CN117917398A

CN117917398A - Ionizable lipid compound, nucleic acid drug molecule delivery system and application

Info

Publication number: CN117917398A
Application number: CN202311365006.4A
Authority: CN
Inventors: 崔艳芳; 吉帅洁; 张宝倩
Original assignee: Central China Normal University
Current assignee: Central China Normal University
Priority date: 2022-10-21
Filing date: 2023-10-20
Publication date: 2024-04-23

Abstract

The invention provides an ionizable lipid compound, a nucleic acid drug molecule delivery system and application. In order to provide more good nucleic acid vaccine delivery vehicles capable of meeting the requirements, the present invention has developed an ionizable lipid compound and compositions thereof suitable for use in nucleic acid drugs or vaccines in the form of mRNA, siRNA, ASO, linear DNA or circular plasmid DNA, and the like. The formulation formed by the ionizable lipid compound and the composition thereof can effectively encapsulate several forms of nucleic acids such as mRNA and the like to form nano particles, and deliver the nano particles to cells in vivo or in vitro to realize high-level protein translation. The nucleic acid delivery has high in vivo delivery efficiency and good tissue/organ targeting, can effectively excite the activity of an immune system, and can be used for protein substitution therapy, preventive or therapeutic vaccines and the like.

Description

Ionizable lipid compound, nucleic acid drug molecule delivery system and application

Technical Field

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

Background

Nucleic acid drugs, in particular messenger RNA (mRNA), have great potential for use in the field of protein replacement therapies, infectious diseases or tumor vaccines. Successful delivery and efficient expression of nucleic acid drug molecules to cells in vivo is one of the currently urgent techniques for achieving therapeutic or prophylactic purposes. Preferred nucleic acid vectors in the form of mRNA, etc., are highly efficient in delivery to avoid toxic side effects due to increased doses of the drug; the ability to specifically target a tissue/organ in practical use is an advantageous property even for a particular cell type; furthermore, for the development of therapeutic or prophylactic vaccines, it is also important that the delivery vehicle of the nucleic acid has adjuvant properties at the same time to effectively trigger an innate immune response and thereby assist the body in establishing an acquired immune efficacy. Therefore, the development of a nucleic acid delivery system with small toxic and side effects, high nucleic acid delivery efficiency, good targeting and proper immunogenicity has important practical significance.

Disclosure of Invention

The invention aims to provide a delivery system which is suitable for nucleic acid drug delivery and mainly consists of an ionizable lipid compound, can efficiently deliver nucleic acid drug molecules into a body, can realize efficient expression of nucleic acid, has tissue/organ targeting and/or proper immunogenicity, and is suitable for vaccine development.

The invention adopts the following technical scheme:

An ionizable lipid compound represented by general formula (1), general formula (2), general formula (3), general formula (4), general formula (5), general formula (6), general formula (7), general formula (8):

Wherein R ₁、R₂、R₃、R₄ or R ₅ is independently selected from

R ₆、R₇ is each independently H, -CH ₃ or-CH ₃CH₂;

x is an integer between 0 and 8, for example 0, 1,2, 3,4, 5, 6, 7, 8;

y is an integer between 0 and 8, for example 0, 1,2, 3,4, 5, 6, 7, 8;

m is an integer between 0 and 7, for example 0, 1,2, 3, 4, 5, 6, 7;

n is an integer between 0 and 7, for example 0, 1,2, 3, 4, 5, 6, 7;

p is an integer between 0 and 4, for example 0, 1,2, 3, 4.

Preferably, R ₆、R₇ is-CH ₃, respectively.

Preferably, none of said x, y, m, n, P is 0.

Preferably, the nucleic acid drug molecule is one or more of mRNA, siRNA, ASO or pDNA (plasmid DNA).

Further preferred is an ionizable lipid compound which is:

The invention also provides a nucleic acid drug molecule delivery system comprising an ionizable lipid compound that is one or more of the above-described ionizable lipid compounds.

Preferably, the nucleic acid drug molecule is one or more of mRNA, siRNA, ASO, linear DNA, or circular plasmid DNA.

Preferably, the mass ratio of the nucleic acid drug molecule to the nucleic acid drug molecule delivery system is 1 (5-50), more preferably 1 (5-40), still more preferably 1 (5-30), still more preferably 1 (5-20).

Further preferably, the nucleic acid drug molecule delivery system is a nanolipid particle having an average size of 50nm to 200nm, such as 50nm, 80nm, 100nm, 120nm, 140nm, 160nm, 180nm 200nm.

More preferably, the polydispersity index of the nano-lipid particles is less than or equal to 0.4.

Further, the average size of the nano lipid particles is 50 nm-150 nm.

Further, the polydispersity index of the nano lipid particles is less than or equal to 0.3.

Preferably, the ionizable lipid compound may optionally be modified with a targeting agent, including but not limited to a ligand that is a receptor molecule, such as folic acid, mannose, etc., and one or more of a monoclonal or polyclonal antibody, single chain antibody, nanobody, aptamer, polypeptide, or peptide analog, wherein the modification of the ionizable lipid compound by the targeting agent includes covalent coupling, non-covalent bonding, mixing, or/and bonding with other chemical bonds, such as coordination bonds.

Preferably, the nucleic acid drug molecule delivery system further comprises an auxiliary molecule optionally modified with a targeting agent, the ionizable lipid compound and the auxiliary molecule being fed in a molar ratio of (0.1-1): (0.1 to 1), more preferably (0.5 to 1): (0.5-1).

Such auxiliary molecules include auxiliary lipids or lipid molecules commonly used in the art.

