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

Abstract

The present application provides an ionizable lipid compound, a nucleic acid drug molecule delivery system and application. In order to provide more good nucleic acid vaccine delivery carriers that can meet the requirements, the present application develops an ionizable lipid compound and its composition suitable for mRNA, siRNA, ASO, linear DNA or circular plasmid DNA and other forms of nucleic acid drugs or vaccines. The formulation formed by the ionizable lipid compound and its composition 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-level protein translation. Its delivery of nucleic acids has high in vivo delivery efficiency, good tissue / organ targeting, and can effectively stimulate immune system activity, and can be used for protein replacement therapy, preventive or therapeutic vaccines and other purposes.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid drug delivery carrier technology, specifically relating to an ionizable lipid compound, a nucleic acid drug molecule delivery system, and its applications. Background Technology

[0002] Nucleic acid drugs, especially messenger RNA (mRNA), have great potential applications in protein replacement therapy, infectious disease vaccines, and tumor vaccines. Successfully delivering nucleic acid drug molecules into cells and achieving efficient expression to achieve therapeutic or preventative purposes is currently a crucial technology. Preferred nucleic acid vectors, such as those in the form of mRNA, should have high delivery efficiency to avoid increased dosage and toxic side effects. In practical applications, the ability to specifically target a particular tissue / organ, or even a specific cell type, is also an advantage. Furthermore, for the development of therapeutic or preventative vaccines, it is essential that the nucleic acid delivery vector also possesses adjuvant properties to effectively trigger an innate immune response, thereby helping the body establish acquired immunity. Therefore, developing nucleic acid delivery systems with low toxicity, high delivery efficiency, good targeting, and appropriate immunogenicity is of significant practical importance. Summary of the Invention

[0003] The purpose of this invention is to provide a delivery system for nucleic acid drug delivery, which is mainly composed of ionizable lipid compounds. The nucleic acid drug molecule delivery system can efficiently deliver nucleic acid drug molecules into the body and achieve efficient nucleic acid expression. It also has tissue / organ targeting and / or suitable immunogenicity, making it suitable for vaccine development.

[0004] The present invention adopts the following technical solution:

[0005] An ionizable lipid compound, wherein the ionizable lipid compound is a compound represented by general formula (1), general formula (2), general formula (3), general formula (4), general formula (5), general formula (6), general formula (7), or general formula (8):

[0006]

[0007]

[0008] Wherein, R1, R2, R3, R4, or R5 are independently selected from...

[0009] R6 and R7 are independently H, -CH3, or -CH3CH2, respectively;

[0010] x is an integer between 0 and 8, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0011] y is an integer between 0 and 8, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8;

[0012] m is an integer between 0 and 7, for example, 0, 1, 2, 3, 4, 5, 6, 7;

[0013] n is an integer between 0 and 7, for example, 0, 1, 2, 3, 4, 5, 6, 7;

[0014] p is an integer between 0 and 4, such as 0, 1, 2, 3, 4.

[0015] Preferably, R6 and R7 are -CH3.

[0016] Preferably, x, y, m, n, and P are all non-zero.

[0017] Preferably, the nucleic acid drug molecule is one or more of mRNA, siRNA, ASO, or pDNA (plasmid DNA).

[0018] More preferably, an ionizable lipid compound, wherein the ionizable lipid compound is:

[0019]

[0020] The present invention also provides a nucleic acid drug molecule delivery system, wherein the nucleic acid drug molecule delivery system comprises an ionizable lipid compound, wherein the ionizable lipid compound is one or more of the above-mentioned ionizable lipid compounds.

[0021] Preferably, the nucleic acid drug molecule is one or more of mRNA, siRNA, ASO, linear DNA, or circular plasmid DNA.

[0022] 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), even more preferably 1:(5-30), and even more preferably 1:(5-20).

[0023] More preferably, the nucleic acid drug delivery system is a nanolipid particle, and the average size of the nanolipid particle is 50nm to 200nm, for example, 50nm, 80nm, 100nm, 120nm, 140nm, 160nm, 180nm and 200nm.

[0024] More preferably, the polydispersity index of the nanolipid particles is ≤0.4.

[0025] Furthermore, the average size of the nanolipid particles is 50 nm to 150 nm.

[0026] Furthermore, the polydispersity index of the nanolipid particles is ≤0.3.

[0027] Preferably, the ionizable lipid compound may be selectively modified with a targeting substance, which includes, but is not limited to, a ligand of a receptor molecule such as folic acid or mannose, and one or more of monoclonal or polyclonal antibodies, single-chain antibodies, nanobodies, aptamers, peptides or peptide analogs. The modification of the ionizable lipid compound by the targeting substance includes covalent coupling, mixing of non-covalent bonds, and / or binding of other chemical bonds such as coordination bonds.

[0028] Preferably, the nucleic acid drug delivery system further includes an auxiliary molecule selectively modified with a target substance, wherein the molar ratio of the ionizable lipid compound to the auxiliary molecule is (0.1-1):(0.1-1), more preferably (0.5-1):(0.5-1).

