Biodegradable amino acid derived ionizable lipids, methods of making and using the same

Abstract

The application belongs to the field of biological medicine, and provides a biodegradable amino acid derivative ionizable lipid, a preparation method and application thereof. The lipid structure comprises an amine group head group, an amino acid core connecting part and a hydrophobic lipid tail chain. The prepared lipid nanoparticle formula comprises the above amino acid lipid, a phospholipid, a PEG lipid, an auxiliary lipid and a nucleic acid drug, can safely and efficiently transfect the nucleic acid into cells, and has a wide application prospect in the field of nanometer nucleic acid vaccines, nucleic acid drug preparations and other gene therapies.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an amino acid derivative ionizable lipid as well as a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission that the information forms part of the prior art already known to a person of ordinary skill in the art.

[0003] Therapeutic nucleic acids including small interfering RNA (siRNA), messenger RNA (mRNA), microRNA (miRNA), antisense oligonucleotide, plasmid, etc. are developed for correcting genetic disorders or acquired diseases caused by abnormal gene expression profiles. Although nucleic acid drugs have high specificity, they face challenges such as short half-life, poor stability, easy hydrolysis by endogenous nucleases, etc. during clinical application, and their negative electric phosphate skeleton is difficult to interact with the negative electric cell membrane to enter the intracellular. Therefore, developing safe and efficient nucleic acid delivery vectors to improve the stability and the ability to penetrate the cell membrane of nucleic acid drugs is a continuous medical challenge.

[0004] Non-viral nucleic acid delivery vectors are mainly lipid nanoparticles (LNP) or polymer nanoparticles, which utilize the charge interaction between ionizable or cationic lipids and negatively charged nucleic acids to deliver nucleic acid drugs to the intracellular to exert therapeutic effects. As a new type of drug delivery system, lipid nanoparticles (including ionizable lipids, auxiliary lipids, phospholipids and polyethylene glycol-lipids, etc.) have been widely used in nucleic acid delivery and clinical research due to their advantages such as controllable preparation, large carrier capacity, high transport efficiency, good biocompatibility, no risk of integrating host genome, etc. Ionizable lipids, as the core structure of LNP, usually contain one or more ionizable amine groups in the molecular structure, and the apparent pKa is a key attribute for the in vivo delivery of nucleic acid drugs. There are great differences in the chemical space of ionizable lipid structure and in the cellular uptake and endosome escape, so it is of great significance to develop ionizable lipids with different chemical space, biodegradability and high transfection efficiency to promote the in-depth development and clinical transformation of nucleic acid drugs. SUMMARY

[0005] In order to solve the problems in the prior art, the present application aims to provide a biodegradable amino acid derivative ionizable lipid as well as a preparation method and application thereof. The amino acid derivative ionizable lipid provided by the present application has the advantages of mild preparation conditions and easy separation and purification, and exhibits good biodegradability and high in vitro and in vivo transfection efficiency, and can be used as a novel ionizable lipid for in vivo delivery of nucleic acid drugs.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] In a first aspect, the present application provides a biodegradable amino acid derived ionizable lipid, which is a compound of Formula (I), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof;

[0008]

[0009] wherein R1, R2are the same or different from each other, and each is independently C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 alkynyl, C6-C 24 alkyl substituted with a substituent group, C6-C 24 alkenyl substituted with a substituent group, C6-C 24 alkynyl, including or excluding one or more heteroatoms;

[0010] R3, R4are the same or different from each other, and each is independently C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 alkynyl, C6-C 24 alkyl substituted with a substituent group, C6-C 24 alkenyl substituted with a substituent group, C6-C 24 alkynyl substituted with a substituent group, C6-C 10 alkynyl, including or excluding one or more heteroatoms;

[0011] R1, R2are the same or different from R3, R4;

[0012] R5is independently selected from hydrogen, C1-C 10 alkyl, C1-C 10 alkyl substituted with a substituent group;

[0013] X1, X2are selected from oxygen or nitrogen or sulfur;

[0014] a is selected from a positive integer from 1-3;

[0015] b is selected from a positive integer from 1-3;

[0016] c is selected from an integer from 0-2;

[0017] d is selected from an integer from 0-2.

[0018] In some embodiments, c=d=0, and the amino acid is alpha- aminoacetic acid;

[0019] In some embodiments, c=d=1, and the amino acid is beta-alanine;

[0020] In some embodiments, c = d = 2, the amino acid is γ-aminobutyric acid.

[0021] In some embodiments, R1, R2, R3, R4, are the same or different from each other and are independently selected from C6-C 24 alkyl; R1 = R2, R3 = R4; R5 is hydrogen or methyl; X is nitrogen; a = b, c = d.

