CN117623978A - Biodegradable amino acid-derived ionizable lipids and preparation methods and applications thereof - Google Patents

Abstract

The invention belongs to the field of biological medicine, and provides biodegradable amino acid derived ionizable lipid, a preparation method and application thereof, wherein a lipid structure comprises an amino head group, a connecting part with amino acid as a core and a hydrophobic lipid tail chain; the prepared lipid nanoparticle formula comprises the amino acid lipid, phospholipid, PEG lipid, auxiliary lipid and nucleic acid drugs, can safely and efficiently transfect nucleic acid into cells, and has wide application prospects in the gene therapy fields of nano nucleic acid vaccines, nucleic acid drug preparations and the like.

Description

Biodegradable amino acid-derived ionizable lipids and preparation methods and applications thereof

Technical field

The invention belongs to the field of biomedicine technology, and specifically relates to amino acid-derived ionizable lipids and their preparation methods and applications.

Background technique

The information in this Background section is disclosed solely for the purpose of increasing understanding of the general background of the invention and is not necessarily considered to be an admission or in any way implying that the information constitutes prior art that is already known to a person of ordinary skill in the art.

Therapeutic nucleic acids including small interfering RNA (siRNA), messenger RNA (mRNA), microRNA (miRNA), antisense oligonucleotides, plasmids, etc. are developed to correct genetic disorders or acquired diseases caused by abnormal gene expression profiles. Although nucleic acid drugs are highly specific, they face problems such as short half-life, poor stability, and easy hydrolysis by endogenous nucleases during clinical application. Moreover, their negatively charged phosphate backbone is difficult to interact with negatively charged cell membranes and enter the cell. Inside. Therefore, developing safe and efficient nucleic acid delivery vehicles to improve the stability and ability of nucleic acid drugs to penetrate cell membranes is an ongoing medical challenge.

Non-viral nucleic acid delivery carriers 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 into cells to exert therapeutic effects. As a new type of drug delivery system, lipid nanoparticles (including ionizable lipids, auxiliary lipids, phospholipids and polyethylene glycol-lipids) have the advantages of controllable preparation, large carrier capacity, high transport efficiency, It has the advantages of good biocompatibility and no risk of integration into the host genome, and has been widely used in nucleic acid delivery and clinical research. As the core structure of LNP, ionizable lipids usually contain one or more ionizable amine groups in their molecular structure, and their apparent pKa is a key attribute for the in vivo delivery of nucleic acid drugs. There are huge differences in the chemical space of ionizable lipid structures and their cellular uptake and endosomal escape. Therefore, the development of ionizable lipids with different chemical spaces, biodegradability, and high transfection efficiency is of great significance in promoting nucleic acid drugs. Development and clinical translation are of great significance.

Contents of the invention

In order to solve the deficiencies of the existing technology, the present invention aims to provide a biodegradable amino acid-derived ionizable lipid and its preparation method and application. The amino acid-derived ionizable lipid provided by the present invention has the advantages of mild preparation conditions, easy separation and purification, good biodegradability and high in vitro and in vivo transfection efficiency, and can be used as a new type of ionizable lipid for delivering nucleic acid drugs in the body. quality.

In order to achieve the above objects, the present invention adopts the following technical solutions:

A first aspect of the present invention provides a biodegradable amino acid-derived ionizable lipid, which is a compound of formula (I), or its N-oxide, solvate, pharmaceutically acceptable salt or stereo isomer;

Wherein, R ₁ and R ₂ are the same or different from each other, and each is independently a C ₆ -C ₂₄ alkyl group, a C ₆ -C ₂₄ alkenyl group, a C ₆ -C ₂₄ alkynyl group, or a C ₆ -C substituted by a substituent group. ₂₄ alkyl, C ₆ -C ₂₄ alkenyl substituted by a substituent group, C ₆ -C ₂₄ alkynyl substituted by a substituent group, including or excluding one or more heteroatoms;

R ₃ and R ₄ are the same as or different from each other, and each is independently a C ₆ -C ₂₄ alkyl group, a C ₆ -C ₂₄ alkenyl group, a C ₆ -C ₂₄ alkynyl group, or a C ₆ -C ₂₄ alkyl group substituted by a substituent group. base, a C ₆ -C ₂₄ alkenyl group substituted by a substituent group, a C ₆ -C ₂₄ alkynyl group substituted by a substituent group, including or excluding one or more heteroatoms;

R ₁ and R ₂ are the same as or different from R ₃ and R ₄ ;

R ₅ is independently selected from hydrogen, C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ alkyl substituted by a substituent group;

X ₁ and X ₂ are selected from oxygen, nitrogen or sulfur;

a is a positive integer selected from 1-3;

b is a positive integer selected from 1-3;

c is selected from an integer from 0-2;

d is selected from an integer from 0-2.