[0029] The auxiliary molecules include auxiliary lipids or lipid molecules commonly used in the art.

[0030] Preferably, the auxiliary molecules include, but are not limited to, cholesterol, calcipotriol, stigmasterol, β-sitosterol, betulinol, lupeol, ursolic acid, oleanolic acid, dioleoylphosphatidylcholine, distearylphosphatidylcholine, 1-stearoyl-2-oleoyllecithin, dioleoylphosphatidylethanolamine, (1,2-dioleoyloxypropyl)trimethylammonium chloride, didecyldimethylammonium bromide, 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine, dipalmitoylphosphatidylethanolamine-methoxy polyethylene glycol 5000, and distearylphosphatidylethanolamine-polyethylene glycol 2000, or one or more of these.

[0031] Preferably, the targeting substance is a ligand of a receptor molecule, such as folic acid or mannose, or one or more of any form of antibody, aptamer, or polypeptide.

[0032] In this invention, conventional methods in the art can be used to target and modify ionizable lipid compounds or auxiliary molecules.

[0033] According to some implementation methods, Mal-PEG2000-DSPE and peptides can be dissolved in ultrapure water, stirred and reacted for 48 hours, and then concentrated by dialysis to obtain peptide-PEG2000-DSPE, thus obtaining a targeted auxiliary molecule.

[0034] Furthermore, ionizable lipid molecules: cholesterol: DOPE: polypeptide-PEG2000-DSPE are dissolved in anhydrous ethanol and mixed with DNA or RNA to form polypeptide-modified liposomes.

[0035] Preferably, the nucleic acid drug delivery system is an injectable formulation.

[0036] Preferably, the nucleic acid drug delivery system further includes additives, which include stabilizers and / or diluents.

[0037] Preferably, the amount of the additive added is 1% to 20% of the total mass of the injection.

[0038] The additives mentioned can be those commonly used in the art.

[0039] Preferably, the stabilizer includes, but is not limited to, sucrose or trehalose.

[0040] 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, and Tris hydrochloride buffer.

[0041] Preferably, the nucleic acid drug delivery system further includes the addition of other immune adjuvants. These adjuvants include, but are not limited to, self-developed or commercially available adjuvants, such as QS-21, MPL, CpG, and RNA-based adjuvants.

[0042] Preferably, the nucleic acid drug delivery system is administered via local intramuscular, subcutaneous, endothelial, or intratumoral injection or perfusion, or via intravenous injection.

[0043] The nucleic acid drug delivery system of this invention can deliver nucleic acid drug molecules such as mRNA, siRNA, or pDNA into the body via various administration methods, including local intramuscular, subcutaneous, endothelial, intratumoral, and perfusion, as well as systemic administration methods such as intravenous injection. It can even target the spleen / lungs, effectively expressing therapeutic protein drugs or antigens in cells to prevent or treat diseases. This nucleic acid drug delivery system significantly reduces the hepatic accumulation effect of liposomes. By selecting an appropriate administration method, nucleic acids can be efficiently delivered to the spleen or lungs and effectively translated into target molecules. Therefore, the potential of this nucleic acid drug delivery system lies in its suitability for delivering nucleic acid drugs into the body to function as preventative or therapeutic vaccines.

[0044] The present invention also provides the use of the ionizable lipid compound or a combination thereof or the nucleic acid drug molecule delivery system in nucleic acid drugs or nucleic acid drug delivery.

[0045] An mRNA vaccine delivery system comprising the ionizable lipid compound or a combination thereof, the mRNA vaccine delivery system being capable of delivering mRNA molecules into the body to achieve antigen expression.

[0046] Preferably, the mRNA vaccine delivery system has tissue / organ targeting and / or appropriate immune system activation properties.

[0047] The nucleic acid delivery system of the present invention is suitable for various drug administration methods such as intramuscular injection. It has low liver toxicity and high nucleic acid delivery efficiency, while also having good tissue and organ targeting and / or good immunogenicity.

[0048] Compared with the prior art, the present invention has the following advantages:

[0049] The delivery system formed by the ionizable lipid compounds and their compositions of this invention can effectively encapsulate several forms of nucleic acids, such as mRNA, into nanoparticles and deliver them to cells in vivo or in vitro to achieve high-level protein translation. Its nucleic acid delivery offers advantages such as high in vivo delivery efficiency and good tissue / organ targeting, while also effectively stimulating immune system activity. Therefore, these ionizable lipid compounds and their compositions can be used for applications such as protein replacement therapy, preventative or therapeutic vaccines, etc. Attached Figure Description

[0050] Figure 1 This is the mass spectrum of compound 1;

[0051] Figure 2 The NMR spectrum of compound 1;

[0052] Figure 3 This is the mass spectrum of compound 2;

[0053] Figure 4 The NMR spectrum of compound 2;

[0054] Figure 5 This is the mass spectrum of compound 3;

[0055] Figure 6 The NMR spectrum of compound 3;

[0056] Figure 7 This is the mass spectrum of compound 4;

[0057] Figure 8 The NMR spectrum of compound 4 is shown below.

