional conditions in the industry. The technical features of the various embodiments of the present invention may be combined with each other as long as they do not collide with each other.

In order to reduce the production cost of nucleic acid drug delivery vehicles and to improve the in vivo delivery and expression effects of nucleic acid drugs, the inventors have conducted a great deal of research and experimental verification to develop a novel ionizable lipid compound, specifically an ionizable lipid compound represented by general formula (A), general formula (B), general formula (C),

The nucleic acid vector composed of the ionizable lipid compound and other auxiliary molecules has high encapsulation efficiency on nucleic acid molecules, can efficiently deliver and express nucleic acid drugs in vivo, and can realize non-liver targeting and efficient delivery on spleen or lung organs, and the toxic and side effects of a nucleic acid delivery system on the liver are greatly reduced due to extremely low expression at the liver part. Some exhibit pronounced lung organ targeting, which would provide a new nucleic acid delivery vehicle for the treatment of diseases of lung organs.

Wherein the non-liver targeted ionizable lipid compound comprises compounds represented by general formula (I), general formula (II), general formula (IV), general formula (VI), general formula (VII), general formula (VIII) and general formula (IX)

A is an integer of 1 to 10, b is an integer of 1 to 10, g is an integer of 0 to 20, h is an integer of 1 to 10, i is an integer of 1 to 10, f is an integer of 1 to 10, j is an integer of 1 to 5, and m is an integer of 1 to 10.

The carrier prepared by the ionizable lipid compound can reduce toxic and side effects generated by the expression of nucleic acid molecules accumulated in liver organs, and can form a nucleic acid drug delivery carrier really taking high transfection efficiency, high expression effect and low toxicity into consideration.

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

In the following examples, the experimental materials used were purchased from conventional biochemical reagent manufacturers, unless otherwise specified.

Example 1

Synthetic route for compound 1:

synthesis of Compound 1-1:

8-bromooctanoic acid (11.44 g,51.25 mmol), 3, 7-dimethyloct-6-octen-1-ol (7 g,51.25 mmol) and 150mL of methylene chloride were sequentially added to a 250mL reaction flask, and after stirring and dissolution, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (9.8 g,51.25 mmol) and 4-dimethylaminopyridine (12.5 g,122.5 mmol) were added, reacted at room temperature for 48h, saturated NaHCO ₃ solution was washed with water 3 times, saturated NaCl solution was washed with water 3 times, dried with anhydrous Na ₂SO₄, and after spin-drying the solvent, compound 1-1 (5 g, yield: 40%) was obtained by purification with a silica gel column (petroleum ether: ethyl acetate=100/1).

Synthesis of Compound 1:

Compound 1-1 (0.4 g,2 mmol) and 1, 4-bis (3-aminopropyl) piperazine (3.32 g,10 mmol) were added to the reaction flask and dissolved in THF/CH ₃ CN (1:1, 20 mL) followed by DIPEA (1.67 g,10 mmol). The reaction was stirred at 63 ℃ for 72h. TLC detects the end of the reaction, the crude product was washed 3 times with ethyl acetate and saturated NaHCO ₃ and saturated NaCl solution, the combined organic layers were dried over anhydrous Na ₂SO₄ and the crude product was purified by silica gel chromatography (dichloromethane/methanol=80/1) to give compound 1 as a yellow oil (369 mg, yield: 14%). Mass spectra of compound 1 are shown in figure 1.

Example 2

Synthetic route for compound 2:

synthesis of Compound 2-1:

8-bromooctanoic acid (22.88 g,103 mmol), 3, 7-dimethyl-2, 6-octadien-1-ol (15.78 g,103 mmol) and 300mL of methylene chloride were sequentially added into a 500mL reaction flask, and after stirring and dissolution, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (19.6 g,103 mmol) and 4-dimethylaminopyridine (25 g, 248 mmol) were added thereto, and the reaction was carried out at room temperature for 48 hours, and the product was washed with ethyl acetate and saturated NaHCO ₃ and saturated NaCl solution for 3 times, dried over anhydrous Na ₂SO₄, and purified by silica gel chromatography (petroleum ether: ethyl acetate=100 to 200:1) to give compound 2-1 (9 g, yield: 38%).

Synthesis of Compound 2:

1, 4-bis (3-aminopropyl) piperazine (0.8 g,4 mmol) and compound 2-1 (7.16 g,20 mmol) were added to the reaction flask and dissolved in THF/CH ₃ CN (1:1, 40 mL) followed by DIPEA (3.33 g,20 mmol). The reaction was stirred at 63 ℃ for 72h. TLC detects the end of the reaction, the crude product was washed 3 times with ethyl acetate and saturated NaHCO ₃ and saturated NaCl, the combined organic layers were dried over anhydrous Na ₂SO₄ and the crude product was purified by silica gel chromatography (dichloromethane/methanol=70/1) to give compound 2 as a yellow oil (789 mg, yield: 15%). Mass spectra of compound 2 are shown in figure 2.

