Iitaconic acid mother nucleus-based ionizable lipid as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, relates to a drug delivery technology, and particularly relates to an ionizable lipid based on an itaconic acid mother nucleus or a pharmaceutically acceptable salt, an isomer, a solvate, a preparation method and application thereof. Experimental results show that the lipid nanoparticles prepared from the ionizable lipid based on the itaconic acid mother nucleus or pharmaceutically acceptable salts, isomers and solvates of the ionizable lipid are higher in transfection efficiency, good in encapsulation effect and high in drug delivery capacity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to drug delivery technology, specifically to a class of ionizable lipids based on an itaconic acid nucleus or pharmaceutically acceptable salts, isomers, solvates thereof, and preparation methods and applications thereof. Background Art

[0002] In recent years, research on RNA therapies, represented by mRNA vaccines, has made significant progress. However, RNA itself is sensitive to nucleases and has characteristics such as large size and negative charge, which makes it difficult for RNA to directly penetrate the cell membrane and enter the cell. The emergence of lipid nanoparticles (LNPs) has effectively solved the RNA delivery problem. LNPs are typically composed of four components: ionizable lipids, structural lipids, neutral lipids, and polymer lipids. Ionizable lipids play a crucial role in protecting RNA and facilitating its cytoplasmic transport.

[0003] Ionizable lipids are positively charged in acidic environments, allowing them to electrostatically bind to negatively charged RNA and aggregate it within lipid nanoparticles (LNPs). At physiological pH, however, they are neutral, minimizing toxicity. Following cellular uptake, LNPs are protonated within acidic endosomes, forming incompatible conical ion pairs with anionic phospholipids of the endosomal membrane, promoting membrane fusion and disruption, endosomal escape, and RNA release. Ionizable lipids are the core of LNP delivery systems, and their structure plays a decisive role in LNP delivery efficiency, targeting, and formulation stability.

[0004] Lipid nanoparticles based on ionizable lipids can be used to deliver a variety of nucleic acids, including small interfering RNA (siRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), microRNA (microRNA), and circular mRNA. Patent CN116082179A discloses ionizable lipids based on endogenous dicarboxylic acids, their preparation methods, and applications. These ionizable lipids are used to prepare lipid nanoparticles for effective nucleic acid delivery, enabling targeted gene therapy. However, the transfer rate of these ionizable lipids is not high enough, necessitating the design of novel ionizable lipids and the development of lipid nanoparticles with low toxicity, safe and efficient delivery, for the delivery of nucleic acid drugs to facilitate gene therapy. Summary of the Invention

[0005] In order to address the deficiencies of the prior art, the purpose of the present invention is to provide a class of ionizable lipids based on an itaconic acid core, and a preparation method and application thereof. The ionizable lipids based on an itaconic acid core provided by the present invention are used to prepare lipid nanoparticles to achieve efficient delivery of biologically active drugs such as nucleic acid drugs.

[0006] In order to solve the technical problems of the present invention, the present invention provides the following technical solutions: The first aspect of the technical solution of the present invention is to provide an ionizable lipid based on an itaconic acid nucleus represented by general formula (I) or a pharmaceutically acceptable salt, isomer or solvate thereof. Wherein, n is 2 or 3, and R is selected from a straight-chain alkyl group having 9 to 16 carbon atoms.

[0007] Preferably, in the above-mentioned ionizable lipid based on itaconic acid nucleus or its pharmaceutically acceptable salt, isomer or solvate, the structural formula of the ionizable lipid based on itaconic acid nucleus is:

[0008] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention formed with an acid or base that is suitable for pharmaceutical use. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts are salts formed with a compound of the present invention and an acid. Suitable acids for forming salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid.

[0009] Another preferred salt is a salt of the compound of the present invention formed with a base, such as an alkali metal salt (e.g., sodium salt or potassium salt), an alkaline earth metal salt (e.g., magnesium salt or calcium salt), an ammonium salt (e.g., lower alkanolammonium salt and other pharmaceutically acceptable amine salts), for example, methylamine salt, ethylamine salt, propylamine salt, dimethylamine salt, trimethylamine salt, diethylamine salt, triethylamine salt, tert-butylamine salt, ethylenediamine salt, hydroxyethylamine salt, dihydroxyethylamine salt, trihydroxyethylamine salt, and amine salts formed from morpholine, piperazine, and lysine, respectively.