In some embodiments, c=d=0, and the amino acid is α-aminoacetic acid;

In some embodiments, c=d=1, and the amino acid is β-alanine;

In some embodiments, c=d=2 and the amino acid is gamma-aminobutyric acid.

In some embodiments, R ₁ , R ₂ , R ₃ , and R ₄ are the same as or different from each other and are independently selected from C ₆ -C ₂₄ alkyl; R ₁ =R ₂ , R ₃ =R ₄ ; R ₅ is hydrogen or Methyl; X is nitrogen; a=b, c=d.

In some embodiments, R ₁ , R ₂ , R ₃ , and R ₄ are independently selected from C ₈ -C ₁₈ alkyl, R ₅ is methyl, and c=d=2.

In some embodiments, the biodegradable amino acid derived ionizable lipid is selected from one of the following compounds:

A second aspect of the present invention provides a method for preparing the above-mentioned biodegradable amino acid-derived ionizable lipid, including:

The step of obtaining the compound represented by formula (I) through an esterification reaction or amidation reaction from a compound represented by formula (I1) and a compound represented by formula (III) in an organic solvent in the presence of a catalyst;

Among them, in formulas (II) and (III), R ₁ , R ₂ , R ₅ , X ₁ , X ₂ , a, b and c have the same meanings as in the compound of formula (I).

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

In some embodiments, when R ₅ is methyl, X is nitrogen, a=b=2 or 3, R ₁ and R ₂ are independently selected from C _6-24 alkyl, the preparation method of carboxylic acid (II) It includes steps: in acetonitrile, under the action of potassium carbonate and potassium iodide, the amino acid tert-butyl ester and the brominated alkane are reacted to obtain intermediate a; under the action of trifluoroacetic acid, the dichloromethane solution of intermediate a is reacted to obtain Intermediate b is the compound represented by formula (II).

In some preferred embodiments, the molar ratio of amino acid tert-butyl ester to acetonitrile is 0.01-1 mol/L; the molar ratio of potassium carbonate and amino acid tert-butyl ester is 1-3:1; potassium iodide and amino acid tert-butyl ester The molar ratio of amino acid tert-butyl ester and brominated alkanes is 1:2-2.5; the reaction temperature of amino acid tert-butyl ester and brominated alkanes is 60-100°C, and the reaction time is 60- 80h; the molar ratio of intermediate 1 to trifluoroacetic acid is 1-3:1; the reaction temperature of intermediate 1 is an ice bath, and the reaction time is 2-8h.

In some embodiments, the organic solvent is selected from the group consisting of methanol, ethanol, isopropyl alcohol, benzene, toluene, xylene, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene, chlorobenzene, Dichlorobenzene, methylene chloride, diethyl ether, propylene oxide, acetone, methyl butyl ketone, methyl isobutyl ketone, acetonitrile, pyridine, phenol, styrene, perchlorethylene, trichlorethylene, ethylene glycol ether, One or a combination of two or more of N,N-dimethylformamide or triethanolamine;

In some preferred embodiments, the solvent can be methanol, dichloromethane, acetonitrile, petroleum ether, ethyl acetate, isopropyl alcohol, N,N-diisopropylethylamine, N,N-dimethyl Formamide, etc.; the molar ratio of carboxylic acid (II) to the volume ratio of organic solvent is 0.01-10mol/L.

In some embodiments, the catalyst is selected from N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3- Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), 2-(7-azobenzotriazole)-N, N,N',N'-tetramethylurea hexafluorophosphate (HATU), O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), 4-dimethylaminopyridine (DMAP), Or one or a combination of two or more O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU).

In some preferred embodiments, the catalyst may be 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT ), catalyst and carboxylic acid (II) are 2-3:1.