[0022] In some embodiments, R1, R2, R3, R4, are the same or different from each other and are independently selected from C6-C 18 alkyl, R5 is methyl, c = d = 2.

[0023] In some embodiments, the biodegradable amino acid derivative ionizable lipid is selected from one of the following compounds:

[0024]

[0025]

[0026] In a second aspect of the present application, a method for preparing the biodegradable amino acid derivative ionizable lipid as described above is provided, comprising:

[0027] a step of obtaining the compound of formula (I) from a compound of formula (I1) and a compound of formula (III) by esterification or amidation in the presence of a catalyst in an organic solvent;

[0028]

[0029] wherein, in formula (II) and (III), R1, R2, R5, X1, X2, a, b and c have the same meaning as in the compound of formula (I).

[0030] In some embodiments, the carboxylic acid (II) and the organic amine (III) are commercially available or prepared according to existing methods.

[0031] In some embodiments, when R5 is methyl, X is nitrogen, a = b = 2 or 3, R1, R2 are independently selected from C 6-24 alkyl, the method for preparing the carboxylic acid (II) comprises the steps of: reacting a tert-butyl ester of amino acid and a bromoalkane in the presence of potassium carbonate and potassium iodide in acetonitrile to obtain an intermediate a; reacting a dichloromethane solution of the intermediate a in the presence of trifluoroacetic acid to obtain the intermediate b, i.e. the compound of formula (II).

[0032] In some preferred embodiments, the molar ratio of the tertiary butyl ester of amino acid and acetonitrile is 0.01-1 mol / L; the molar ratio of potassium carbonate and the tertiary butyl ester of amino acid is 1-3:1; the molar ratio of potassium iodide and the tertiary butyl ester of amino acid is 0.1-1:1; the molar ratio of the tertiary butyl ester of amino acid and the bromoalkane is 1:2-2.5; the reaction temperature of the tertiary butyl ester of amino acid and the bromoalkane is 60-100℃, and the reaction time is 60-80 h; the molar ratio of the intermediate 1 and trifluoroacetic acid is 1-3:1; the reaction temperature of the intermediate 1 is ice bath, and the reaction time is 2-8 h.

[0033] In some embodiments, the organic solvent is selected from one or more than two combinations of methanol, ethanol, isopropanol, benzene, toluene, xylene, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene cyclohexanone, chlorobenzene, dichlorobenzene, dichloromethane, diethyl ether, propylene oxide, acetone, methyl butanone, methyl isobutyl ketone, acetonitrile, pyridine, phenol, styrene, perchloroethylene, trichloroethylene, ethylene glycol ether, N,N-dimethylformamide, or triethanolamine.

[0034] In some preferred embodiments, the solvent can be methanol, dichloromethane, acetonitrile, petroleum ether, ethyl acetate, isopropanol, N,N-diisopropylethylamine, N,N-dimethylformamide, etc.; the molar ratio of carboxylic acid (II) and organic solvent is 0.01-10 mol / L.

[0035] In some embodiments, the catalyst is selected from one or more than two combinations of N-hydroxysuccinimide (NHS), dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), 4-dimethylaminopyridine (DMAP), or O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU).

[0036] In some preferred embodiments, the catalyst can be 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), the molar ratio of the catalyst and carboxylic acid (II) is 2-3:1.

[0037] In some embodiments, the molar ratio of carboxylic acid (II) and organic amine (III) is 2-2.1:1-1.1.

[0038] In some embodiments, the reaction temperature is room temperature, and the reaction time is 10-30 h.

[0039] In some embodiments, the method for post-treatment of the reaction solution obtained by reacting carboxylic acid (II) and organic amine (III) comprises the steps of adding saturated sodium chloride solution to the reaction solution, extracting with dichloromethane, drying the organic phase with anhydrous sodium sulfate, filtering, evaporating under reduced pressure, and separating by silica gel column chromatography to obtain the lipid; the eluent used in the silica gel column chromatography is a mixture of dichloromethane and methanol, DCM / MeOH = 100:0-10:1.

[0040] The third aspect of the present application also provides the use of the biodegradable amino acid derivative ionizable lipid described above in a drug delivery carrier.

[0041] The fourth aspect of the present application provides a lipid nanoparticle or a lipid nanoparticle composition comprising a lipid, in particular an ionizable lipid as defined herein. The nanoparticle composition can further comprise a helper lipid, a phospholipid, a PEG lipid and a drug.

[0042] In some embodiments, the helper lipid is selected from the group consisting of a steroid, cholesteryl hemisuccinate, cholesterol and an alkyl resorcinol. Preferably, the helper lipid is cholesterol.