[0010] The term "solvate" refers to a complex in which the compound of the present invention is coordinated with solvent molecules to form a specific ratio.

[0011] The second aspect of the technical solution of the present invention is to provide a method for preparing the above-mentioned ionizable lipid based on itaconic acid core, comprising the following steps: ; Wherein, n is 2 or 3, and R is selected from a straight-chain alkyl group having 9 to 16 carbon atoms.

[0012] The above-mentioned method for preparing the ionizable lipid based on itaconic acid core is as follows: the compound represented by formula (II) and triethylamine are dissolved in dichloromethane and mixed evenly, the compound represented by formula (III) is dissolved in dichloromethane, the above mixture is added, and the reaction is carried out at 0°C for 15 minutes.

[0013] The third aspect of the technical solution of the present invention is to provide a lipid nanoparticle for drug delivery, which comprises the ionizable lipid based on the itaconic acid nucleus described in the first aspect or its pharmaceutically acceptable salt, isomer, solvate, neutral lipid, polyethylene glycol lipid and steroid lipid.

[0014] The neutral lipid is selected from at least one of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), palmitoyloleoylphosphatidylcholine phosphatidylcholine (POPC), dioleoylphosphatidylglycerol (DOPG), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylcholine (DLPC), diethyl pyrocarbonate (DEPC), dimyristoylphosphatidylcholine (DMPC) and egg yolk lecithin (EPC).

[0015] The polyethylene glycol lipid is at least one selected from the group consisting of dipalmitoylphosphatidylethanolamine-PEG (DPPE-PEG), 1,2-distearoyl-rac-glyceryl-3-methoxy-PEG (DSG-PEG), diacylglycerol-PEG (DAG-PEG), dioleoylphosphatidylethanolamine-PEG (DOPE-PEG), 2-[(polyethylene glycol)-2000]-N,N-diformylacetamide (ALC-0159), distearoylphosphatidylethanolamine-PEG (DSPE-PEG), dimyristylglycerol-PEG (DMG-PEG), and dimethacrylate-PEG (DMA-PEG). Preferably, dimyristylglycerol-PEG (DMG-PEG) is used.

[0016] The polyethylene glycol lipid is selected from at least one of dipalmitoylphosphatidylethanolamine-PEG (DPPE-PEG), 1,2-distearoyl-rac-glyceryl-3-methoxy-PEG (DSG-PEG), diacylglycerol-PEG (DAG-PEG), dioleoylphosphatidylethanolamine-PEG (DOPE-PEG), 2-[(polyethylene glycol)-2000]-N,N-diformylacetamide (ALC-0159), distearoylphosphatidylethanolamine-PEG (DSPE-PEG), dimyristylglycerol-PEG (DMG-PEG), and dimethacrylate-PEG (DMA-PEG). Preferably, dimyristylglycerol-PEG (DMG-PEG) is selected from the group consisting of:

[0017] In the above-mentioned lipid nanoparticles for drug delivery, the molar ratio of the ionizable lipid based on the itaconic acid nucleus or its pharmaceutically acceptable salt, its isomer, its solvate, neutral lipid, polyethylene glycol lipid and steroid lipid is (10-60):(10-50):(0.5-15):(20-60).

[0018] The fourth aspect of the technical solution of the present invention is to provide the use of the above-mentioned lipid nanoparticles for drug delivery as a delivery carrier for nucleic acid drugs.

[0019] The fifth aspect of the technical solution of the present invention is to provide a pharmaceutical composition, which includes an active ingredient and a carrier, wherein the active ingredient is a nucleic acid drug, and the carrier is a lipid nanoparticle for drug delivery described in the third aspect.

[0020] The pharmaceutical composition described above, wherein the mass ratio of the active ingredient to the carrier is 1:1-50.

[0021] The sixth aspect of the technical solution of the present invention is to provide the use of the pharmaceutical composition described in the fifth aspect in the preparation of gene drugs, wherein the nucleic acid drug is siRNA, mRNA, tRNA, rRNA, cDNA, ASO, plasmid DNA, microRNA or long non-coding RNA.