Preferably, the auxiliary molecules include, but are not limited to, one or more of cholesterol, calcipotriol, stigmasterol, beta-sitosterol, betulin, lupeol, 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-glycerol-3-ethylphosphocholine, dipalmitoyl phosphatidylethanolamine-methoxypolyethylene glycol 5000, distearoyl phosphatidylethanolamine-polyethylene glycol 2000.

Preferably, the targeting agent is a ligand for a receptor molecule, such as folic acid, mannose, etc., and one or more of any form of antibody, aptamer, or polypeptide.

In the present invention, modification of the ionizable lipid compound or the helper molecule with the targeting agent may be performed using methods conventional in the art.

According to some embodiments, mal-PEG2000-DSPE and polypeptide are dissolved in ultrapure water, stirred and reacted for 48 hours, and dialyzed and concentrated to obtain polypeptide-PEG 2000-DSPE, thus obtaining the auxiliary molecule with targeting property.

Further, the ionizable lipid molecules are: cholesterol: DOPE: the polypeptide-PEG 2000-DSPE is dissolved in absolute ethyl alcohol and mixed with DNA or RNA to form polypeptide modified liposome.

Preferably, the nucleic acid drug molecule delivery system is an injection.

Preferably, the nucleic acid drug molecule delivery system further comprises an additive, wherein the additive comprises a stabilizer and/or a diluent.

Preferably, the additive is added in an amount of 1-20% of the total mass of the injection.

The additives may be additives commonly used in the art.

Preferably, the stabilizer includes, but is not limited to, sucrose or trehalose.

Preferably, the diluent includes, but is not limited to, buffers commonly used in the art, including, but not limited to, one or more of phosphate buffer, acetate buffer, tris hydrochloride buffer.

Preferably, the nucleic acid drug molecule delivery system further comprises the addition of other immunoadjuvants. Such adjuvants include, but are not limited to, self-grinding or commercial adjuvants, such as QS-21, MPL, cpG, RNA-type adjuvants, and the like.

Preferably, the nucleic acid drug molecule delivery system is administered by local intramuscular, subcutaneous, endothelial, intratumoral injection or infusion, or by intravenous injection.

The nucleic acid drug molecule delivery system can deliver mRNA, siRNA or pDNA and other nucleic acid drug molecules into a body through a plurality of administration modes such as local muscle, subcutaneous, endothelial, intratumoral and perfusion and the like, can also deliver the mRNA, siRNA or pDNA and other nucleic acid drug molecules into the body through systemic administration modes such as intravenous injection and the like, can even target spleen/lung, can effectively express therapeutic protein drugs or antigens in cells in the body, and plays a role in preventing or treating diseases. The nucleic acid drug molecule delivery system of the present invention can significantly reduce the liver accumulation effect of liposomes, and by selecting a suitable administration mode, nucleic acids can be efficiently delivered into the spleen or lung and effectively translated into target molecules, so that the nucleic acid drug molecule delivery system of the present invention has the potential of being suitable for delivering nucleic acid drugs into the body to perform the function of a prophylactic or therapeutic vaccine.

The invention also provides the application of the ionizable lipid compound or the composition thereof or the nucleic acid drug molecule delivery system in nucleic acid drug or nucleic acid drug delivery.

An mRNA vaccine delivery system comprising said ionizable lipid compound or combination thereof, said mRNA vaccine delivery system capable of delivering mRNA molecules into the body for antigen expression.

Preferably, the mRNA vaccine delivery system has tissue/organ targeting and/or appropriate immune system activation properties.

The nucleic acid delivery system is suitable for various administration modes such as intramuscular injection and the like, has small toxic and side effects on livers, has high nucleic acid delivery efficiency, and has good tissue organ targeting and/or good immunogenicity.

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

The delivery system formed by the ionizable lipid compounds and compositions thereof of the present invention can effectively encapsulate several forms of nucleic acids, such as mRNA, to form nanoparticles and deliver them to cells in vivo or in vitro to achieve high levels of protein translation. The nucleic acid delivery has the advantages of high in vivo delivery efficiency and good tissue/organ targeting, and can effectively excite the activity of an immune system. Thus, the ionizable lipid compounds and compositions thereof are useful for protein replacement therapy, prophylactic or therapeutic vaccine applications, and the like.

Drawings

FIG. 1 is a mass spectrum of Compound 1;

FIG. 2 is a nuclear magnetic resonance diagram of Compound 1;

FIG. 3 is a mass spectrum of Compound 2;

FIG. 4 is a nuclear magnetic resonance diagram of Compound 2;

FIG. 5 is a mass spectrum of Compound 3;

FIG. 6 is a nuclear magnetic resonance diagram of Compound 3;

FIG. 7 is a mass spectrum of Compound 4;

FIG. 8 is a nuclear magnetic resonance diagram of Compound 4;

FIG. 9 is a mass spectrum of Compound 5;

FIG. 10 is a nuclear magnetic resonance diagram of Compound 5;

FIG. 11 is a mass spectrum of Compound 6;

FIG. 12 is a nuclear magnetic resonance diagram of Compound 6;

FIG. 13 is a mass spectrum of Compound 7;

FIG. 14 is a nuclear magnetic resonance diagram of Compound 7;

FIG. 15 is a mass spectrum of Compound 8;

FIG. 16 is a nuclear magnetic resonance plot of Compound 8;

FIG. 17 is a representative LNPs particle size test chart and transmission electron microscope chart;

FIG. 18 is a graph showing in vivo delivery efficiency and Fluc expression in various organs of a representative LNPs mouse;

FIG. 19 is a schematic diagram of the principle of antibody coupling LNPs and the particle size change after coupling;

FIG. 20 is a graph of organ targeting effects of LNPs after antibody coupling;

FIG. 21 is a graph of adjuvant versus LNP particle size change;

FIG. 22 is a diagram showing the flow of BALB/c mouse immunization and effect analysis.