[0043] In some embodiments, the phospholipid is selected from the group consisting of one or more than two combinations of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), diethylpyrocarbonate (DEPC), dilauroylphosphatidylcholine (DLPC), phosphatidylcholine (POPC), egg phosphatidylcholine (EPC), hydrogenated soy phosphatidylcholine (HSPC), sphingomyelin (SM) and dimyristoylphosphatidylcholine (DMPC); preferably, the phospholipid is dioleoylphosphatidylethanolamine (DOPE) and dipalmitoylphosphatidylcholine (DPPC).

[0044] In some embodiments, the PEG lipid is selected from the group consisting of one or more than two combinations of DSPE-PEG, DMG-PEG, DPPE-PEG or DMA-PEG; preferably, the PEG lipid is DSPE-PEG.

[0045] In some embodiments, the molar ratio of the amino acid derivative ionizable lipid, the helper lipid, the phospholipid and the PEG lipid is 20-50:20-60:10-40:0.5-10; the mass ratio of the amino acid derivative ionizable lipid to the drug is 1-100:1.

[0046] In some embodiments, the diameter of the lipid nanoparticle is in the range of 1 nm to 1000 nm. For example, the diameter of the particle is in the range of 20 nm to 800 nm, or in the range of 50 nm to 500 nm, or in the range of 50 nm to 200 nm, or in the range of 1 nm to 100 nm. When the diameter of the lipid nanoparticle is in the range of 1 nm to 1000 nm, it is a nanoparticle as commonly described in the art.

[0047] According to the present application, the lipid nanoparticle can be prepared using any method known in the art. These methods include, but are not limited to, liposome extrusion, thin film hydration, nanoprecipitation, microfluidics, and impinging stream mixing, and other methods well known to those of ordinary skill in the art. Preferably, the method for preparing the lipid nanoparticle comprises the steps of: dissolving the ionizable lipid, the helper lipid, the phospholipid, and the PEG lipid in ethanol to obtain a lipid mixture ethanol phase; dispersing the drug in a citric acid buffer with pH = 4 to obtain a drug water phase; rapidly mixing the lipid mixture ethanol phase and the drug water phase using microfluidics to prepare a solution containing the lipid nanoparticle; and then preparing the lipid nanoparticle through steps such as dialysis and ultrafiltration.

[0048] In some embodiments, the drug comprises one or more than two combinations of a biological drug or a chemical drug; the biological drug comprises one or more than two combinations of a nucleic acid drug, a protein drug, a polypeptide drug, or a polysaccharide drug; further preferably, the nucleic acid drug comprises one or more than two combinations of small interfering RNA (siRNA), messenger RNA (mRNA), MicroRNA (miRNA), circular mRNA, long non-coding RNA (lncRNA), plasmid DNA, mini circle DNA (mcDNA), antisense oligonucleotide (ASOs), small activating RNA (saRNA), or nucleic acid aptamer (Aptamer); the chemical drug comprises one or more than two combinations of a small molecule drug, a fluorescent dye, or a contrast agent. Most preferably, the drug is mRNA, which includes linear mRNA and circular mRNA.

[0049] In some embodiments, a targeting molecule can be modified on the lipid nanoparticle to have a targeting function to target specific cells, tissues, or organs. The targeting molecule can be in the entire lipid nanoparticle or can only be located on the surface of the lipid nanoparticle. The targeting molecule can be a protein, a peptide, a glycoprotein, a lipid, a small molecule, a nucleic acid, etc., examples of which include, but are not limited to, an antibody, an antibody fragment, low-density lipoprotein (LDL), transferrin, asialycoprotein, a receptor ligand, sialic acid, an aptamer, etc.

[0050] The fifth aspect of the present application provides the use of the amino acid derivative ionizable lipid according to the first aspect of the present application and the lipid nanoparticle according to the fourth aspect of the present application in the preparation of a gene drug, wherein the gene drug comprises an active ingredient and a delivery carrier, the active ingredient is a nucleic acid drug, and the delivery carrier is the above-mentioned composition.

[0051] The lipid nanoparticles of the present application can be used for the prevention and treatment of various diseases of humans and / or animals in oral, rectal, intravenous, intramuscular, intravaginal, intranasal, subcutaneous, intraperitoneal, buccal, or oral, injection or inhalation forms.