[0022] Compared with the prior art, the present invention has the following main advantages: 1. Experimental results show that the lipid nanoparticles prepared by the present invention using ionizable lipids based on an itaconic acid core or its pharmaceutically acceptable salts, isomers, or solvates have higher transfection efficiency, better encapsulation effect, and stronger drug delivery ability.

[0023] 2. The synthesis route of the ionizable lipid based on itaconic acid nucleus of the present invention is simple, the reaction conditions are mild, and the raw materials are simple and easily available. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the particle size measurement result of the lipid nanoparticles of the present invention.

[0025] Figure 2 This is the measurement result of the encapsulation efficiency of the lipid nanoparticles of the present invention.

[0026] Figure 3 This is the result of measuring the cell transfection ability of the lipid nanoparticles of the present invention. DETAILED DESCRIPTION

[0027] The embodiments of the present invention specifically describe methods for preparing the ionizable lipids based on an itaconic acid core according to the present invention, but these specific methods do not constitute any limitation to the present invention. The ionizable lipids based on an itaconic acid core according to the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art. Such combinations can be readily performed by those skilled in the art.

[0028] Unless otherwise specified, the raw materials and reagents used in the preparation process of the compounds of the present invention and the reagents used in the pharmacological experiments of the present invention can be purchased through commercial channels.

[0029] In the following examples, the experimental methods without specific conditions are generally based on conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0031] The first exemplary embodiment of the present invention provides an ionizable lipid having an itaconic acid core represented by the general formula (I) or a pharmaceutically acceptable salt, isomer, or solvate thereof: Wherein, n is 2 or 3, and R is selected from a straight-chain alkane having 9 to 16 carbon atoms.

[0032] In some embodiments, n is an integer from 2 to 5, preferably 2 or 3.

[0033] In some embodiments, R is selected from a linear alkane having 9 to 16 carbon atoms, and R is selected from 、 .

[0034] In some embodiments, the following compounds are included:

[0035] A second embodiment of the present invention provides a method for preparing the above-mentioned ionizable lipid based on an itaconic acid core, comprising obtaining a compound represented by formula (I) according to the following reaction scheme: Wherein, n is 2 or 3, and R is selected from a straight-chain alkane having 9 to 16 carbon atoms.

[0036] In some embodiments, the compound represented by formula (II) and triethylamine are dissolved in dichloromethane and mixed uniformly, the compound represented by formula (III) is dissolved in dichloromethane, and the above mixture is added and reacted at 0°C for 15 minutes.

[0037] In one or more embodiments, the molar ratio of the compound represented by formula (III), the compound represented by formula (II), and triethylamine is 1.0:(2.0-2.3):(2.5-3.0): In one or more embodiments, the organic solvent in the reaction system is at least one of dichloromethane, tetrahydrofuran, N,N-dimethylacetamide, dimethyl sulfoxide, chloroform, methanol, ether, pyridine, ethylene glycol dimethyl ether, benzene or toluene, preferably dichloromethane.

[0038] In one or more embodiments, after the reaction, silica gel column chromatography is performed for purification, wherein the mobile phase of the silica gel column chromatography is a mixed solution of dichloromethane and methanol or dichloromethane, methanol and water.

[0039] The third embodiment of the present invention provides a lipid nanoparticle, which is composed of the above-mentioned ionizable lipid based on the itaconic acid nucleus or its pharmaceutically acceptable salt, its isomer, its solvate, neutral lipid, polyethylene glycol lipid and steroid lipid.

[0040] The lipid nanoparticles of the present invention can be in the form of particles. The particle diameter can be in the range of 1 nm to 1000 nm, or in the range of 20 nm to 800 nm, or in the range of 50 nm to 500 nm, or in the range of 80 nm to 200 nm, or in the range of 1 nm to 100 nm, or in the range of 1 nm to 10 nm. When the particle size range of the particles is in the range of 1 nm to 1000 nm, they are nanoparticles commonly described in the art.