[0058] Figure 9 This is the mass spectrum of compound 5;

[0059] Figure 10 The NMR spectrum of compound 5;

[0060] Figure 11 This is the mass spectrum of compound 6;

[0061] Figure 12 The NMR spectrum of compound 6;

[0062] Figure 13 This is the mass spectrum of compound 7;

[0063] Figure 14 The NMR spectrum of compound 7;

[0064] Figure 15 This is the mass spectrum of compound 8;

[0065] Figure 16 The NMR spectrum of compound 8;

[0066] Figure 17 Particle size distribution and transmission electron microscopy (TEM) images of representative LNPs;

[0067] Figure 18 The graph shows the in vivo delivery efficiency of representative LNPs in mice and the expression of Fluc in various organs.

[0068] Figure 19 This diagram illustrates the principle of antibody-conjugated LNPs and shows the particle size changes after conjugation.

[0069] Figure 20 This is a diagram showing the organ targeting effect of LNPs after antibody conjugation.

[0070] Figure 21 This is a graph showing the particle size variation of LNP with the adjuvant.

[0071] Figure 22 A diagram illustrating the immunization administration process and efficacy analysis for BALB / c mice. Detailed Implementation

[0072] The present invention will be further described below with reference to embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0073] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the experimental materials used, unless otherwise specified, were purchased from conventional biochemical reagent manufacturers.

[0074] Example 1

[0075] Synthetic route of compound 1:

[0076]

[0077] Synthesis of compound 1-1:

[0078] 6-Bromhexanoic acid (10 g, 51.27 mmol) and (±)-β-citronellol, 3,7-dimethyl-6-octen-1-ol (7.0 g, 51.28 mmol) were dissolved in DCM (140 ml) in a reaction flask. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (9.79 g, 51.27 mmol) and 4-dimethylaminopyridine (1.25 g, 10.25 mmol) were added sequentially. The mixture was stirred at room temperature for 72 h. The reaction was stopped by TLC. The solvent DCM in the reaction solution was concentrated to dryness, dissolved in ethyl acetate, and washed three times with saturated sodium bicarbonate aqueous solution and saturated NaCl solution. The combined organic layers were dried over anhydrous Na₂SO₄ to obtain the crude product. The crude product was purified by silica gel column chromatography (elution ratio: petroleum ether / ethyl acetate = 120 / 1) to give a colorless oily compound 1-1 (9.74 g, yield: 57%).

[0079] Synthesis of compounds 1-2:

[0080] 8-Bromooctanoic acid (10 g, 44.82 mmol) and heptadecano-9-ol (14.9 g, 58.27 mmol) were dissolved in DCM (140 ml) in a reaction flask, followed by the sequential addition of 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 48 h. The reaction was stopped by TLC. The solvent DCM in the reaction solution was concentrated to dryness, dissolved in ethyl acetate, and washed three times with saturated sodium bicarbonate aqueous solution and saturated NaCl solution. The combined organic layers were dried over anhydrous Na₂SO₄ to obtain the crude product. The crude product was purified by silica gel column chromatography (elution ratio: petroleum ether / ethyl acetate = 150 / 1) to give a colorless oily compound 1-2 (7.20 g, yield: 34.8%).

[0081] Synthesis of compounds 1-3:

[0082] 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). The mixture was stirred at 63 °C for 24 h. After the reaction was completed by TLC, the solvent ethanol was concentrated, dissolved in ethyl acetate, and then extracted three times with water. The mixture was washed three times with saturated brine. The combined organic layers were dried over anhydrous Na2SO4. The crude product was purified by silica gel chromatography (elution ratio: dichloromethane / methanol = 100 / 1) to give a pale yellow oily compound 1-3 (1.50 g, yield: 50.6%).

[0083] Synthesis of Compound 1:

[0084] Compounds 1-3 (1.40 g, 3.07 mmol) and 1-1 (2.04 g, 6.14 mmol) were dissolved in EtOH (2 mL) in a reaction flask, followed by the addition of DIPEA (0.79 g, 6.14 mmol). The mixture was stirred at 63 °C for 24 h. After TLC detection, the reaction was completed. The solvent ethanol was concentrated, dissolved in ethyl acetate, and then washed three times with saturated sodium bicarbonate aqueous solution and saturated brine. The combined organic layers were dried over anhydrous Na₂SO₄. The crude product was purified by silica gel chromatography (elution buffer volume ratio: dichloromethane / methanol = 1:10 / 1) to give compound 1 (0.54 g, yield: 25%), a pale yellow oil. The mass spectrum of compound 1 is shown below. Figure 1 See MRI Figure 2 , 1 H 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=79.1 Hz, 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).