[0052] Further, the nucleic acid drug is used for the prevention and / or treatment of cancer, inflammation, fibrotic diseases, autoimmune diseases, infections, mental disorders, blood diseases, chromosomal diseases, genetic diseases, connective tissue diseases, digestive diseases, ear-nose-throat diseases, endocrine diseases, eye diseases, reproductive diseases, heart diseases, kidney diseases, lung diseases, metabolic disorders, oral diseases, musculoskeletal diseases, neonatal screening, nutritional diseases, parasitic diseases, and skin diseases.

[0053] Advantages of the present application

[0054] 1. The present application provides a class of amino acid derivative ionizable lipids, which have the characteristics of biodegradability and high transfection efficiency, and the amino acid components are derived from naturally occurring amino acids (such as glycine, beta-alanine, gamma-aminobutyric acid, etc.). The synthetic raw materials of the ionizable lipids are cheap and easy to obtain, the design is reasonable, the operation is convenient, and the lipid nanoparticle delivery system composed of the ionizable lipids can be widely used for delivering nucleic acid drugs.

[0055] 2. The amino acid derivative ionizable lipids provided by the present application are not charged under physiological conditions (pH = 7.4), but are positively charged under acidic conditions, and are combined with negatively charged nucleic acid macromolecules in the form of electrostatic interaction. Under the acidic conditions of the intracellular endosome, protonation occurs to be positively charged, interact with negatively charged lipids, easily form unstable inverse hexagonal phase, promote LNP fusion with endosome membrane, or realize endosome escape of LNP through "proton sponge effect".

[0056] 3. The structure and proportion of the ionizable lipids in the lipid nanoparticle formulation of the present application affect the stability of the lipid nanoparticle and the nucleic acid transfection efficiency; in order to achieve high-efficiency nucleic acid transfection, the molar ratio of the amino acid derivative ionizable lipids, auxiliary lipids, phospholipids and PEG lipids in the present application is 20-50:20-60:10-40:0.5-10, and the prescription is optimized. The optimized ratio can achieve high-efficiency transfection of nucleic acid drugs; in some embodiments, in order to further achieve high-efficiency pulmonary delivery of nucleic acid drugs, DPPC is added to the phospholipid component, and the molar ratio of DOPE to DPPC is 0.2-5. The mass ratio of the amino acid derivative ionizable lipids to the drug is 1-100:1.

[0057] 4. The amino acid derived ionizable lipid provided by the present application can effectively deliver nucleic acid drugs, can realize efficient transfection of nucleic acids in vitro and in vivo, has a transfection effect comparable to that of marketed products (composed of DLin-MC3), and has good biocompatibility, and the lipid compound has a wide application prospect in the delivery of nucleic acid drugs such as mRNA. BRIEF DESCRIPTION OF DRAWINGS

[0058] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The embodiments of these drawings are set forth to explain the application and are not intended to limit the application.

[0059] Figure 1 is a synthetic route map of the lipid nanoparticles of the present application.

[0060] Figure 2 is the particle size and PDI of the lipid nanoparticles of Example 1 of the present application before and after atomization.

[0061] Figure 3 is the Zeta potential characterization of the lipid nanoparticles in Example 1 of the present application before and after atomization.

[0062] Figure 4 is the transmission electron microscopy graph of the lipid nanoparticles in Example 1 of the present application before and after atomization.

[0063] Figure 5 is the encapsulation efficiency investigation result of the lipid nanoparticles in Example 1 of the present application.

[0064] Figure 6 is the transfection efficiency investigation result of the lipid nanoparticles in Example 2 of the present application. DETAILED DESCRIPTION

[0065] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0066] Terminology:

[0067] The term "alkyl", by itself or as part of another substituent, means a fully saturated alkane of the formula C x H 2x+1 wherein x is a number greater than or equal to 1. Typically, alkyl groups of the present application contain from 1 to 24 carbon atoms. Alkyl groups can be straight-chained or branched, and can be substituted with one or more groups selected from halogen, hydroxyl, amino, oxo, alkoxycarbonyl, acylamino, alkylamido, dialkylamido, nitro, aminyl, alkylaminyl, dialkylaminyl, carboxyl, thio, and thioalkyl.

[0068] Unless otherwise stated, the terms "alkenyl" or "olefin" as used herein refer to a straight-chain, cyclic, or branched hydrocarbon group containing at least one carbon-carbon double bond. In some embodiments, the alkenyl group has 6-24 carbons and is also referred to as C 6-24 Alkenyl groups include, for example, vinyl, propenyl, n-butenyl, isobutenyl, etc. The alkenyl group can be unsubstituted or substituted with one or more substituents (e.g., 1, 2, 3, or 4), said substituents being selected from those substituents defined above for substituted alkyl groups.