[0041] The lipid nanoparticles of the present invention can be prepared using any method known in the art, including but not limited to conventional liposome extrusion, thin film hydration, pipette mixing, vortex mixing, and microfluidic mixing methods, by mixing an aqueous solution of a bioactive substance with an organic solvent solution of a lipid compound.

[0042] In some embodiments, the molar content of the ionizable lipid based on an itaconic acid core, or a pharmaceutically acceptable salt, isomer, or solvate thereof, is 10-60%, preferably 15-45%, and more preferably 28-36%. The total molar content is the sum of the molar amounts of the ionizable lipid based on an itaconic acid core, or a pharmaceutically acceptable salt, isomer, or solvate thereof, the neutral lipid, the polyethylene glycol lipid, and the steroidal lipid.

[0043] In some embodiments, the neutral lipid is selected from at least one of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylglycerol (DOPG), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylcholine (DLPC), diethyl pyrocarbonate (DEPC), dimyristoylphosphatidylcholine (DMPC), and egg yolk phosphatidylcholine (EPC). Preferably, dioleoylphosphatidylcholine (DOPE) is used. The molar content of the neutral lipid is 10-50%, preferably 20-40%, and more preferably 30-35%.

[0044] In some embodiments, the polyethylene glycol lipid is selected from at least one of dipalmitoylphosphatidylethanolamine-PEG (DPPE-PEG), 1,2-distearoyl-rac-glyceryl-3-methoxy-PEG (DSG-PEG), diacylglycerol-PEG (DAG-PEG), dioleoylphosphatidylethanolamine-PEG (DOPE-PEG), 2-[(polyethylene glycol)-2000]-N,N-diformylacetamide (ALC-0159), distearoylphosphatidylethanolamine-PEG (DSPE-PEG), dimyristylglycerol-PEG (DMG-PEG), and dimethacrylate-PEG (DMA-PEG). Preferably, dimyristylglycerol-PEG (DMG-PEG) is used. The molar content of the polyethylene glycol lipid is 0.5-15%, preferably 0.5-5.0%, and more preferably 2.0-3.0%.

[0045] In some embodiments, the steroidal lipid is selected from at least one of cholesterol, avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, melanosterol, epicholesterol, ergosterol, fucoxosterol, hexahydroluminosterol, hydroxycholesterol; lanosterol, luminosterol, trehalosterol, sitostanol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, sulfocholic acid, deoxycholic acid, and lithocholic acid. Preferably, the steroidal lipid has a molar content of 20-60%, preferably 25-35%, and more preferably 30-35%.

[0046] In some embodiments, the molar ratio of the ionizable lipid based on itaconic acid core or its pharmaceutically acceptable salt, isomer, solvate, neutral lipid, polyethylene glycol lipid and steroid lipid is (10-60): (10-50): (0.5-15): (20-60), preferably (15-45): (20-40): (0.5-5.0): (25-35).

[0047] In some embodiments, an aqueous solvent is included, and the aqueous solvent is an acidic buffer. Furthermore, the buffer comprises at least one selected from citric acid, sodium citrate, acetic acid, sodium acetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, tris(hydroxymethyl)aminomethane-hydrochloric acid, potassium dihydrogen phosphate-sodium hydroxide, boric acid-borax, glycine-hydrochloric acid, phthalic acid-hydrochloric acid, potassium hydrogen phthalate, and sodium dihydrogen phosphate-citric acid, preferably citric acid.

[0048] In some embodiments, an organic solvent is included. The organic solvent is selected from at least one of methanol, ethanol, isopropanol, benzene, toluene, xylene, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene-cyclohexanone, chlorobenzene, dichlorobenzene, dichloromethane, ethyl ether, propylene oxide, acetone, methyl butyl ketone, methyl isobutyl ketone, acetonitrile, pyridine, phenol, styrene, perchloroethylene, trichloroethylene, ethylene glycol ether, and triethanolamine, preferably ethanol or isopropanol.

[0049] A fourth embodiment of the present invention provides a use of the lipid nanoparticles as a delivery vehicle for nucleic acid drugs.

[0050] A fifth embodiment of the present invention provides a pharmaceutical composition comprising an active ingredient and a carrier, wherein the active ingredient is a nucleic acid drug and the carrier is the lipid nanoparticles described above.