Example 3

Synthetic route for compound 3:

synthesis of Compound 3-1:

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), DMAP (1.3 g,10.74 mmol) was added. The mixture was stirred at ambient temperature for 48h. TLC detects the end of the reaction and the crude product is washed 3 times with ethyl acetate and saturated NaHCO ₃ and saturated NaCl. The combined organic layers were dried over anhydrous Na ₂SO₄, and the crude product was purified by silica gel chromatography (petroleum ether/ethyl acetate=100/1) to give compound 3-1 (15.15 g, yield: 75%) as a pale yellow oil.

Synthesis of Compound 3:

1, 4-bis (3-aminopropyl) piperazine (0.8 g,4 mmol) and compound 3-1 (7.56 g,20 mmol) were added to the reaction flask and dissolved in THF/CH ₃ CN (1:1, 40 mL) followed by DIPEA (3.33 g,20 mmol). The reaction was stirred at 63 ℃ for 72h. TLC checked the end of the reaction, the crude product was washed 3 times with ethyl acetate and saturated NaHCO ₃ and saturated NaCl, the combined organic layers were dried over anhydrous Na ₂SO₄ and the crude product was purified by silica gel chromatography (dichloromethane/methanol=70/1) to give compound 3 as a yellow oil (831 mg, yield: 15%). Mass spectra of compound 3 are shown in figure 3.

Example 4

Synthetic route for compound 4:

synthesis of Compound 4-1:

N-decylamine (9.4 g,60 mmol) and triethylamine (7.8 g,78 mmol) were added to a round bottom flask containing 240mL of dichloromethane, 8mL of acryloyl chloride was slowly added under ice-bath conditions and reacted at 0℃for 24h. After the reaction, dichloromethane was suspended, the white solid was removed by suction filtration, and the organic phase was suspended after three times of extraction with ethyl acetate and saturated NaCl and drying over anhydrous Na ₂SO₄ to give a pale yellow liquid. The crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate=5/1) to give 4-1 (4.0 g, yield: 32%) as a colorless oily product.

Synthetic route for compound 4:

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 reaction stirred at 80℃for 48h. TLC checked the end of the reaction, the crude product was washed 3 times with ethyl acetate and saturated NaHCO ₃ and saturated NaCl, the combined organic layers were dried over anhydrous Na ₂SO₄ and the crude product was purified by silica gel chromatography (dichloromethane/methanol=70/1) to give compound 4 as a yellow oil (626 mg, yield: 15%). Mass spectra of compound 4 are shown in figure 4.

Example 5

Synthetic route for compound 5:

Synthesis of Compound 5-1:

Octadecylamine (8.01 g,30 mmol) and triethylamine (3.9 g,39 mmol) were added to a round bottom flask containing 240mL of methylene chloride, 4mL of acryloyl chloride was slowly added under ice bath conditions and reacted at 0℃for 24h. After the reaction, dichloromethane was suspended, the white solid was removed by suction filtration, and the organic phase was suspended after three times of extraction with ethyl acetate and saturated NaCl and drying over anhydrous Na ₂SO₄ to give a pale yellow liquid. The crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate=5/1) to give 5-1 (3.0 g, yield: 32%) as a colorless oily product.

Synthesis of Compound 5:

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 reaction stirred at 80℃for 48h. TLC checked the end of the reaction, the crude product was washed 3 times with ethyl acetate and saturated NaHCO ₃ and saturated NaCl, the combined organic layers were dried over anhydrous Na ₂SO₄ and the crude product was purified by silica gel chromatography (dichloromethane/methanol=60/1) to give compound 5 as a yellow oil (446 mg, yield: 15%). Mass spectra of compound 5 are shown in figure 5.

Example 6

Synthetic route to compound 6:

synthesis of Compound 6-1:

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), DMAP (1.3 g,10.74 mmol) was added. The mixture was stirred at ambient temperature for 48h. TLC detects the end of the reaction and the crude product is washed 3 times with ethyl acetate and saturated NaHCO ₃ and saturated NaCl. The combined organic layers were dried over anhydrous Na ₂SO₄ and the crude product was purified by silica gel chromatography (petroleum ether/ethyl acetate=100/1) to give compound 6-1 (14.15 g, 72%) as a pale yellow oil.

Synthesis of Compound 6:

1, 4-bis (3-aminopropyl) piperazine (0.8 g,4 mmol) and compound 6-1 (9 g,24 mmol) were added to the reaction flask and dissolved in THF/CH ₃ CN (1:1, 30 mL) followed by DIPEA (4 g,24 mmol). The reaction was stirred at 63 ℃ for 72h. TLC checked the end of the reaction, the crude product was washed 3 times with ethyl acetate and saturated NaHCO ₃ and saturated NaCl, the combined organic layers were dried over anhydrous Na ₂SO₄ and the crude product was purified by silica gel chromatography (dichloromethane/methanol=70/1) to give compound 6 as a yellow oil (887 mg, yield: 16%). Mass spectrum of compound 6 is shown in figure 6.