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

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

In some embodiments, the post-treatment method of the reaction liquid obtained from the reaction of carboxylic acid (II) and organic amine (III) includes the steps of: adding saturated sodium chloride solution to the reaction liquid, extracting with dichloromethane, and the organic phase is anhydrous Dry with sodium sulfate, filter, evaporate under reduced pressure, and separate by silica gel column chromatography to obtain lipids; the eluent used for silica gel column chromatography separation is a mixture of dichloromethane and methanol, DCM/MeOH=100:0-10:1.

A third aspect of the present invention also provides the use of the above-mentioned biodegradable amino acid-derived ionizable lipid in drug delivery carriers.

A fourth aspect of the invention provides a lipid nanoparticle or lipid nanoparticle composition comprising a lipid, in particular an ionizable lipid as defined herein. The nanoparticle composition may further comprise helper lipids, phospholipids, PEG lipids and drugs.

In some embodiments, the accessory lipid is selected from the group consisting of steroids, cholesterol hemisuccinates, cholesterol, and alkylresorcinols. Cholesterol is preferred.

In some embodiments, the phospholipid is selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), diethyl pyrocarbonate ( DEPC), dilauroylphosphatidylcholine (DLPC), phosphatidylcholine (POPC), egg yolk lecithin (EPC), hydrogenated soybean phosphatidylcholine (HSPC), sphingomyelin (SM) or dimyristoyl phospholipid One or a combination of two or more acylcholines (DMPC); preferably dioleoylphosphatidylethanolamine (DOPE) and dipalmitoylphosphatidylcholine (DPPC).

In some embodiments, the PEG lipid is selected from one or a combination of two or more of DSPE-PEG, DMG-PEG, DPPE-PEG or DMA-PEG; preferably, the PEG lipid is DSPE-PEG PEG.

In some embodiments, the molar ratio of amino acid-derived ionizable lipids, auxiliary lipids, phospholipids and PEG lipids is 20-50:20-60:10-40:0.5-10; amino acid-derived ionizable lipids and drugs The mass ratio is 1-100:1.

In some embodiments, the diameter of the lipid nanoparticles ranges from 1 nm to 1000 nm. For example, the particle diameter 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 lipid nanoparticles is in the range of 1 nm to 1000 nm, they are generally referred to as nanoparticles in the art.

According to the present invention, lipid nanoparticles can be prepared using any method known in the art. These methods include, but are not limited to, liposome extrusion, thin film differentiation, nanoprecipitation, microfluidics, and impact jet mixing, as well as other methods well known to those of ordinary skill in the art. Preferably, the preparation method of lipid nanoparticles includes the steps of: dissolving ionizable lipids, auxiliary lipids, phospholipids and PEG lipids in ethanol to obtain a lipid mixed ethanol phase; fully dispersing the drug in citric acid with pH=4 The drug aqueous phase is obtained in the buffer; the lipid mixed ethanol phase and the drug aqueous phase are rapidly mixed using microfluidics to prepare a solution containing lipid nanoparticles; and then the lipid nanoparticles are obtained through dialysis, ultrafiltration and other steps.

In some embodiments, the drugs include one or a combination of two or more of biological drugs or chemical drugs; the biological drugs include one or more of nucleic acid drugs, protein drugs, polypeptide drugs or polysaccharide drugs. Combinations; further preferably, the nucleic acid drugs include small interfering RNA (siRNA), messenger RNA (mRNA), MicroRNA (miRNA), circular mRNA, long non-coding RNA (lncRNA), plasmid DNA, mini circle DNA ( mcDNA), antisense oligonucleotides (ASOs), small activating RNA (saRNA) or a combination of two or more; the chemical drugs include small molecule drugs, fluorescein or One or a combination of two or more developers. Most preferably, the drug is mRNA, which includes linear mRNA and circular mRNA.

In some embodiments, targeting molecules can be modified on lipid nanoparticles to provide targeting functions to target specific cells, tissues or organs. The targeting molecule can be throughout the lipid nanoparticle or can be located only on the surface of the lipid nanoparticle. Targeting molecules can be proteins, peptides, glycoproteins, lipids, small molecules, nucleic acids, etc. Examples include (but are not limited to) antibodies, antibody fragments, low-density lipoproteins (LDL), transferrin, Sialyl glycoprotein (asialycoprotein), receptor ligands, sialic acid, aptamers, etc.

The fifth aspect of the present invention provides the application of the amino acid-derived ionizable lipid according to the first aspect of the present invention and the lipid nanoparticles described in the fourth aspect in the preparation of genetic medicines. The genetic medicines include active ingredients and delivery carriers. The active ingredient is a nucleic acid drug, and the delivery carrier is the above composition.