[0051] The nucleic acid drugs of the present invention include but are not limited to siRNA, linear mRNA, tRNA, rRNA, cDNA, ASO, plasmid DNA, circular RNA, microRNA, long non-coding RNA and other nucleic acid molecules and their analogs.

[0052] In some embodiments, the mass ratio of the ionizable lipid based on the itaconic acid core or its pharmaceutically acceptable salt, isomer, or solvate thereof to the nucleic acid drug in the lipid nanoparticles is (1-50):1, preferably (5-15):1, and further preferably 10:1.

[0053] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0054] Example 1 Synthesis of ionizable lipid compound IAR2-9 Step 1: Synthesis of intermediate 1b.

[0055] Imidazole (30 mmol) and tert-butyldimethylsilyl chloride (16.5 mmol) were dissolved in dichloromethane, and compound 1a (15 mmol) was slowly added. The mixture was stirred at room temperature for 3 h until the reaction was almost complete. The reaction solution was extracted with dichloromethane, washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain intermediate 1b (yield 90%).

[0056] Step ②: Synthesis of intermediate 1c.

[0057] Bromononane (9.65 mmol) and potassium carbonate (10.5 mmol) were dissolved in acetonitrile and mixed thoroughly. Compound 1b (4.5 mmol) was dissolved in acetonitrile and slowly added dropwise to the reaction mixture. The mixture was stirred and heated in an oil bath at 80°C. The reaction was allowed to react for 3 days until the reaction was essentially complete. The solvent was removed by rotary evaporation under reduced pressure. The resulting mixture was dissolved in 20 ml of dichloromethane, washed three times with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated and purified by silica gel column chromatography to obtain intermediate 1c (yield 50.1%).

[0058] Step ③: Synthesis of intermediate 1d.

[0059] Compound 1c (0.3 mmol) and tetrabutylammonium fluoride trihydrate (0.33 mmol) were separately dissolved in 2 ml of tetrahydrofuran. The mixture was stirred at 0°C for 1 hour, mixed thoroughly, and then warmed to room temperature and allowed to react for 4 hours until nearly complete. The solvent was removed by rotary evaporation under reduced pressure. The resulting mixture was dissolved in 20 ml of ethyl acetate, washed three times with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to provide intermediate 1d (yield 87%).

[0060] Step ④: Synthesis of intermediate 1e.

[0061] Place phosphorus pentachloride in a 25ml reaction flask and mix with itaconic acid. Place the mixture in a condenser reflux system equipped with an HCl absorber and stir to combine the reactants. The initial reaction is quite vigorous, rapidly releasing HCl. After the intense reaction, heat the reaction system to 130°C and reflux phosphorus oxychloride until the phosphorus pentachloride is completely dissolved. Continue heating for 15 minutes, then stop heating and cool. Vacuum distillation is performed to collect the yellow oily product to yield Intermediate 1e.

[0062] Step ⑤: Synthesis of the final product IAR2-9.

[0063] Compound 1e (0.2 mmol) and triethylamine (0.3 mmol) were dissolved in 6 ml of dichloromethane and stirred at 0°C overnight. Compound 1d (0.2 mmol) was weighed and dissolved in 6 ml of anhydrous dichloromethane. The mixture was added dropwise to the reaction system and allowed to react overnight at room temperature. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography to obtain the final product, IAR2-9 (yield: 65%).

[0064] 1 HNMR(400MHz,Chloroform-d)δ6.29(d,J=2.1Hz,2H),3.55(t,J=6.8Hz,2H),2.58(t,J=6.8Hz, 4H),2.40(t,J=7.4Hz,6H),2.06(d,J=1.8Hz,6H),1.48–0.96(m,56H),0.88(t,J=6.7Hz,12H). Example 2 Synthesis of ionizable lipid compound IAR2-16.

[0065] Step ①: Synthesis of intermediate 3a.