Example 7

Synthetic route for compound 7:

synthesis of Compound 7-1:

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), DMAP (1.4 g,11.75 mmol) was added. The mixture was stirred at ambient temperature for 48h. TLC detects the end of the reaction and the crude product is washed 3 times with ethyl acetate and saturated NaHCO ₃ and saturated NaCl. The combined organic layers were dried over anhydrous Na ₂SO₄, and the crude product was purified by silica gel chromatography (petroleum ether/ethyl acetate=100/1) to give compound 7-1 (15.9 g, yield: 75%) as a pale yellow oil.

Synthesis of Compound 7:

1, 4-bis (3-aminopropyl) piperazine (0.8 g,4 mmol) and compound 7-1 (8.64 g,24 mmol) were added to the reaction flask and dissolved in THF/CH ₃ CN (1:1, 40 mL) followed by DIPEA (4 g,24 mmol). The reaction was stirred at 63 ℃ for 72h. TLC detected the end of the reaction, the crude product was washed with ethyl acetate and saturated NaHCO ₃ and brine, the combined organic layers were dried over anhydrous Na ₂SO₄, and the crude product was purified by silica gel chromatography (dichloromethane/methanol=60/1) to give compound 7 as a yellow oil (825 mg, yield: 16%). Mass spectra of compound 7 are shown in figure 7.

Example 8

Synthetic route for compound 8:

synthesis of Compound 8-1:

Linolenic alcohol (8.01 g,30 mmol) and triethylamine (3.99 g,39 mmol) were added to the flask in an ice-water bath, dichloromethane (180 mL) was added, acryloyl chloride (3.3 g,39 mmol) was dissolved in dichloromethane (66 mL) and slowly added dropwise to the flask, the reaction was continued for 10min, the reaction was maintained below 10℃and finally ice-bath removed, and the reaction solution was reacted at room temperature for 2 hours. Washing with saturated NaCl gave a crude product, purifying the crude product by chromatography (silica gel column, eluent petroleum ether containing 0.5% EA by volume) and evaporating the pure product to give compound 8-1 (5.19 g, yield: 50%) as a pale yellow oil

Synthesis of Compound 8:

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 reaction stirred at 80℃for 48h. TLC checked the end of the reaction, the crude product was washed 3 times with ethyl acetate and saturated NaHCO ₃ and saturated NaCl, the combined organic layers were dried over anhydrous Na ₂SO₄ and the crude product was purified by silica gel chromatography (dichloromethane/methanol=60/1) to give compound 8 as a yellow oil (446 mg, yield: 18%). Mass spectra of compound 8 are shown in figure 8.

Example 9

Synthetic route for compound 9:

synthesis of Compound 9-1:

8-Bromocaprylic acid (11.2 g,50 mmol) and (E) -3, 7-dimethyl-2, 6-octadien-1-ol (11 g,50 mmol) were dissolved in dichloromethane (140 mL) and EDC (9.55 g,50 mmol), DMAP (1.22 g,10 mmol) was added. The mixture was stirred at ambient temperature for 48h. TLC detects the end of the reaction and the crude product is washed 3 times with ethyl acetate and saturated NaHCO ₃ and saturated NaCl. The combined organic layers were dried over anhydrous Na ₂SO₄, and the crude product was purified by silica gel chromatography (petroleum ether/ethyl acetate=100/1) to give compound 9-1 (14.0 g, yield: 78%) as a pale yellow oil.

Synthesis of compound 9:

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 the reaction flask and dissolved in THF/CH ₃ CN (1:1, 30 mL) followed by DIPEA (1.74 g,13.5 mmol). The reaction was stirred at 63 ℃ for 72h. TLC checked the end of the reaction, the crude product was washed 3 times with ethyl acetate and saturated NaHCO ₃ and saturated NaCl, the combined organic layers were dried over anhydrous Na ₂SO₄ and the crude product was purified by silica gel chromatography (dichloromethane/methanol=70/1) to give compound 9 as a yellow oil (510 mg, yield: 15%). Mass spectra of compound 9 are shown in figure 9.

Example 10:

Liposome preparation, particle size, potential and encapsulation efficiency measurement.

(1) According to the ionized compound DSPC, DMG-PEG2000 and cholesterol in the molar ratio of 50:10:1.5:38.5, preparing liposome solution by using absolute ethyl alcohol as solvent, controlling the concentration sum of the components to be 50mM, dissolving and mixing uniformly, and then standing at-20 ℃ for storage.