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.

The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used, unless specified, are all purchased from conventional biochemical reagent manufacturers.

Example 1

Synthetic route for compound 1:

Synthesis of Compound 1-1:

6-Bromohexanoic acid (10 g,51.27 mmol) and (+ -) - β -citronellol, 3, 7-dimethyl-6-octen-1-ol (7.0 g,51.28 mmol) were added to a reaction flask and dissolved in DCM (140 ml), followed by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (9.79 g,51.27 mmol) and 4-dimethylaminopyridine (1.25 g,10.25 mmol). The mixture was stirred at room temperature for 72h. After completion of the reaction by TLC, the solvent DCM in the reaction mixture was concentrated to dryness, dissolved in ethyl acetate, washed 3 times with saturated aqueous sodium bicarbonate and saturated NaCl solution, and the combined organic layers were dried over anhydrous Na ₂SO₄ to give the crude product. The crude product was purified by silica gel chromatography (volume ratio of eluent: petroleum ether/ethyl acetate=120/1) to give compound 1-1 (9.74 g, yield: 57%) as a colorless oil.

Synthesis of Compounds 1-2:

8-Bromooctanoic acid (10 g,44.82 mmol) and heptadecan-9-ol (14.9 g,58.27 mmol) were added to a reaction flask and dissolved in DCM (140 ml), followed by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (11.17 g,58.27 mmol), DMAP (1.09 g,8.96 mmol) and DIPEA (23.17 g,179.28 mmol). The mixture was stirred at room temperature for 48h. After completion of the reaction by TLC, the solvent DCM in the reaction mixture was concentrated to dryness, dissolved in ethyl acetate, washed 3 times with saturated aqueous sodium bicarbonate and saturated NaCl solution, and the combined organic layers were dried over anhydrous Na ₂SO₄ to give the crude product. The crude product was purified by silica gel chromatography (volume ratio of eluent: petroleum ether/ethyl acetate=150/1) to give compound 1-2 (7.20 g, yield: 34.8%) as a colorless oil.

Synthesis of Compounds 1-3:

Compounds 1-2 (3.0 g,6.5 mmol) and 3-amino-1-propanol (14.67 g,195.3 mmol) were added to a reaction flask and dissolved in EtOH (6 mL) and the mixture stirred at 63℃for 24h. After the completion of the TLC detection, the solvent ethanol was concentrated, dissolved in ethyl acetate, extracted 3 times with water, washed 3 times with saturated brine, and the combined organic layers were dried over anhydrous Na ₂SO₄, and the crude product was purified by silica gel chromatography (eluent volume ratio: dichloromethane/methanol=100/1) to give compound 1-3 (1.50 g, yield: 50.6%) as pale yellow oil.

Synthesis of Compound 1:

Compound 1-3 (1.40 g,3.07 mmol) and compound 1-1 (2.04 g,6.14 mmol) were added to a reaction flask and dissolved in EtOH (2 mL) followed by DIPEA (0.79 g,6.14 mmol). The mixture was stirred at 63℃for 24h. After the completion of the TLC detection, the solvent ethanol was concentrated, dissolved in ethyl acetate, and then washed 3 times with saturated aqueous sodium bicarbonate and saturated brine, and the combined organic layers were dried over anhydrous Na ₂SO₄, and the crude product was purified by silica gel chromatography (volume ratio of eluent: dichloromethane/methanol=110/1) to give compound 1 (0.54 g, yield: 25%) as a pale yellow oil. Mass spectrum of compound 1 is shown in figure 1, and nuclear magnetism is shown in figure 2,¹H NMR(400MHz,CDCl₃)δ5.08(s,1H),4.85(s,1H),4.09(t,J＝8.4Hz,2H),3.81(t,J＝6.8Hz,2H),2.70(d,J＝79.1Hz,6H),2.29(ddd,J＝11.1,7.4,3.8Hz,5H),1.70–1.45(m,23H),1.28(d,J＝26.3Hz,36H),0.93–0.84(m,9H).

Example 2

Synthetic route for compound 2:

synthesis of Compound 2-1: compound 2-1 and compound 1-1 are the same compound, and the synthesis method is described with reference to the synthesis method of compound 1-1 in example 1.

Synthesis of Compound 2-2: the compound 2-2 and the compound 1-2 are the same compound, and the synthetic method refers to the synthetic method of the compound 1-2 in the embodiment 1, and is not repeated here.

Synthesis of Compound 2-3:

Compound 2-2 (3.0 g,6.5 mmol) and ethanolamine (11.91 g,195.3 mmol) were added to a reaction flask and dissolved in EtOH (6 mL) and the mixture stirred at 63℃for 24h. After the completion of the TLC detection, the solvent ethanol was concentrated, dissolved in ethyl acetate, extracted 3 times with water, washed 3 times with saturated brine, and the combined organic layers were dried over anhydrous Na ₂SO₄, and the crude product was purified by silica gel chromatography (eluent volume ratio: dichloromethane/methanol=60/1) to give compound 2-3 (1.80 g, yield: 62.5%) as a pale yellow oil.