[0069] Unless otherwise stated, the terms "alkynyl" or "alkynylene" as used herein refer to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon triple bond. In some embodiments, the alkynyl group has 6-24 carbons and is also referred to as C0. 6-24 Alkynyl. Alkynyl groups include, for example, ethynyl, 2-propynyl (propynyl), 1-propynyl, etc. The alkynyl group can be unsubstituted or substituted with one or more substituents (e.g., 1, 2, 3, or 4), said substituents being selected from those substituents defined above for substituted alkyl groups.

[0070] "Substitution" refers to the independent replacement of one or more hydrogen atoms in a group by a corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions, and those skilled in the art can determine (through experiment or theory) possible substitutions without much effort.

[0071] In the context of this invention, the alkyl, olefin, and alkyne portions as defined herein may further include one or more heteroatoms, for example, carbon in the alkyl, olefin, or alkyne chain may be replaced by heteroatoms such as those selected from nitrogen, oxygen, or sulfur.

[0072] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0073] Example 1

[0074] Synthesis of intermediate tert-butyl 4-N,N'-didecylaminobutyrate (GA10a):

[0075] To a solution of 4-amino butyric acid tert-butyl ester hydrochloride (978.45 mg, 5 mmol) in acetonitrile (20 mL) was added 1-bromodecane (2.4 g, 11 mmol), potassium carbonate (1.3 g, 10 mmol) and potassium iodide (166 mg, 1 mmol). The reaction mixture was stirred at 85 °C under reflux for 48 h. The reaction was monitored by thin layer chromatography. The reaction mixture was cooled to room temperature, filtered to remove potassium carbonate and potassium iodide and concentrated under reduced pressure. After drying, the oily residue was purified by column chromatography (200-300 mesh silica gel, eluent: petroleum ether / ethyl acetate = 50:1-20:1) to give 4-N,N' didecylaminobutyric acid tert-butyl ester (1.32 g, yield 60.03 %).

[0076] Synthesis of intermediate 4-N,N' didecylaminobutyric acid (GA10b):

[0077] To a solution of GA10a (439.77 mg, 1 mmol) in dry dichloromethane (6 mL) was added trifluoroacetic acid (2.5 mL) and stirred for 6 h. To the reaction mixture was added saturated sodium bicarbonate solution (5 mL). The organic layer was separated, washed with saturated sodium bicarbonate (3 x 10 mL) and saturated sodium chloride solution (3 x 10 mL), dried over anhydrous sodium sulfate and the solvent was removed on a rotary evaporator to give intermediate GA10b.

[0078] Example 2

[0079] Synthesis of intermediate 4-[bis(dodecyl)amino]butyric acid-2-methylprop-2-yl ester (GA12a) and 4-[bis(dodecyl)amino]butyric acid (GA12b) was carried out as described in Example 1 except that 1-bromodecane was replaced by 1-bromododecane (11 mol); other steps and conditions were same as in Example 1. The single step yield of intermediate GA12a was 38.9 %.

[0080] Example 3

[0081] Synthesis of intermediate (GA14a) and 4-[bis(tetradecyl)amino]butyric acid (GA14b) was carried out as described in Example 1 except that 1-bromodecane was replaced by 1-bromotetradecane (11 mol); other steps and conditions were same as in Example 1. The single step yield of intermediate GA14a was 64.73 %.

[0082] Example 4

[0083] Synthesis of N-(11-decyl-3-methyl-7-oxo-3,6,11-triazahenicosan-1-yl)-4- (didecylamino)butyramide (GAE10)

[0084] GA10b (314.6 mg, 0.84 mmol) was dissolved in 4 mL of dichloromethane. N- methyl-diaminoethylamine (46.9 mg, 0.4 mmol), l-(3-dimethylamino propyl)-3- ethylcarbodiimide hydrochloride (EDCI) (184.03 mg, 0.96 mmol), 1- hydroxybenzotriazole (HOBT) (129.72 mg, 0.96 mmol), N,N-diisopropylethylamine (DIPEA) (115.12 μl, 1.2 mmol) were added to the above solution and stirred at room temperature for 24 h. The reaction was monitored by thin layer chromatography. The reaction mixture was washed with saturated brine (3 x 10 mL) and the organic phase was dried over anhydrous sodium sulfate. The oil residue after concentration on a rotary evaporator was purified by column chromatography (column: 200-300 mesh silica gel; eluent: DCM / MeOH = 100:0-10:1) to give GAE10 (111.3 mg, yield 43.76%). 1 H NMR (400 MHz, CDC13) δ 8.37 (s, 2H), 3.33 (s, 4H), 3.07 (s, 4H), 3.00 (s, 8H), 2.58 (d, J = 17.71 Hz, 8H), 2.26 (s, 3H), 2.04 (s, 4H), 1.69 (s, 8H), 1.22 (d, J = 27.68, 56H), 0.80 (t, J = 5.12 Hz, 12H).