(2) MRNA was dissolved in 25mM sodium acetate buffer having a pH of about 5.2, and a nucleic acid preparation having a final concentration of about 0.17mg/mL was prepared.

(3) The liposome solution and the nucleic acid preparation are mixed uniformly at a two-phase volume ratio of about 15:1, and under the condition that the total speed of the two-phase solution is 12mL/min, the two-phase solution is rapidly mixed uniformly by a Nano Assemblr microfluidic system or by a vortex method, so that uniform and stable liposome nanoparticles are formed. After liposome formation, the liposome environment is rapidly changed from pH 4.0 to 7.2-7.4. Specifically, the liposome is diluted by PBS buffer solution with pH of 7.2 or sodium acetate buffer solution with pH of 7.4 for 20 times, concentrated by an ultrafiltration tube with 50KD, the rotation speed of a centrifuge does not exceed the maximum rotation speed limit of the ultrafiltration tube, after 4-5 times of liquid exchange, the pH of the solution environment of the liposome is about 7.2-7.4, and the liposome is concentrated to a final concentration of about 200mM and is placed in an environment with 4 ℃ for standby.

The particle size and PDI of the liposomes were measured using Zetasizer Nano ZS (Malvern, worcestershire, UK). Particle size was measured by diluting the liposome solution 50 times with 1 XPBS, and Zeta potential was measured by diluting the liposomes in 15mM purified water. The encapsulation efficiency is determined by using a Quant-It RiboGreenRNA quantitative detection kit on a modular microporous multifunctional detector. The particle size, PDI, encapsulation efficiency and potential were measured as shown in table 1.

TABLE 1 physical parameters of representative LNP-mRNA nanolipid particles

Nanometer lipid particle (LNP preparation)	Particle size (nm)	Encapsulation efficiency (%)	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

Table 1 shows that the average particle size of each of the assembled LNPs was 80 to 100nm, the encapsulation efficiency was 90% or more, and the potentials of LNP-2, LNP-3, LNP-6 and LNP-7 were positive, and the potentials of LNP-1, LNP-4, LNP-5, LNP-8 and LNP-9 were negative.

Example 11 Liposome in vivo transfection experiments:

nano-lipid particles were prepared according to the preparation method of example 4 using mRNA expressing Luciferase fluorescent protein, wherein the amount of mRNA was 20 μg, and the total amount of ionizable liposome compound, DSPC, DMG-PEG2000 and cholesterol was 200 μg, and the liposome environment was rapidly switched using 200 μl of neutral PBS buffer.

The nano-lipid particles prepared above were rapidly injected into the inner muscle of hind limbs of 6-8 week female Babl/c mice by intramuscular injection of 30 μg mRNA from the left and right hind limbs, respectively. And observing the expression condition of luciferase in the mice after injection through a small animal imager respectively in different time periods after injection.

The nanolipid particles prepared above were rapidly injected into the body of 6-8 week female Babl/c mice via tail vein (IV) at an mRNA injection amount of 30 μg. And observing the expression condition of luciferase in the mice after injection through a small animal imager respectively in different time periods after injection. After 4h, the heart, liver, spleen, lung, kidney of the mice were individually subjected to fluorescence imaging.

The fluorescence expression intensity of 4 hours after intravenous injection of each liposome is shown in fig. 10 and 11, the fluorescence intensity in the circle is integrated to obtain a fluorescence intensity value, the fluorescence intensity value is used for quantitatively representing the expression amount of luciferase and drawing a bar graph, the fluorescence intensity in the graph is closely related to the delivery efficiency of the liposome, and the higher the fluorescence intensity is, the higher the delivery efficiency of the liposome is represented. The fluorescence expression intensity of LNP-5 is highest and reaches 1.3X10 ¹¹, and the fluorescence expression intensity of LNP-3 is 2.5X10 ¹⁰, and the fluorescence intensity of other liposome is about 10 ⁸～10⁹.

The fluorescent expression profile in each organ of the mice after intravenous injection of each LNP is shown in fig. 12. The results showed that LNP-1, LNP-2 and LNP-6 showed significant lung organ targeting, LNP-3 showed significant liver organ targeting, LNP-5 and LNP-8 were mainly liver targeting, but both spleen and lung had fluorescent expression, and LNP-4, LNP-7 and LNP-9 showed significant spleen targeting.

The present invention has been described in detail with the purpose of enabling those skilled in the art to understand the contents of the present invention and to implement the same, but not to limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.

Claims

1. An ionizable lipid compound represented by general formula (A), general formula (B) or general formula (C),

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,

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 linear 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 vector 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 is characterized in that the delivery vector 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 source 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 phosphatidylcholine, 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 vector 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 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 comprises 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 locally administered by microneedle, injection or perfusion through muscle, subcutaneous, endothelial, or intratumoral administration, or administered by intravenous injection.