Synthesis of Compound 2:

Compound 2-3 (1.50 g,3.4 mmol) and compound 2-1 (2.26 g,6.8 mmol) were added to a reaction flask and dissolved in EtOH (2 mL) followed by DIPEA (0.88 g,6.8 mmol). The mixture was stirred at 63℃for 24h. After the completion of the TLC detection, the solvent ethanol was concentrated, dissolved in ethyl acetate, and then washed 3 times with saturated aqueous sodium bicarbonate and saturated brine, and the combined organic layers were dried over anhydrous Na ₂SO₄, and the crude product was purified by silica gel chromatography (dichloromethane/methanol=65/1) to give compound 30 (0.78 g, yield: 22%) as a pale yellow oil. Mass spectrum of compound 2 is shown in figure 3, and nuclear magnetism is shown in figure 4,¹H NMR(400MHz,CDCl₃)δ4.10(dd,J＝13.4,6.7Hz,2H),3.99(s,2H),3.15(s,2H),3.05(s,3H),2.30(dt,J＝18.5,7.1Hz,4H),1.83(d,J＝21.5Hz,7H),1.71–1.57(m,17H),1.54–1.48(m,5H),1.46–1.40(m,3H),1.36(s,5H),1.27(d,J＝15.8Hz,25H),0.93–0.85(m,8H).

Example 3

Synthetic route for compound 3:

Synthesis of Compound 3-1:

6-Bromohexanoic acid (10 g,51.27 mmol) and (E) -3, 7-dimethyl-2, 6-octadien-1-ol (7.9 g,51.28 mmol) were added to a reaction flask and dissolved in DCM (140 ml), followed by 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (9.8 g,51.28 mmol), DMAP (1.25 g,10.26 mmol) and stirred at room temperature for 72h. After TLC detection of the end of the reaction, the solvent was concentrated to dryness, washed 3 times with saturated aqueous NaHCO ₃ and saturated aqueous NaCl, the combined organic layers were dried over anhydrous Na ₂SO₄ and the crude product was purified by column chromatography on silica gel (volume ratio of eluent: petroleum ether/ethyl acetate=120/1) to give compound 3-1 (9.91 g, yield: 58%).

Synthesis of Compound 3-2: the compound 3-2 and the compound 1-2 are the same compound, and the synthetic method is described with reference to the synthetic method of the compound 1-2 in example 1.

Synthesis of Compound 3-3: the compound 3-3 and the compound 1-3 are the same compound, and the synthetic method refers to the synthetic method of the compound 1-3 in example 1.

Synthesis of Compound 3:

Compound 3-3 (0.3 g,0.66 mmol) and compound 3-1 (0.44 g,1.32 mmol) were added to a reaction flask and dissolved in EtOH (1 mL) followed by DIPEA (0.17 g,1.32 mmol). The mixture was stirred at 63℃for 24h. After the completion of the TLC detection, the solvent ethanol was concentrated, dissolved in ethyl acetate, and then, saturated aqueous NaHCO ₃ and saturated brine were added to wash 3 times, the combined organic layers were dried over anhydrous Na ₂SO₄, and the crude product was purified by preparative thin layer chromatography (Prep TLC) (eluent volume ratio: dichloromethane/methanol=7/1) to give compound 3 (0.16 g, yield: 34%) as a pale yellow oil. Mass spectrum of compound 3 is shown in figure 5, and nuclear magnetism is shown in figure 6,¹H NMR(400MHz,CDCl₃)δ5.35–5.29(m,3H),5.08(t,J＝5.7Hz,1H),4.85(t,J＝6.2Hz,1H),4.59(d,J＝7.1Hz,2H),3.83(t,J＝5.2Hz,2H),3.11–2.94(m,4H),2.31(dt,J＝22.5,7.4Hz,4H),2.03(dd,J＝13.6,7.4Hz,4H),1.69(d,J＝8.0Hz,18H),1.50(d,J＝5.8Hz,5H),1.36(s,5H),1.25(s,27H),0.88(t,J＝6.8Hz,7H).

Example 4

Synthetic route for compound 4:

Synthesis of Compound 4-1: the compound 4-1 and the compound 1-2 are the same compound, and the synthetic method is described with reference to the synthetic method of the compound 1-2 in example 1.

Synthesis of Compound 4:

4-aminocyclohexanol (0.30 g,2.61 mmol) and compound 4-1 (3.0 g,6.53 mmol) were added to a reaction flask and dissolved in EtOH (2 mL), followed by DIPEA (1.01 g,7.83 mmol). The mixture was stirred at 63℃for 24h. After the completion of the TLC detection, the solvent ethanol was concentrated, dissolved in ethyl acetate, and then, saturated aqueous sodium bicarbonate and saturated brine were added to wash for 3 times, the combined organic layers were dried over anhydrous Na ₂SO₄, and the crude product was purified by preparative thin layer chromatography (Prep TLC) (eluent volume ratio: dichloromethane/methanol=7/1) to give compound 4 (0.25 g, yield: 11%) as a brown oil. Mass spectrum of compound 4 is shown in fig. 7, and nuclear magnetism is shown in fig 8,¹H NMR(400MHz,CDCl₃)δ4.86(p,J＝6.0Hz,2H),3.02(dt,J＝12.3,5.6Hz,2H),2.96–2.82(m,2H),2.28(t,J＝7.5Hz,4H),2.05(s,1H),1.66–1.58(m,14H),1.50(d,J＝5.7Hz,8H),1.36(s,12H),1.28(d,J＝15.3Hz,52H),0.88(t,J＝6.8Hz,12H).

Example 5

Synthetic route for compound 5:

Synthesis of Compound 5-1: the compound 5-1 and the compound 1-2 are the same compound, and the synthetic method is described with reference to the synthetic method of the compound 1-2 in example 1.