[0085] Example 2

[0086] Synthetic route of compound 2:

[0087]

[0088] Synthesis of compound 2-1: Compound 2-1 is the same compound as compound 1-1, and the synthesis method is the same as that of compound 1-1 in Example 1.

[0089] Synthesis of compound 2-2: Compound 2-2 is the same compound as compound 1-2. The synthesis method is the same as that of compound 1-2 in Example 1, and will not be repeated here.

[0090] Synthesis of compounds 2-3:

[0091] 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). The mixture was stirred at 63 °C for 24 h. After the reaction was completed by TLC, the solvent ethanol was concentrated, dissolved in ethyl acetate, and then extracted three times with water. The mixture was washed three times with saturated brine. The combined organic layers were dried over anhydrous Na2SO4. The crude product was purified by silica gel chromatography (elution ratio: dichloromethane / methanol = 60 / 1) to give a pale yellow oily compound 2-3 (1.80 g, yield: 62.5%).

[0092] Synthesis of compound 2:

[0093] Compounds 2-3 (1.50 g, 3.4 mmol) and 2-1 (2.26 g, 6.8 mmol) were dissolved in EtOH (2 mL) in a reaction flask, followed by the addition of DIPEA (0.88 g, 6.8 mmol). The mixture was stirred at 63 °C for 24 h. After TLC detection, the reaction was complete. The solvent ethanol was concentrated, dissolved in ethyl acetate, and then washed three times with saturated sodium bicarbonate aqueous solution and saturated brine. The combined organic layers were dried over anhydrous Na₂SO₄. The crude product was purified by silica gel chromatography (dichloromethane / methanol = 65 / 1) to give a pale yellow oily compound 30 (0.78 g, yield: 22%). The mass spectrum of compound 2 is shown below. Figure 3 See MRI Figure 4 , 1 HNMR (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).

[0094] Example 3

[0095] Synthetic route of compound 3:

[0096]

[0097] Synthesis of compound 3-1:

[0098] 6-Bromhexanoic acid (10 g, 51.27 mmol) and (E)-3,7-dimethyl-2,6-octadien-1-ol (7.9 g, 51.28 mmol) were dissolved in DCM (140 ml) in a reaction flask. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (9.8 g, 51.28 mmol) and DMAP (1.25 g, 10.26 mmol) were added sequentially, and the mixture was stirred at room temperature for 72 h. After the reaction was completed by TLC, the solvent was concentrated to dryness, and the mixture was washed three times with saturated NaHCO3 aqueous solution and saturated NaCl aqueous solution. The combined organic layers were dried over anhydrous Na2SO4. The crude product was purified by silica gel chromatography (elution ratio: petroleum ether / ethyl acetate = 120 / 1) to give compound 3-1 (9.91 g, yield: 58%).

[0099] Synthesis of compound 3-2: Compound 3-2 is the same compound as compound 1-2, and the synthesis method is the same as that of compound 1-2 in Example 1.

[0100] Synthesis of compound 3-3: Compound 3-3 is the same compound as compound 1-3. The synthesis method is the same as that of compound 1-3 in Example 1.

[0101] Synthesis of compound 3:

[0102] 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 the addition of DIPEA (0.17 g, 1.32 mmol). The mixture was stirred at 63 °C for 24 h. After TLC detection, the reaction was completed. The solvent ethanol was concentrated, dissolved in ethyl acetate, and then washed three times with saturated NaHCO3 aqueous solution and saturated brine. The combined organic layers were dried over anhydrous Na2SO4. The crude product was purified by preparative thin-layer chromatography (Prep TLC) (elution buffer volume ratio: dichloromethane / methanol = 7 / 1) to obtain a pale yellow oily compound 3 (0.16 g, yield: 34%). The mass spectrum of compound 3 is shown below. Figure 5 See MRI 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,1 8H), 1.50 (d, J = 5.8Hz, 5H), 1.36 (s, 5H), 1.25 (s, 27H), 0.88 (t, J = 6.8Hz, 7H).

[0103] Example 4

[0104] Synthetic route of compound 4:

[0105]

[0106] Synthesis of compound 4-1: Compound 4-1 is the same compound as compound 1-2, and the synthesis method is the same as that of compound 1-2 in Example 1.

[0107] Synthesis of compound 4:

[0108] 4-Aminocyclohexanol (0.30 g, 2.61 mmol) and compound 4-1 (3.0 g, 6.53 mmol) were dissolved in EtOH (2 mL) in a reaction flask, followed by the addition of DIPEA (1.01 g, 7.83 mmol). The mixture was stirred at 63 °C for 24 h. After TLC detection, the reaction was completed. The solvent ethanol was concentrated, dissolved in ethyl acetate, and then washed three times with saturated sodium bicarbonate aqueous solution and saturated brine. The combined organic layers were dried over anhydrous Na₂SO₄. The crude product was purified by preparative thin-layer chromatography (Prep TLC) (elution buffer volume ratio: dichloromethane / methanol = 7 / 1) to obtain a brown oily compound 4 (0.25 g, yield: 11%). The mass spectrum of compound 4 is shown below. Figure 7 See MRI Figure 8 , 1 H 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).