[0085] Example 5

[0086] Synthesis of 4-[bis(dodecyl)amino]-N-(3-methyl-7-oxoimino-11-dodecyl-3,6,11- triazatricosan-1-yl)butyramide (GAE12)

[0087] GAE12 was prepared as described in Example 4, except that GA10b (0.84 mmol) was replaced by GA12b (0.63 mmol); other steps and conditions and the ratio of each reagent were consistent with Example 4. GAE12 (55.3 mg, 19.2%) was obtained. 1 H NMR (400 MHz, CDC13) δ 8.37 (s, 2H), 3.33 (s, 4H), 3.07 (s, 4H), 3.00 (s, 8H), 2.58 (d, J = 17.71 Hz, 8H), 2.26 (s, 3H), 2.04 (s, 4H), 1.69 (s, 8H), 1.22 (d, J = 27.68, 56H), 0.80 (t, J = 5.12 Hz, 12H).

[0088] Example 6

[0089] 4-[Di(tetradecyl)amino]-N-(3-methyl-7-oxoimino-11-tetradecyl-3,6,11- triazapentacosan-1-yl)butyramide (GAE14) synthesis

[0090] GAE14 was prepared as described in Example 4, except that GAE10b (0.84 mmol) was replaced by GAE14b (0.84 mmol); the other steps and conditions were identical to those of Example 4. GAE14 (85.47 mg, 21.63% yield) was obtained. 1 H NMR (400 MHz, CDC13) δ 8.37 (s, 2H), 3.44 (s, 4H), 3.13 (s, 4H), 3.00 (d, J = 8.00, 8H), 2.84 (d, J = 8.19 Hz, 4H), 2.62 (s, 4H), 2.44 (s, 3H), 2.11 (s, 4H), 1.75 (s, 12H), 1.28 (m, 88H), 0.87 (t, J = 6.50 Hz, 12H).

[0091] Example 7

[0092] 4-[Di(tetradecyl)amino]-N-(3-methyl-7-oxoimino-11-tetradecyl-3,6,11- triazapentacosan-1-yl)butyramide (GAE14) synthesis

[0093] GAP12 was prepared as described in Example 4, except that GA10b (0.84 mmol) was replaced by GA12b (0.84 mmol) and N-methyl-diaminoethylamine was replaced by N,N-bis(3- aminopropyl)methylamine (0.4 mmol); the other steps and conditions were identical to those of Example 4. GAP12 (58.3 mg, 14.75%) was obtained. 1 H NMR (400 MHz, CDC13) δ 7.43 (s, 2H), 3.29 (dd, Ji = 6.15 Hz, J2 = 12.13 Hz, 4H), 2.63 (d, J = 25.78 Hz, 12H), 2.39 (t, J = 6.33 Hz, 4H), 2.31 (t, J = 6.83 Hz, 4H), 2.18 (s, 3H), 1.87 (m, 4H), 1.66 (m, 4H), 1.53 (s, 8H), 1.25 (s, 72H), 0.87 (t, J = 6.64 Hz, 12H).

[0094] Example 8

[0095] N-(13-decyl-4-methyl-9-oxoimino-4,8,13-triazatricosan-1-yl)-4-(didecylamino)butyramide (GAP10) synthesis.

[0096] GAP10 was prepared as described in Example 4, except that N-methyl- diaminomethylamine (0.4 mmol) was replaced by N,N-bis(3-aminopropyl)methylamine (0.4 mmol); other steps and conditions and molar ratios of reagents were consistent with Example 4. GAP10 was obtained (56.5 mg, yield 16.11%). 1 H NMR (400 MHz, CDC13) δ 7.15 (s, 2H), 3.29 (dd, Ji = 6.24 Hz, J2 = 6.25 Hz, 4H), 2.44 (m, 8H), 2.37 (s, J = 6.46 Hz, 4H), 2.24 (d, J = 7.06 Hz, 8H), 2.17 (s, 3H), 1.78 (m, 4H), 1.64 (m, 4H), 1.44 (s, 8H), 1.24 (s, 56H), 0.87 (t, J = 6.65 Hz, 12H).