Synthesis of Compound 5:

1- (3-aminopropyl) imidazole (0.30 g,2.4 mmol) and compound 5-1 (2.77 g,6.0 mmol) were added to a reaction flask and dissolved in EtOH (2 mL) followed by DIPEA (0.93 g,7.2 mmol). The mixture was stirred at 63℃for 24h. After the completion of the TLC detection, the solvent ethanol was concentrated, dissolved in ethyl acetate, and then, saturated aqueous sodium bicarbonate and saturated brine were added to wash for 3 times, the combined organic layers were dried over anhydrous Na ₂SO₄, and the crude product was purified by preparative thin layer chromatography (Prep TLC) (eluent volume ratio: dichloromethane/methanol=7/1) to give compound 5 (0.32 g, yield: 15%) as a brown oil. The mass spectrum of the compound 5 is shown in figure 9, and the nuclear magnetism is shown in figure 10,¹H NMR(400MHz,CDCl₃)δ4.88–4.82(m,2H),2.28(t,J＝7.5Hz,4H),1.79(s,3H),1.63(s,19H),1.50(d,J＝4.3Hz,9H),1.35(s,12H),1.26(s,46H),0.88(t,J＝6.4Hz,12H).

Example 6

Synthetic route to compound 6:

synthesis of Compound 6-1:

8-Bromooctanoic acid (11.44 g,51.28 mmol) and (+ -) - β -citronellol, 3, 7-dimethyl-6-octen-1-ol (7.0 g,51.28 mmol) were added to a reaction flask and dissolved in DCM (140 ml), followed by 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (9.8 g,51.28 mmol) and DMAP (1.25 g,10.26 mmol). The mixture was stirred at room temperature for 72h. After completion of the TLC, the solvent DCM in the reaction solution was concentrated, and washed 3 times with saturated aqueous sodium bicarbonate and saturated brine, and the combined organic layers were dried over anhydrous Na ₂SO₄ to give a crude product. The crude product was purified by silica gel chromatography (volume ratio of eluent: petroleum ether/ethyl acetate=100/1) to give compound 6-1 (9.8 g, yield: 53%) as a colorless oil.

Synthesis of Compound 6-2: compound 6-2 and compound 1-2 are the same compound, and the synthesis method is described with reference to the synthesis method of compound 1-2 in example 1.

Synthesis of Compound 6-3:

Compound 6-2 (3 g,6.50 mmol) and 4-aminocyclohexanol (22.43 g,195.0 mmol) were added to a reaction flask and dissolved in EtOH (6 mL) and the mixture was stirred at 63℃for 24h. After the completion of the TLC detection, the solvent ethanol was concentrated, dissolved in ethyl acetate, extracted 3 times with water, washed 3 times with saturated brine, and the combined organic layers were dried over anhydrous Na ₂SO₄, and the crude product was purified by silica gel chromatography (eluent volume ratio: dichloromethane/methanol=40/1) to give compound 6-3 (1.45 g, yield: 45%) as a pale yellow oil.

Synthesis of Compound 6:

Compound 6-3 (0.60 g,1.21 mmol) and compound 6-1 (0.87 g,2.42 mmol) were added to a reaction flask and dissolved in EtOH (1 mL) followed by DIPEA (0.31 g,2.42 mmol). The mixture was stirred at 63℃for 24h. After the completion of the TLC detection, the solvent ethanol was concentrated, dissolved in ethyl acetate, and then, saturated aqueous sodium bicarbonate and saturated brine were added to wash for 3 times, the combined organic layers were dried over anhydrous Na ₂SO₄, and the crude product was purified by preparative thin layer chromatography (Prep TLC) (eluent volume ratio: dichloromethane/methanol=6/1) to give Compound 6 (0.19 g, yield: 20%) as a pale yellow oil. Mass spectrum of compound 6 is shown in fig. 11, and nuclear magnetism is shown in fig 12,¹H NMR(400MHz,CDCl₃)δ5.12(t,J＝7.8Hz,1H),4.89(t,J＝6.0Hz,1H),4.18–4.09(m,2H),2.32(td,J＝7.4,4.1Hz,4H),2.02(d,J＝16.9Hz,5H),1.72(s,4H),1.68–1.58(m,26H),1.39(s,12H),1.31(d,J＝15.2Hz,29H),0.98–0.87(m,9H).

Example 7

Synthetic route for compound 7:

Synthesis of Compound 7-1: the compound 7-1 and the compound 6-1 were the same compound, and the synthesis method was referred to as the synthesis method of the compound 6-1 in example 6.

Synthesis of Compound 7-2: the compound 7-2 and the compound 1-2 are the same compound, and the synthetic method is described with reference to the synthetic method of the compound 1-2 in example 1.

Synthesis of Compound 7-3:

Compound 7-2 (3 g,6.50 mmol) and 1- (3-aminopropyl) imidazole (24.38 g,195.0 mmol) were added to a reaction flask and dissolved in EtOH (6 mL) and the mixture stirred at 63℃for 24h. After the completion of the TLC detection, the solvent ethanol was concentrated, dissolved in ethyl acetate, extracted 3 times with water, washed 3 times with saturated brine, and the combined organic layers were dried over anhydrous Na ₂SO₄, and the crude product was purified by passing through a silica gel column (eluent volume ratio: dichloromethane/methanol=60/1) to give compound 7-3 (1.50 g, yield: 46%) as a pale yellow oil.