[0109] Example 5

[0110] Synthetic route of compound 5:

[0111]

[0112] Synthesis of compound 5-1: Compound 5-1 is the same compound as compound 1-2, and the synthesis method is the same as that of compound 1-2 in Example 1.

[0113] Synthesis of compound 5:

[0114] 1-(3-aminopropyl)imidazole (0.30 g, 2.4 mmol) and compound 5-1 (2.77 g, 6.0 mmol) were dissolved in EtOH (2 mL) in a reaction flask, followed by the addition of DIPEA (0.93 g, 7.2 mmol). The mixture was stirred at 63 °C for 24 h. After TLC detection, the reaction was completed. The solvent ethanol was concentrated, dissolved in ethyl acetate, and then washed three times with saturated sodium bicarbonate aqueous solution and saturated brine. The combined organic layers were dried over anhydrous Na₂SO₄. The crude product was purified by preparative thin-layer chromatography (Prep TLC) (elution buffer volume ratio: dichloromethane / methanol = 7 / 1) to obtain a brown oily compound 5 (0.32 g, yield: 15%). The mass spectrum of compound 5 is shown in [Figure number missing]. Figure 9 See MRI Figure 10 , 1 H 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).

[0115] Example 6

[0116] Synthetic route of compound 6:

[0117]

[0118] Synthesis of compound 6-1:

[0119] 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 dissolved in DCM (140 ml) in a reaction flask, followed by the sequential addition of 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 72 h. The reaction was stopped by TLC. The solvent DCM in the reaction solution was concentrated, washed three times with saturated sodium bicarbonate aqueous solution and saturated brine, and the combined organic layers were dried over anhydrous Na₂SO₄ to obtain the crude product. The crude product was purified by silica gel column chromatography (elution ratio: petroleum ether / ethyl acetate = 100 / 1) to give a colorless oily compound 6-1 (9.8 g, yield: 53%).

[0120] Synthesis of compound 6-2: Compound 6-2 is the same compound as compound 1-2, and the synthesis method is the same as that of compound 1-2 in Example 1.

[0121] Synthesis of compound 6-3:

[0122] 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). The mixture was stirred at 63 °C for 24 h. After the reaction was completed by TLC, the solvent ethanol was concentrated, dissolved in ethyl acetate, and then extracted three times with water. The mixture was washed three times with saturated brine, and the combined organic layers were dried over anhydrous Na2SO4. The crude product was purified by silica gel chromatography (elution buffer volume ratio: dichloromethane / methanol = 40 / 1) to give a pale yellow oily compound 6-3 (1.45 g, yield: 45%).

[0123] Synthesis of compound 6:

[0124] Compound 6-3 (0.60 g, 1.21 mmol) and compound 6-1 (0.87 g, 2.42 mmol) were dissolved in EtOH (1 mL) in a reaction flask, followed by the addition of DIPEA (0.31 g, 2.42 mmol). The mixture was stirred at 63 °C for 24 h. After TLC detection, the reaction was completed. The solvent ethanol was concentrated, dissolved in ethyl acetate, and then washed three times with saturated sodium bicarbonate aqueous solution and saturated brine. The combined organic layers were dried over anhydrous Na₂SO₄. The crude product was purified by preparative thin-layer chromatography (Prep TLC) (elution buffer volume ratio: dichloromethane / methanol = 6 / 1) to obtain a pale yellow oily compound 6 (0.19 g, yield: 20%). The mass spectrum of compound 6 is shown below. Figure 11 See MRI Figure 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).

[0125] Example 7

[0126] Synthetic route of compound 7:

[0127]

[0128] Synthesis of compound 7-1: Compound 7-1 is the same compound as compound 6-1, and the synthesis method is the same as that of compound 6-1 in Example 6.

[0129] Synthesis of compound 7-2: Compound 7-2 is the same compound as compound 1-2, and the synthesis method is the same as that of compound 1-2 in Example 1.

[0130] Synthesis of compound 7-3:

[0131] 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). The mixture was stirred at 63 °C for 24 h. After the reaction was completed by TLC, the solvent ethanol was concentrated, dissolved in ethyl acetate, and then extracted three times with water. The mixture was washed three times with saturated brine, and the combined organic layers were dried over anhydrous Na2SO4. The crude product was purified by silica gel chromatography (elution ratio: dichloromethane / methanol = 60 / 1) to give a pale yellow oily compound 7-3 (1.50 g, yield: 46%).