[0097] Example 9

[0098] Lipid nanoparticles were prepared using the representative compound GAE14. First, the ionizable lipid (GAE14), cholesterol, DOPE, DSPE-PEG2k used for preparing the lipid nanoparticles were dissolved in ethanol to prepare the ethanol phase solution at a molar ratio of 35:25:30:0.5. Then, EGFP (or Luciferase) mRNA was added to 10-50 mM citrate buffer (pH = 4) to obtain the mRNA aqueous phase solution, and mRNA-lipid nanocomplexes were prepared by rapidly mixing the ethanol phase solution with the aqueous phase solution. The weight ratio of ionizable lipid to mRNA was 10:1. The mRNA-encapsulated lipid nanoparticles were obtained by dialysis, ultrafiltration, and the like.

[0099] Further formulation optimization was carried out to achieve good aerosol stability and deep lung delivery of the lipid nanoparticles. The natural lung surfactant component DPPC was added to the single phospholipid component, and an optimized formulation was further screened. The molar ratio of ionizable lipid, cholesterol, DOPE, DPPC, DSPE-PEG2k included but not limited to 35:35:6:4:1.5. Lipid nanoparticles were prepared according to the above operation and formulation ratio, and were labeled as NGAE14 for further characterization.

[0100] Experimental Example 1

[0101] Characterization of ionizable lipid nanoparticles:

[0102] The lipid nanoparticles prepared in Example 9 were further characterized. The morphology of the above lipid nanoparticles was characterized by transmission electron microscopy, and the nanometer size, polydispersity coefficient PDI and Zeta potential were characterized by dynamic light scattering laser particle size instrument (Malvern Zetasizer Nano ZS), and the encapsulation rate was determined by Quant-iT RiboGreen RNA Assay Kit RNA quantitative detection kit, and the results are shown in Table 1 (only show the prescription: ionizable lipid, cholesterol, DOPE, DPPC, DSPE-PEG2k molar ratio 35:35:6:4:1.5 prepared lipid nanoparticles GAE14L). Figures 2-5 The lipid nanoparticles were spheroidal, uniform in particle size (PDI <0.3), close to neutral in potential, good in encapsulation efficiency, and stable in performance before and after atomization.

[0103] Experimental Example 2

[0104] In vitro transfection efficiency of ionizable lipid nanoparticles:

[0105] MLE12 cells in the logarithmic growth phase were inoculated in a 96-well plate containing DMEM / F12 medium, and the plating density was 1*10 5 cells per well. After the cells adhered, they were ready for transfection (37°C, about 12 hours). 0.2 μg of mRNA containing lipid nanoparticles were added to each well, and 3 replicate wells were set for each group. After 24 hours of transfection, the fluorescence proportion of cells in each group was detected using a fluorescence microscope or a flow cytometer.

[0106] The overall experimental results show that the particle sizes of LNP prepared by different ionizable lipids of different prescriptions are basically maintained within 200 nm, but there are great differences in delivery efficiency. Further optimization of the prescription to prepare high-efficiency transfection lipid nanoparticles can meet the needs of different disease treatments while reducing the risk of multiple drug administration. Part of the flow cytometry experimental results are shown in Table 2 (only show GAE14L lipid nanoparticles), wherein the positive control is a commercially available DLin-MC3 prepared lipid nanoparticle. The transfection efficiency of the remaining lipids GAE12, GAE10, GAP10, GAP12 prepared by the same prescription is 84.7%, 73.8%, 54.3%, and 67.5%, respectively. Figure 6

[0107] The above only describes some embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.​

Claims

1. The application of a biodegradable amino acid-derived ionizable lipid in the preparation of a pulmonary drug delivery carrier, characterized in that, The biodegradable amino acid-derived ionizable lipid is a compound of formula (I); the compound of formula (I) is selected from one of compounds GAE10, GAE12, GAE14, GAP10, and GAP12: (Ⅰ) R1 and R2 may be the same or different from each other, and each is independently C6-C. 24 Alkyl, C6-C 24 alkenyl, C6-C 24 C6-C substituted with alkynyl or substituent groups 24 C6-C substituted with alkyl or substituent groups 24 alkenyl groups, substituents substituted C6-C 24 The alkynyl group may or may not include one or more heteroatoms; R3 and R4 may be the same or different from each other, and each is independently C6-C. 24 Alkyl, C6-C 24 alkenyl, C6-C 24 C6-C substituted with alkynyl or substituent groups 24 C6-C substituted with alkyl or substituent groups 24 alkenyl groups, substituents substituted C6-C 24 The alkynyl group may or may not include one or more heteroatoms; R1, R2 are the same as or different from R3, R4; R5 is independently selected from hydrogen, C1-C 10 C1-C substituted with alkyl or substituent groups 10 alkyl; X1 and X2 are selected from oxygen, nitrogen, or sulfur; a is a positive integer selected from 1 to 3; b is a positive integer selected from 1 to 3; c is an integer between 0 and 2; d is an integer selected from 0 to 2; GAE10 GAE12 GAE14 GAP10 GAP12.