Synthesis of Compound 7:

Compound 7-3 (0.60 g,1.20 mmol) and compound 7-1 (0.86 g,2.40 mmol) were added to a reaction flask and dissolved in EtOH (1 mL) followed by DIPEA (0.31 g,2.40 mmol). The mixture was stirred at 63℃for 24h. After the completion of the TLC detection, the solvent ethanol was concentrated, dissolved in ethyl acetate, and then, saturated aqueous sodium bicarbonate and saturated brine were added to wash for 3 times, the combined organic layers were dried over anhydrous Na ₂SO₄, and the crude product was purified by preparative thin layer chromatography (Prep TLC) (eluent volume ratio: dichloromethane/methanol=6/1) to give compound 7 (0.23 g, yield: 24%) as a pale yellow oil. Mass spectrum of compound 7 is shown in fig. 13, and nuclear magnetism is shown in fig 14,¹H NMR(400MHz,CDCl₃)δ5.08(t,J＝7.1Hz,1H),4.91–4.77(m,1H),4.25–4.00(m,4H),3.38–2.89(m,3H),2.72(d,J＝18.5Hz,1H),2.28(dd,J＝12.9,5.9Hz,4H),2.08–1.87(m,5H),1.80(s,3H),1.61(t,J＝26.7Hz,21H),1.54–1.40(m,6H),1.40–1.14(m,34H),0.99–0.80(m,8H).

Example 8

Synthetic route for compound 8:

Synthesis of Compound 8-1: compound 8-1 and compound 6-1 are the same compound, and the synthesis method is described with reference to the synthesis method of compound 6-1 in example 6.

Synthesis of Compound 8-2: compound 8-2 and compound 1-2 are the same compound, and the synthesis method is described with reference to the synthesis method of compound 1-2 in example 1.

Synthesis of Compound 8-3:

compound 8-2 (3.0 g,6.5 mmol) and ethanolamine (11.91 g,195.3 mmol) were added to a reaction flask and dissolved in EtOH (6 mL) and the mixture stirred at 63℃for 24h. After the completion of the TLC detection, the solvent ethanol was concentrated, dissolved in ethyl acetate, extracted 3 times with water, washed 3 times with saturated brine, and the combined organic layers were dried over anhydrous Na ₂SO₄, and the crude product was purified by silica gel chromatography (eluent volume ratio: dichloromethane/methanol=60/1) to give 8-3 (1.90 g, yield: 65.5%) as pale yellow oily compound.

Synthesis of Compound 8:

compound 8-3 (1.70 g,3.4 mmol) and compound 2-1 (2.26 g,6.8 mmol) were added to a reaction flask and dissolved in EtOH (2 mL) followed by DIPEA (0.88 g,6.8 mmol). The mixture was stirred at 63℃for 24h. After the completion of the TLC detection, the solvent ethanol was concentrated, dissolved in ethyl acetate, and then washed 3 times with saturated aqueous sodium bicarbonate and saturated brine, and the combined organic layers were dried over anhydrous Na ₂SO₄, and the crude product was purified by silica gel chromatography (dichloromethane/methanol=65/1) to give compound 30 (0.78 g, yield: 22%) as a pale yellow oil. Mass spectrum of compound 8 is shown in figure 15, and nuclear magnetism is shown in figure 16,¹H NMR(400MHz,CDCl₃)δ5.09(t,J＝7.1Hz,1H),4.86(p,J＝6.3Hz,1H),4.15–4.04(m,2H),2.28(td,J＝7.5,3.4Hz,4H),1.68(s,4H),1.65–1.56(m,11H),1.50(d,J＝5.7Hz,9H),1.38–1.17(m,43H),0.96–0.80(m,10H).

Example 9

With reference to the above reaction principle, compounds 9 to 34 were prepared according to the following synthetic routes.

Synthetic route for compound 9:

Synthetic route for compound 10:

synthetic route for compound 11:

Synthetic route for compound 12:

synthetic route for compound 13:

synthetic route to compound 14:

synthetic route for compound 15:

synthetic route for compound 16:

synthetic route to compound 17:

synthetic route to compound 18:

Synthetic route to compound 19:

synthetic route for compound 20:

Synthetic route for compound 21:

synthetic route for compound 22:

synthetic route for compound 23:

Synthetic route to compound 24:

Synthetic route to compound 25:

Synthetic route to compound 26:

Synthetic route to compound 27:

Synthetic route to compound 28:

synthetic route to compound 29:

synthetic route for compound 30:

Synthetic route to compound 31:

synthetic route to compound 32:

synthetic route to compound 33:

synthetic route to compound 34:

example 10: preparation of LNP-mRNA nanolipid particles, determination of particle size and potential:

The compound prepared in the example is dissolved and mixed with DSPC, DMG-PEG2000 and cholesterol respectively according to the mol ratio of 50:10:1.5:38.5 by using absolute ethyl alcohol as a solvent to obtain a liposome raw material solution, the concentration of each component is controlled to be 50mM, and the liposome raw material solution is stored at the temperature of minus 20 ℃ after being completely dissolved and mixed.

MRNA expressing Luciferase fluorescent protein was dissolved in 25mM sodium acetate buffer at pH of about 5.2 to prepare a nucleic acid preparation having a final concentration of about 0.1 mg/mL.

The liposome raw material solution and the nucleic acid preparation prepared above are mixed uniformly by a Nano Assemblr microfluidic system under the conditions that the volume ratio of two phases is about 4:1 and the total speed of the two-phase solution is 12mL/min, uniform and stable nano liposome particles are formed by a vortex method, and then the environment of the nano liposome particles is quickly changed from pH 5.2 to 7.0-7.4. Specifically, the solution is diluted by a PBS buffer solution with pH of 7.2 or a sodium acetate buffer solution with pH of 7.4 for 20 times, concentrated by an ultrafiltration tube with 10KD, the rotation speed of a centrifuge does not exceed the maximum rotation speed limit of the ultrafiltration tube, after 2-3 times of liquid exchange, the pH of the solution environment of the nano liposome particles is about 7.2-7.4, concentrated to a final concentration of about 200mM, and placed in an environment of 4 ℃ for standby.