[0066] Hexadecane bromide (9.5 mmol) and potassium carbonate (10.33 mmol) were dissolved in acetonitrile and mixed evenly. Compound 1b (4.42 mmol) was dissolved in acetonitrile and slowly added dropwise to the reaction mixture with stirring. The mixture was heated in an oil bath at 80°C for 3 days. When the reaction was almost complete, the solvent was removed by rotary evaporation under reduced pressure. The resulting mixture was dissolved in 20 ml of dichloromethane, washed three times with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated and purified by silica gel column chromatography to obtain intermediate 3a (yield 48.7%).

[0067] Step ②: Synthesis of intermediate 3b.

[0068] Compound 3a (0.3 mmol) and tetrabutylammonium fluoride trihydrate (0.33 mmol) were separately dissolved in 2 ml of tetrahydrofuran. The mixture was stirred at 0°C for 1 hour, mixed thoroughly, and then warmed to room temperature and allowed to react for 4 hours until nearly complete. The solvent was removed by rotary evaporation under reduced pressure. The resulting mixture was dissolved in 20 ml of ethyl acetate, washed three times with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to obtain intermediate 3b (yield 81.3%).

[0069] Step ③: Synthesis of the final product IAR3-16.

[0070] Compound 1e (0.2 mmol) and triethylamine (0.3 mmol) were dissolved in 6 ml of dichloromethane and stirred at 0°C overnight. Compound 3b (0.2 mmol) was weighed and dissolved in 6 ml of anhydrous dichloromethane. The mixture was added dropwise to the reaction system and allowed to react until nearly complete. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography to obtain the final product, IAR2-16 (yield 60%).

[0071] 1 H NMR (400 MHz, Chloroform-d) δ 6.05 (d, J = 2.8 Hz, 2H), 3.34 (t, J= 6.5 Hz, 2H), 2.93 (td, J = 6.81, 0.8 Hz, 4H), 2.52 (t, J = 6.1 Hz, 6H), 1.51 – 1.40 (m, 6H), 1.36 – 1.22 (m, 112H), 0.93 – 0.85 (t, J=6.9Hz, 12H). Example 3 Synthesis of ionizable lipid compound IAR3-9.

[0072] Step ①: Synthesis of intermediate 2a 3-Amino-1-propanol (5.0 mmol) and decanal (11.0 mmol) were dissolved in 25 mL of dichloromethane and stirred at room temperature for 3 hours. Sodium triacetoxyborohydride (11.0 mmol) was added and the resulting mixture was stirred at room temperature for 4 days until the reaction was nearly complete. After concentration, the mixture was purified by silica gel column chromatography to obtain intermediate 2a as a light yellow viscous liquid (yield 78.1%).

[0073] Step ②: Synthesis of the final product IAR3-9.

[0074] Compound 1e (0.2 mmol) and triethylamine (0.3 mmol) were separately dissolved in 6 ml of dichloromethane and stirred at 0°C. Compound 2a (0.2 mmol) was weighed and dissolved in 6 ml of anhydrous dichloromethane. The mixture was added dropwise to the reaction system and allowed to react until nearly complete. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography to obtain the final product, IAR3-9 (yield 60.7%).

[0075] 1 HNMR(400MHz,Chloroform-d)δ6.09(t,J=1.0Hz,2H),4.15(td,J=6.7,2.2Hz,4H),3.28(t,J=1.4Hz,2H),2.65(t,J =6.7Hz,4H),2.37(t,J=6.1Hz,8H),1.87(pd,J=6.7,4.1Hz,4H),1.49(p,J=6.5Hz,8H),1.36(m,48H),0.85(m,12H). Example 4 Synthesis of ionizable lipid compound IAR3-16.

[0076] Step ①: Synthesis of intermediate 4a 3-Amino-1-propanol (5.0 mmol) and hexadecanal (11.0 mmol) were dissolved in 25 mL of dichloromethane and stirred at room temperature for 3 hours. Sodium triacetoxyborohydride (11.0 mmol) was added and the resulting mixture was stirred at room temperature for 4 days until the reaction was essentially complete. After concentration, the reaction was purified by silica gel column chromatography to obtain intermediate 4a as a light yellow viscous liquid (yield 63.8%).

[0077] Step ②: Synthesis of the final product IAR3-16.