[0132] Synthesis of compound 7:

[0133] Compound 7-3 (0.60 g, 1.20 mmol) and compound 7-1 (0.86 g, 2.40 mmol) were dissolved in EtOH (1 mL) in a reaction flask, followed by the addition of DIPEA (0.31 g, 2.40 mmol). The mixture was stirred at 63 °C for 24 h. After TLC detection, the reaction was completed. The solvent ethanol was concentrated, dissolved in ethyl acetate, and then washed three times with saturated sodium bicarbonate aqueous solution and saturated brine. The combined organic layers were dried over anhydrous Na₂SO₄. The crude product was purified by preparative thin-layer chromatography (Prep TLC) (elution buffer volume ratio: dichloromethane / methanol = 6 / 1) to obtain a pale yellow oily compound 7 (0.23 g, yield: 24%). The mass spectrum of compound 7 is shown below. Figure 13 See MRI Figure 14 , 1 H 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).

[0134] Example 8

[0135] Synthetic route of compound 8:

[0136]

[0137] Synthesis of compound 8-1: Compound 8-1 is the same compound as compound 6-1, and the synthesis method is the same as that of compound 6-1 in Example 6.

[0138] Synthesis of compound 8-2: Compound 8-2 is the same compound as compound 1-2, and the synthesis method is the same as that of compound 1-2 in Example 1.

[0139] Synthesis of compound 8-3:

[0140] 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). The mixture was stirred at 63 °C for 24 h. After the reaction was completed by TLC, the solvent ethanol was concentrated, dissolved in ethyl acetate, and then extracted three times with water. The mixture was washed three times with saturated brine, and the combined organic layers were dried over anhydrous Na2SO4. The crude product was purified by silica gel chromatography (elution buffer volume ratio: dichloromethane / methanol = 60 / 1) to give a pale yellow oily compound 8-3 (1.90 g, yield: 65.5%).

[0141] Synthesis of compound 8:

[0142] Compound 8-3 (1.70 g, 3.4 mmol) and compound 2-1 (2.26 g, 6.8 mmol) were dissolved in EtOH (2 mL) in a reaction flask, followed by the addition of DIPEA (0.88 g, 6.8 mmol). The mixture was stirred at 63 °C for 24 h. After TLC detection, the reaction was completed. The solvent ethanol was concentrated, dissolved in ethyl acetate, and then washed three times with saturated sodium bicarbonate aqueous solution and saturated brine. The combined organic layers were dried over anhydrous Na₂SO₄. The crude product was purified by silica gel chromatography (dichloromethane / methanol = 65 / 1) to give a pale yellow oily compound 30 (0.78 g, yield: 22%). The mass spectrum of compound 8 is shown below. Figure 15 See MRI Figure 16 , 1 HNMR(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).

[0143] Example 9

[0144] Based on the above reaction principle, compounds 9 to 34 were prepared according to the following synthetic route.

[0145] Synthetic route of compound 9:

[0146]

[0147] Synthetic route of compound 10:

[0148]

[0149] Synthetic route of compound 11:

[0150]

[0151] Synthetic route of compound 12:

[0152]

[0153] Synthetic route of compound 13:

[0154]

[0155] Synthetic route of compound 14:

[0156]

[0157] Synthetic route of compound 15:

[0158]

[0159] Synthetic route of compound 16:

[0160]

[0161] Synthetic route of compound 17:

[0162]

[0163] Synthetic route of compound 18:

[0164]

[0165] Synthetic route of compound 19:

[0166]

[0167] Synthetic route of compound 20:

[0168]

[0169] Synthetic route of compound 21:

[0170]

[0171] Synthetic route of compound 22:

[0172]

[0173] Synthetic route of compound 23:

[0174]

[0175] Synthetic route of compound 24:

[0176]

[0177] Synthetic route of compound 25:

[0178]

[0179] Synthetic route of compound 26:

[0180]

[0181] Synthetic route of compound 27:

[0182]

[0183] Synthetic route of compound 28:

[0184]

[0185] Synthetic route of compound 29:

[0186]

[0187] Synthetic route of compound 30:

[0188]

[0189] Synthetic route of compound 31:

[0190]

[0191] Synthetic route of compound 32:

[0192]

[0193] Synthetic route of compound 33:

[0194]

[0195] Synthetic route of compound 34:

[0196]

[0197] Example 10: Preparation, particle size and potential determination of LNP-mRNA nanolipid particles:

[0198] Using anhydrous ethanol as a solvent, the compounds prepared in the examples were dissolved and mixed with DSPC, DMG-PEG2000 and cholesterol in a molar ratio of 50:10:1.5:38.5 to obtain liposome raw material solutions. The sum of the concentrations of each component was controlled to be 50 mM. After complete dissolution and mixing, the solutions were stored at -20°C.

[0199] The mRNA expressing Luciferase fluorescent protein was dissolved in 25 mM sodium acetate buffer at a pH of approximately 5.2 to prepare a nucleic acid preparation with a final concentration of approximately 0.1 mg / mL.