The particle size, PDI, of the nanoliposome particles was measured using Zetasizer Nano ZS (Malvern, worcestershire, UK). Particle size was measured by diluting nanoliposome particle solution 50 times with 1×PBS, zeta potential was measured by diluting nanoliposome particles in 15mM PBS, and encapsulation efficiency was measured on a modular microporous multifunctional detector using Quant-It RiboGreenRNA quantitative detection kit.

As shown in FIG. 17 and Table 1, representative liposomes were prepared with particle sizes around 100nm, PDI less than 0.2, and LNP-8 particle sizes around 100nm were also shown by transmission electron microscopy (FIG. 17B).

TABLE 1

Nanometer lipid particle (LNP preparation)	Particle size (nm)	Encapsulation efficiency (%)	Potential (mV)
				LNP-1 (containing Compound 1)	89.1	92	-3.3
LNP-2 (containing Compound 2)	93.3	94	3.8
				LNP-3 (containing Compound 3)	86.4	95	6.5
LNP-4 (containing Compound 4)	84.4	96	-5.7
				LNP-5 (containing Compound 5)	91.6	98	-6.9
LNP-6 (containing Compound 6)	86.1	94	6.1
				LNP-7 (containing Compound 7)	89.1	92	8.9
LNP-8 (containing Compound 8)	89.2	98	-3.0

The particle size of the nano lipid particles prepared by the compound 9-34 respectively with reference to the preparation method is in the range of 70-120 nm, the encapsulation efficiency can reach more than 88%, and the potential is between-12 and 10 mV.

Example 11: in vivo targeting experiments of nano lipid particles:

The nano-lipid particles encapsulating mRNA (LNP-1 for Compound 1, LNP-2 for Compound 2, and so on) were prepared by intramuscular injection of nano-lipid particles according to the preparation method of example 10, wherein mRNA was mRNA expressing Luciferase fluorescent protein in an amount of 60. Mu.g, the total amount of ionizable liposome compound, DSPC, DMG-PEG2000 and cholesterol was 600. Mu.g, the liposome environment was rapidly changed by 200. Mu.L of neutral PBS buffer, and rapidly injected into the hindlimb inner muscle of 6-8 week female Babl/c mice, the left and right hindlimbs were controlled to be injected with 30. Mu gmRNA, respectively, or by intravenous injection into 6-8 week female Babl/c mice, and intravenous injection of 50. Mu.g of mRNA was controlled.

The fluorescence expression of mRNA in mice after intravenous injection was integrated with the fluorescence intensity inside the circle and obtained as fluorescence intensity values (FIG. 18), and the fluorescence expression intensities of a part of representative LNPs are shown in Table 2. FIG. 18 also shows that the in vivo distribution of a portion of the representative LNPs Fluc, fluorescence intensity is closely related to the delivery efficiency of liposomes, with stronger fluorescence representing higher delivery efficiency of liposomes. FIG. 18 shows that each mRNA encapsulated nanolipid particle is capable of achieving in vivo mRNA delivery and successful expression, with LNP-5 and LNP-7 exhibiting pulmonary targeting and other lipid molecules exhibiting hepatic targeting.

TABLE 2

The fluorescent expression intensity of mRNA delivered by the nano-lipid particles prepared by the compounds 9 to 34 respectively referring to the preparation method can reach the approximate degree of LNP-1 to LNP-8.

Example 12: experiments of targeting molecules to modify LNP to improve targeting:

Take as an example the purified CD3e or CD4 monoclonal antibody conjugated LNP-1 with T cell targeting. We have invented a modified post-insertion technique, i.e. the antibody is first activated with NHS groups, the NHS or NHS-lipid not conjugated to the antibody is removed by G-25Sephadex Quick Spin Protein column, and then the activated antibody molecule is inserted onto the LNP-1 surface and purified using Sepharose CL-4B gel filtration column. Samples after antibody conjugation were stored at 4 ℃. The schematic diagram of LNP-1 surface antibody modification is shown in FIG. 19, the distribution of LNP-1 in each organ after antibody coupling is shown in FIG. 20, the expression level of LNP-1 in the liver part is reduced, and the expression level in the spleen part is obviously increased.

Example 13: nano-lipid particle addition adjuvant molecules and animal immunization experiments:

preparation of immune nano lipid particles and determination of particle size:

The influenza virus HA antigen protein is used as an immune antigen of animal immune experiments, mRNA thereof is prepared firstly, and then the mRNA is mixed with lipid and adjuvant molecules. The emphasis is on the effective binding together of the adjuvant substance and the lipid component. For fat-soluble adjuvant molecules such as homemade sugar-containing adjuvant molecules, commercial monophosphoryl 3-deacylated lipid a (MPL), etc., we add directly to the formulation as an LNP ingredient. For water-soluble adjuvant molecules such as quillaja saponaria saponin QS-21, oligodeoxynucleotide CpG, etc., when preparing the preparation, we jointly dissolve antigen protein mRNA and adjuvant molecules to prepare the nucleic acid preparation. Then, the mass ratio of lipid components to nucleic acid is controlled, the volume ratio of the two phases is controlled, and the two-phase solution is rapidly and uniformly mixed by a vortex method or other methods such as a microfluidic system, so that uniform and stable LNP-mRNA lipid nanoparticles (parameters such as particle size potential and the like meet the requirements) are formed. Lipid nanoparticles corresponding to the HA antigen protein without adjuvant addition are respectively marked as LNP-HA mRNA; the label with adjuvant addition was LNP-adj-HAmRNA. The particle size change before and after the addition of the adjuvant is shown in FIG. 21, wherein the graph A in FIG. 12 shows the particle size of the mRNA-encapsulated LNP without the addition of the adjuvant, the graph B shows the particle size of the mRNA-encapsulated LNP with the addition of the adjuvant MPLA-HA, and the graph C shows the particle size of the mRNA-encapsulated LNP with the addition of the adjuvant G-MPLA-HA, and it is seen that the addition of the adjuvant MPL increases the particle size of the LNP.