[0078] Compound 1e (0.2 mmol) and triethylamine (0.3 mmol) were separately dissolved in 6 ml of dichloromethane and stirred at 0°C overnight. Compound 4a (0.2 mmol) was weighed and dissolved in 6 ml of anhydrous dichloromethane. The mixture was added dropwise to the reaction system and allowed to react at room temperature until nearly complete. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography to obtain the final product, IAR3-16 (yield 55%).

[0079] 1 H NMR (400 MHz, Chloroform-d) δ 6.05 (t, J = 1.5 Hz, 2H), 4.15 (td,J = 6.8, 1.1 Hz, 4H), 3.36 (t, J = 1.4 Hz, 2H), 2.69 (t, J = 6.7 Hz, 4H), 2.39 (t, J=6.2Hz,8H), 1.86 (dp, J = 10.1, 6.7 Hz, 4H), 1.48 (p, J = 6.5 Hz,8H), 1.35 – 1.22 (m, 104H), 0.95 – 0.85 (m, 12H). Example 5 Preparation of lipid nanoparticles IAR2-9L / IAR3-9L based on ionizable lipid compounds IAR2-9 / IAR3-9 Preparation method ①: pipette mixing method.

[0080] Ionizable lipid compounds IAR2-9 and IAR3-9 were prepared as 10 mg / mL solutions in isopropanol. These solutions were then mixed with ethanolic solutions of DOPE (5 mg / mL), cholesterol (5 mg / mL), and DMG-PEG (5 mg / mL) at a molar ratio of 15:25:1:20 and 15:20:0.5:10, respectively. The isopropanol was diluted to a desired volume to obtain an organic phase. GFP mRNA (EGFPIVT mRNA, purchased from Vector Biosystems, with m1Ψ modification) was then dissolved in citrate buffer to obtain an aqueous phase. Based on a 10:1 mass ratio of ionizable lipid compound to mRNA, appropriate amounts of the aqueous and organic phases were collected. The aqueous and organic phases were mixed, pipetted 70-80 times, and incubated at 0°C for 10 minutes to obtain the lipid nanoparticle solutions IAR2-9L and IAR3-9L.

[0081] Preparation method ②: microfluidic mixing method.

[0082] Ionizable lipid compounds IAR2-9, DOPE, cholesterol, and DMG-PEG were prepared in an isopropanol solution at a molar ratio of 15:25:1:20 (IAR3-9, DOPE, cholesterol, and DMG-PEG were prepared at a molar ratio of 15:20:0.5:10) to obtain an organic phase. mRNA encoding GFP was then dissolved in citrate buffer to obtain an aqueous phase. Based on a mass ratio of ionizable lipid compound to mRNA of 10:1, appropriate amounts of the aqueous and organic phases were collected. The organic and aqueous phases were aspirated using syringes, and the syringe outlets were connected to the sample inlet tubing. The syringe pump flow rate was adjusted, and the device was operated. Once the flow rate stabilized, the liquid was collected to obtain the lipid nanoparticle IAR2-9L or IAR3-9L solution.

[0083] Example 6 Particle size determination of lipid nanoparticles.

[0084] Take 20 μL of the prepared lipid nanoparticle solution, dilute it to 1 mL with 1× PBS solution, and measure the particle size of the lipid nanoparticles using a Malvern laser particle size analyzer. Figure 1 As shown, the average hydrated particle size of lipid nanoparticles IAR2-9L is 130 nm, and the average hydrated particle size of IAR3-9L is 122.7 nm, which proves that the prepared lipid nanoparticles have uniform particle size and good quality.

[0085] Example 7 Determination of lipid nanoparticle encapsulation efficiency.