2. A method for preparing biodegradable amino acid-derived ionizable lipids as described in claim 1, characterized in that, include: The steps of obtaining the compound of formula (I) by esterification or amidation of the compound of formula (II) and the compound of formula (III); (Ⅱ) (Ⅲ) In formulas (II) and (III), R1, R2, R5, X1, X2, a, b, and c have the same meanings as in the compound of formula (I).

3. The method for preparing biodegradable amino acid-derived ionizable lipids as described in claim 2, characterized in that, The method includes one or more of the following conditions: i. The organic solvent is selected from one or more combinations of methanol, ethanol, isopropanol, benzene, toluene, xylene, pentane, hexane, octane, cyclohexane, cyclohexanone, methylcyclohexanone, chlorobenzene, dichlorobenzene, dichloromethane, diethyl ether, propylene oxide, acetone, methyl butyl ketone, methyl isobutyl ketone, acetonitrile, pyridine, phenol, styrene, perchloroethylene, trichloroethylene, ethylene glycol ether, N,N-dimethylformamide, or triethanolamine; ii. The catalyst is selected from one or more of the following: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, N-hydroxysuccinimide, dicyclohexylcarbodiimide, diisopropylcarbodiimide, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, O-benzotriazole-tetramethylurea hexafluorophosphate, 4-dimethylaminopyridine, or O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid; iii. The molar ratio of the compound shown in formula (I1) to the compound shown in formula (III) is 1:1-1.1; iv. The reaction temperature is room temperature, and the reaction time is 10-30 h.

4. The method for preparing biodegradable amino acid-derived ionizable lipids as described in claim 3, characterized in that, The catalysts are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole.

5. A lipid nanoparticle, characterized in that, Includes biodegradable amino acid-derived ionizable lipids, cofactor lipids, phospholipids, and PEG-lipids in the applications described in claim 1.

6. The lipid nanoparticles as described in claim 5, characterized in that, The auxiliary lipids are selected from steroids, cholesterol hemisuccinate, cholesterol, and alkyl resorcinol; Alternatively, the phospholipid is selected from one or more of the following: distearyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylcholine, diethyl pyrocarbonate, dilauroyl phosphatidylcholine, phosphatidylcholine, egg yolk lecithin, hydrogenated soybean phosphatidylcholine, sphingomyelin, or dimyristoyl phosphatidylcholine. Alternatively, the PEG lipid is selected from one or more of DSPE-PEG, DMG-PEG, DPPE-PEG, or DMA-PEG; Alternatively, the molar ratio of biodegradable amino acid-derived ionizable lipids, auxiliary lipids, phospholipids, and PEG lipids is 20-50:20-60:10-40:0.5-10; the mass ratio of ionizable lipids to the drug is 1-100:

1. The drug includes one or more of biological drugs or chemical drugs; the biological drug includes one or more of nucleic acid drugs, protein drugs, polypeptide drugs or polysaccharide drugs. Alternatively, the nucleic acid drug may include one or more of the following: small interfering RNA, messenger RNA, microRNA, circular mRNA, long non-coding RNA, plasmid DNA, mini circle DNA, antisense oligonucleotide, small activating RNA, or nucleic acid aptamers. Alternatively, the chemical drug may include one or more of small molecule drugs, fluorescein, or contrast agents; Alternatively, the drug is mRNA, including linear mRNA and circular mRNA.

7. The method for preparing lipid nanoparticles according to claim 5 or 6, characterized in that, include: Liposome extrusion, thin film differentiation, nanoprecipitation, microfluidics, and impingement jet mixing; Alternatively, the preparation method of lipid nanoparticles includes the following steps: dissolving biodegradable amino acid-derived ionizable lipids, auxiliary lipids, phospholipids and PEG lipids in ethanol to obtain a lipid mixed ethanol phase; fully dispersing the drug in a citrate buffer solution with pH=4-4.5 to obtain a drug aqueous phase; using microfluidics to mix the lipid mixed ethanol phase and the drug aqueous phase to prepare a solution containing lipid nanoparticles; and then obtaining lipid nanoparticles by dialysis and ultrafiltration.

8. The application of the biodegradable amino acid-derived ionizable lipids in the application of claim 1, and the application of the lipid nanoparticles of claim 5 or 6 in the preparation of gene drugs, characterized in that, The gene drug comprises an active ingredient and a delivery vector, wherein the active ingredient is a nucleic acid drug.

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