Animal immunization experiment:

The nucleic acid vaccine LNP-HAmRNA prepared above, LNP-adj-HAmRNA, was immunized by intramuscular injection into 6-8 week BALB/c mice at a dose of 30. Mu.g. The experiment sets an equal volume of normal saline injection group as a negative control, and simultaneously compares the immune effect of the nucleic acid vaccine preparation added with the adjuvant and the nucleic acid vaccine preparation without the adjuvant in parallel. The experiment was evaluated by detecting the content of novel coronavirus neutralizing antibodies in the serum of mice after immunization. With reference to the novel coronavirus vaccine standard, mice serum was withdrawn at day 21 and second immunized after first immunization, 28 days, and serum IgG antibody content was detected by ELISA. The results of neutralizing antibody titres after 7 days of secondary immunization are shown in fig. 22, LNP-HAmRNA shows a significant IgG titre, while LNP-adj-HAmRNA (LNP-HAmRNA-MPLA, LNP-HAmRNA-G-MPLA in the figure) with the addition of an adjuvant can significantly raise IgG titres, indicating that lipid nanoparticles have better immunogenicity, and that they have better immune effects as delivery vehicles for mRNA after the addition of an adjuvant.

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. The ionizable lipid compound is characterized in that the ionizable lipid compound is a compound shown in a general formula (1), a general formula (2), a general formula (3), a general formula (4), a general formula (5), a general formula (6), a general formula (7) and a general formula (8):

Wherein R ₁、R₂、R₃、R₄ or R ₅ is independently selected from

R ₆、R₇ is each independently H, -CH ₃ or-CH ₃CH₂;

x is an integer between 0 and 8;

y is an integer between 0 and 8;

m is an integer between 0 and 7;

n is an integer between 0 and 7;

p is an integer between 0 and 4.

2. An ionizable lipid compound, characterized in that said ionizable lipid compound is:

3. A nucleic acid drug molecule delivery system comprising an ionizable lipid compound that is one or more of the ionizable lipid compounds of claims 1 or 2.

4. The nucleic acid drag molecule delivery system of claim 3, wherein the nucleic acid drag molecule is one or more of mRNA, siRNA, ASO, linear DNA, or circular plasmid DNA;

And/or the mass ratio of the nucleic acid drug molecule to the nucleic acid drug molecule delivery system is 1 (5-50);

And/or the nucleic acid drug molecule delivery system is nano lipid particles, and the average size of the nano lipid particles is 50 nm-200 nm;

And/or the ionizable lipid compound may optionally be modified with a targeting agent comprising one or more of a ligand, monoclonal or polyclonal antibody, single chain antibody, nanobody, aptamer, polypeptide or peptide analog of the receptor molecule, wherein the modification of the ionizable lipid compound by the targeting agent comprises covalent coupling, non-covalent mixing, or/and other chemical bonding.

5. The nucleic acid drag molecule delivery system of claim 4, further comprising an auxiliary molecule optionally modified with a targeting agent, wherein the ionizable lipid compound and the auxiliary molecule are administered in a molar ratio of (0.1-1): (0.1 to 1); the auxiliary molecules comprise one or more of cholesterol, calcipotriol, stigmasterol, beta-sitosterol, betulin, lupeol, ursolic acid, oleanolic acid, dioleoyl phosphatidylcholine, distearoyl phosphatidylcholine, 1-stearoyl-2-oleoyl lecithin, dioleoyl phosphatidylethanolamine, (1, 2-dioleoxypropyl) trimethyl ammonium chloride, bisdecanyl dimethyl ammonium bromide, 1, 2-dimyristoyl-sn-glycero-3-ethyl phosphorylcholine, dipalmitoyl phosphatidylethanolamine-methoxypolyethylene glycol 5000, distearoyl phosphatidylethanolamine-polyethylene glycol 2000; the target substance is one or more of a ligand, an antibody, an aptamer or a polypeptide of a receptor molecule.

6. The nucleic acid drug molecule delivery system of claim 3, wherein the nucleic acid drug molecule delivery system is an injection, the nucleic acid drug molecule delivery system further comprising an additive, the additive comprising a stabilizer and/or a diluent, the additive being added in an amount of 1% to 20% of the total mass of the injection; the stabilizer comprises sucrose or trehalose; the diluent comprises one or more of phosphate buffer, acetate buffer, citrate and tris hydrochloride buffer.

7. The nucleic acid drug molecule delivery system of claim 3, further comprising additional immunoadjuvants.

8. The nucleic acid drug molecule delivery system of any one of claims 3 to 7, wherein the nucleic acid drug molecule delivery system is administered by local intramuscular, subcutaneous, endothelial, intratumoral injection or infusion, or by intravenous injection.

9. Use of an ionizable lipid compound or a composition thereof according to claim 1 or 2 or a nucleic acid drug molecule delivery system according to any one of claims 3 to 8 in nucleic acid drug or nucleic acid drug delivery.

10. An mRNA vaccine delivery system comprising the ionizable lipid compound of claim 1 or 2 or a combination thereof, said mRNA vaccine delivery system being capable of delivering mRNA molecules into the body for antigen expression, and/or said mRNA vaccine delivery system having tissue/organ targeting and/or appropriate immune system activation properties.