[0200] The prepared liposome raw material solution and nucleic acid preparation were mixed rapidly using a Nano Assemblr microfluidic system (either via vortexing or by volume ratio of approximately 4:1) at a total flow rate of 12 mL / min, forming uniform and stable nanoliposome particles. The pH of the nanoliposome particles was then rapidly changed from 5.2 to 7.0–7.4. Specifically, the particles were diluted 20-fold with PBS buffer (pH 7.2) or sodium acetate buffer (pH 7.4), and then concentrated using a 10 kDa ultrafiltration tube. The centrifuge speed should not exceed the maximum speed limit of the ultrafiltration tube. After 2–3 buffer changes, the pH of the nanoliposome solution was approximately 7.2–7.4. The solution was then concentrated to a final concentration of approximately 200 mM and stored at 4°C for later use.

[0201] The particle size and PDI of nanoliposomes were determined using a Zetasizer Nano ZS (Malvern, Worcestershire, UK). Particle size was measured by diluting the nanoliposome solution 50-fold with 1×PBS. Zeta potential was determined by diluting the nanoliposomes in 15 mM PBS. Encapsulation efficiency was determined using the Quant-It RiboGreen RNA Quantification Kit on a Modulus microwell multifunction analyzer.

[0202] like Figure 17 As shown in Table 1, the particle size of the prepared representative liposomes is around 100 nm, and the PDI is less than 0.2. Transmission electron microscopy shows that the particle size of LNP-8 is also around 100 nm. Figure 17 (B)

[0203] Table 1

[0204] 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

[0205] The nanolipid particles prepared by compounds 9 to 34 according to the above preparation method have a particle size in the range of 70 to 120 nm, an encapsulation efficiency of over 88%, and a potential between -12 and 10 mV.

[0206] Example 11: In vivo targeting experiment of nanolipid particles:

[0207] Nanoliposomes encapsulating mRNA were prepared via intramuscular injection according to the preparation method in Example 10 (those containing compound 1 were designated LNP-1, those containing compound 2 were designated LNP-2, and so on). The mRNA was mRNA expressing Luciferase fluorescent protein, and the amount of mRNA was 60 μg. The total amount of ionizable liposome compound, DSPC, DMG-PEG2000, and cholesterol was 600 μg. The liposome environment was rapidly converted using 200 μL of neutral PBS buffer and quickly injected into the medial hind limb muscles of 6-8 week old female Babl / c mice, with 30 μg of mRNA injected into each hind limb, or 50 μg of mRNA was injected intravenously into 6-8 week old female Babl / c mice.

[0208] The fluorescence expression of mRNA in mice after intravenous injection was analyzed by integrating the fluorescence intensity within the circle and obtaining the fluorescence intensity value. Figure 18 The fluorescence expression intensities of some representative LNPs are shown in Table 2. Figure 18 The study also showed the in vivo distribution of Fluorescence in some representative LNPs. Fluorescence intensity was closely related to the delivery efficiency of liposomes; the stronger the fluorescence, the higher the delivery efficiency of liposomes. Figure 18 The results showed that all the nanolipid particles encapsulating mRNA were able to deliver and successfully express mRNA in vivo, with LNP-5 and LNP-7 showing lung targeting and other lipid molecules showing liver targeting.

[0209] Table 2

[0210]

[0211]

[0212] The fluorescence expression intensity of mRNA delivered by the nanolipid particles prepared by compounds 9 to 34 according to the above preparation method can basically reach the same level as that of LNP-1 to LNP-8.

[0213] Example 12: Experiment on improving targeting by modifying LNP with targeting molecules:

[0214] Taking the conjugation of purified CD3e or CD4 monoclonal antibodies with T-cell targeting to LNP-1 as an example, we invented an improved post-insertion technique. First, the antibody is activated with an NHS group. Unconjugated NHS or NHS-lipid is removed using a G-25 Sephadex Quick Spin Protein column. Then, the activated antibody molecule is inserted into the LNP-1 surface and purified using a Sepharose CL-4B gel filtration column. The antibody-conjugated sample is stored at 4°C. A schematic diagram of the antibody modification on the LNP-1 surface is shown below. Figure 19 As shown, after antibody conjugation, the distribution of LNP-1 in various organs is as follows: Figure 20 As shown, its expression level decreases in the liver and increases significantly in the spleen.