[0086] A standard curve solution was prepared according to the instructions for the RiboGreen™ RNA Assay Kit, and relative fluorescence units were measured using a fluorescence spectrophotometer. The lipid nanoparticle sample was diluted, and 10 μL was added to the wells of a cell culture plate. Repeat for 6 wells. 40 μL of 2% Triton-100 was added to 3 wells containing the sample and treated for 5 minutes to rupture the lipid nanoparticles and release mRNA. 40 μL of 1× TE solution was added to the remaining 3 sample wells as a control, and 50 μL of RiboGreen staining working solution was added to 6 sample wells. Fluorescence intensity was read using the Multimode Plate Reader (EnSight) multimode microplate detection system, with the excitation light set to 480 nm and the emission light set to 520 nm. After data processing, the RNA content in the solution before and after lipid nanoparticle demulsification was obtained. The encapsulation efficiency was calculated using the following formula: Encapsulation efficiency (%) = (RNA content after demulsification - RNA content before demulsification) / RNA content after demulsification Test results such as Figure 2 As shown in the figure, the average encapsulation efficiency of lipid nanoparticles IAR2-9L was finally measured to be 78%, and the average encapsulation efficiency of IAR3-9L was 86%, which proved that the prepared lipid nanoparticles can efficiently encapsulate mRNA.

[0087] Example 8 Determination of cell transfection ability of lipid nanoparticles.

[0088] Hep3B cells in the logarithmic growth phase were seeded in a 48-well cell culture plate at a density of 1×10 4 cells. The lipid nanoparticles were placed in an ice bath and diluted with 1× PBS buffer to a mRNA concentration of 10 ng / μL. After the Hep3B cells adhered to the wall of the 48-well plate, the diluted lipid nanoparticles were added, 150 ng mRNA per well, and three replicates were added to each group. An equal amount of PBS was used as a negative control, and the commercially available mRNA transfection reagent LipoSmart mRNA Transfection Reagent (Genetic Biotech) was used as a positive control. 24 hours after transfection, the proportion of GFP-positive cells was detected by flow cytometry. The test results are as follows: Figure 3 As shown, the average transfection efficiency of lipid nanoparticles IAR2-9L was finally measured to be 63.5%, and the average transfection efficiency of IAR3-9L was 78.3%, proving that the prepared lipid nanoparticles can efficiently transfect cells.

[0089] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An ionizable lipid based on an itaconic acid core represented by general formula (I) or a pharmaceutically acceptable salt, isomer or solvate thereof, in, n is 2 or 3, and R is a straight-chain alkyl group having 9 to 16 carbon atoms.

2. The ionizable lipid based on itaconic acid core or its pharmaceutically acceptable salt, isomer or solvate according to claim 1, characterized in that: The structural formula of the ionizable lipid based on itaconic acid core is: .

3. The method for preparing an ionizable lipid based on an itaconic acid core according to claim 1 or 2, characterized in that: The steps include: Wherein, n is 2 or 3, and R is selected from a straight-chain alkyl group having 9 to 16 carbon atoms.

4. The method for preparing an ionizable lipid based on an itaconic acid core according to claim 4, wherein: The compound represented by formula (II) and triethylamine were dissolved in dichloromethane and mixed evenly. The compound represented by formula (III) was dissolved in dichloromethane and added to the above mixture. The mixture was reacted at 0°C for 15 minutes.

5. A lipid nanoparticle for drug delivery, characterized in that The lipid nanoparticles comprise the ionizable lipid based on the itaconic acid core according to any one of claims 1 to 2 or its pharmaceutically acceptable salt, isomer, solvate, neutral lipid, polyethylene glycol lipid and steroid lipid.

6. The lipid nanoparticle for drug delivery according to claim 5, characterized in that The molar ratio of the ionizable lipid based on itaconic acid nucleus or its pharmaceutically acceptable salt, isomer, solvate, neutral lipid, polyethylene glycol lipid and steroid lipid is (10-60): (10-50): (0.5-15): (20-60).

7. Use of the lipid nanoparticles for drug delivery according to claim 5 or 6 as a delivery vehicle for nucleic acid drugs.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an active ingredient and a carrier, wherein the active ingredient is a nucleic acid drug, and the carrier is a lipid nanoparticle for drug delivery according to claim 5 or 6.

9. The pharmaceutical composition according to claim 8, characterized in that The mass ratio of the active ingredient to the carrier is 1:1-50.

10. Use of the pharmaceutical composition according to claim 8 or 9 in the preparation of gene medicine, characterized in that: The nucleic acid drug is siRNA, mRNA, tRNA, rRNA, cDNA, ASO, plasmid DNA, microRNA or long non-coding RNA.

Images