[0215] Example 13: Addition of adjuvant molecules to nanolipid particles and animal immune experiments:

[0216] Preparation and particle size determination of immunolipid nanoparticles:

[0217] Using influenza virus HA antigen protein as the immunogenic antigen in animal immunization experiments, its mRNA was first prepared and then mixed with lipids and adjuvant molecules. The key was to effectively combine the adjuvant and lipid components. For lipid-soluble adjuvant molecules, such as self-made sugar-containing adjuvant molecules and commercially available monophosphoryl 3-deacyl lipid A (MPL), they were directly added as LNP components during formulation preparation. For water-soluble adjuvant molecules, such as saponin QS-21 and oligodeoxynucleotide CpG, the antigen protein mRNA and adjuvant molecules were co-dissolved to form a nucleic acid formulation. Then, by controlling the mass ratio of lipid components to nucleic acids and the volume ratio of the two phases, the two-phase solutions were rapidly mixed using vortexing or other methods such as a microfluidic system to form homogeneous and stable LNP-mRNA lipid nanoparticles (with parameters such as particle size and potential meeting requirements). Lipid nanoparticles without adjuvant addition were labeled LNP-HA mRNA, while those with adjuvant addition were labeled LNP-adj-HA mRNA. Particle size changes before and after adjuvant addition are as follows: Figure 21 As shown, Figure 12 Figure A shows the particle size of LNPs encapsulated with mRNA without adjuvant, Figure B shows the particle size of LNPs encapsulated with mRNA with adjuvant MPLA-HA, and Figure C shows the particle size of LNPs encapsulated with mRNA with adjuvant G-MPLA-HA. It can be seen that the addition of adjuvant MPL increases the particle size of LNPs.

[0218] Animal immunization experiments:

[0219] The prepared nucleic acid vaccines LNP-HAmRNA and LNP-adj-HAmRNA were administered intramuscularly to BALB / c mice at 6-8 weeks of age, at a dose of 30 μg. An equal volume of saline injection was used as a negative control, and the immunogenicity of the vaccine formulations with and without adjuvants was compared in parallel. The experiment was evaluated by detecting the level of SARS-CoV-2 neutralizing antibodies in the serum of immunized mice. Following the standards for SARS-CoV-2 vaccines, a second immunization was performed on day 21 after the first immunization, and serum was collected on day 28 for ELISA detection of serum IgG antibody levels. The neutralizing antibody titer results 7 days after the second immunization are shown below. Figure 22 The results showed that LNP-HAmRNA exhibited a significant IgG titer, and the addition of adjuvants to LNP-adj-HAmRNA (LNP-HAmRNA-MPLA and LNP-HAmRNA-G-MPLA in the figure) significantly increased the IgG titer, indicating that lipid nanoparticles have good immunogenicity and that the addition of adjuvants makes them better immune delivery carriers for mRNA.

[0220] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand and implement the invention. However, this description should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be included within the scope of protection of the invention.

Claims

1. An ionizable lipid compound, characterized in that, The ionizable lipid compound is: or .

2. A nucleic acid drug molecule delivery system, characterized in that, The nucleic acid drug delivery system includes the ionizable lipid compound of claim 1.

3. The nucleic acid drug delivery system according to claim 2, characterized in that, The ionizable lipid compound may be selectively modified with a targeting substance, which includes one or more of the following: ligands of receptor molecules, monoclonal or polyclonal antibodies, single-chain antibodies, nanobodies, aptamers, peptides or peptide analogs. The modification of the ionizable lipid compound by the targeting substance includes covalent coupling, non-covalent mixing, and / or other chemical bond binding.

4. The nucleic acid drug molecule delivery system according to claim 2, characterized in that, The nucleic acid drug delivery system further includes auxiliary molecules selectively modified with targeting substances, wherein the molar ratio of the ionizable lipid compound to the auxiliary molecules is (0.1~1):(0.1~1); the auxiliary molecules include cholesterol, calcipotriol, stigmasterol, β-sitosterol, betulinol, lupeol, ursolic acid, oleanolic acid, dioleoylphosphatidylcholine, distearylphosphatidylcholine, 1-stearoyl-2-oleoyllecithin, dioleoylphosphatidylethanolamine, (1,2-dioleoyloxypropyl)trimethylammonium chloride, didecyldimethylammonium bromide, 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine, dipalmitoylphosphatidylethanolamine-methoxy polyethylene glycol 5000, and distearylphosphatidylethanolamine-polyethylene glycol 2000. One or more of the following; the target substance is one or more of the following: a ligand, antibody, aptamer, or polypeptide of a receptor molecule.

5. The nucleic acid drug molecule delivery system according to claim 2, characterized in that, The nucleic acid drug 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 delivery system is a lipid nanoparticle, and the average size of the lipid nanoparticle is 50 nm to 200 nm.

6. The nucleic acid drug molecule delivery system according to claim 2, characterized in that, The nucleic acid drug delivery system is an injectable formulation. The system further includes additives, which include stabilizers and / or diluents. The amount of additives added is 1% to 20% of the total mass of the injectable formulation. The stabilizers include sucrose or trehalose. The diluents include one or more of phosphate buffer, acetate buffer, citrate, and Tris hydrochloride buffer.

7. The nucleic acid drug molecule delivery system according to claim 2, characterized in that, The described nucleic acid drug delivery system also includes an immune adjuvant.

8. The nucleic acid drug molecule delivery system according to claim 2, characterized in that, The aforementioned nucleic acid drug delivery system delivers the drug via local intramuscular, subcutaneous, endothelial, or intratumoral injection or perfusion, or via intravenous injection.

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