The lipid nanoparticles of the present invention can be used in various forms of human and/or animals via oral, rectal, intravenous, intramuscular injection, intravaginal, intranasal, subcutaneous, intraperitoneal, buccal, oral, injection or inhalation forms. Disease prevention and treatment.

Further, the nucleic acid drugs are used to prevent and/or treat cancer, inflammation, fibrotic diseases, autoimmune diseases, infections, mental disorders, blood diseases, chromosomal diseases, genetic diseases, connective tissue diseases, digestive diseases, ear and nose diseases. Throat diseases, endocrine diseases, eye diseases, reproductive diseases, heart disease, kidney disease, lung disease, metabolic diseases, oral diseases, musculoskeletal diseases, newborn screening, nutritional diseases, parasitic diseases, skin diseases, etc.

Beneficial effects of the invention

1. The present invention provides a class of amino acid-derived ionizable lipids, which are biodegradable and can be efficiently transfected. The amino acid components thereof are derived from naturally occurring amino acids (such as glycine, β-alanine, and γ-aminobutyrate). acid, etc.). The ionizable lipid synthetic raw material is cheap and easy to obtain, has reasonable design and is easy to operate, and the lipid nano-delivery system composed of it can be widely used to deliver nucleic acid drugs.

2. The amino acid-derived ionizable lipid provided by the present invention is not charged under physiological conditions (pH=7.4), but is positively charged under acidic conditions, and binds negatively charged nucleic acid macromolecules in the form of electrostatic interaction. Protonation occurs under the acidic conditions of intracellular endosomes and becomes positively charged. It interacts with negatively charged lipids and easily forms an unstable inverted hexagonal phase, which promotes the fusion of LNP with the endosomal membrane, or through the "proton sponge effect". Achieving endosomal escape of LNP.

3. In the lipid nanoparticle formula provided by the present invention, the structure and proportion of ionizable lipids affect the stability of the lipid nanoparticles and the nucleic acid transfection efficiency; in order to achieve efficient nucleic acid transfection, amino acids are selected in the present invention The molar ratio of derived ionizable lipids, auxiliary lipids, phospholipids and PEG lipids is 20-50:20-60:10-40:0.5-10, and the formula is optimized. The optimized ratio can achieve high efficiency of nucleic acid drugs. Transfection; in some embodiments, in order to further achieve efficient 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-derived ionizable lipid to the drug is 1-100:1.

4. The amino acid-derived ionizable lipid provided by the present invention can effectively deliver nucleic acid drugs, and can achieve efficient transfection of nucleic acids in vivo and in vitro. Its transfection effect is equivalent to that of the marketed product (composed of DLin-MC3), and has good Biocompatible, this lipid compound has broad application prospects in the delivery of nucleic acid drugs such as mRNA.

Description of drawings

The description and drawings that constitute a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

Figure 1 is a synthesis route diagram of lipid nanoparticles of the present invention.

Figure 2 is the particle size and PDI of the lipid nanoparticles before and after atomization in Experimental Example 1 of the present invention.

Figure 3 is the Zeta potential characterization before and after atomization of lipid nanoparticles in Experimental Example 1 of the present invention.

Figure 4 is a transmission electron microscope image of lipid nanoparticles before and after atomization in Experimental Example 1 of the present invention.

Figure 5 is the results of the investigation of the encapsulation efficiency of lipid nanoparticles in Experimental Example 1 of the present invention.

Figure 6 is the result of examining the transfection efficiency of lipid nanoparticles in Experimental Example 2 of the present invention.

Detailed ways

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings commonly understood by one of ordinary skill in the art to which this invention belongs.

Terminology explanation:

The term "alkyl" by itself or as part of another substituent refers to a fully saturated alkane of the formula _{CxH2x +1} , where x is a number greater than or equal to 1. Typically, the alkyl groups of the present invention contain 1 to 24 carbon atoms. Alkyl groups may be straight or branched and may be substituted by one or more selected from halogen, hydroxyl, amino, oxo, alkoxycarbonyl, amide, alkylamido, dialkylamido , nitro, amine, alkylamino, dialkylamino, carboxyl, thio and thioalkyl group substitutions.

Unless otherwise stated, the term "alkenyl" or "alkene" as used herein refers to a linear, cyclic or branched hydrocarbon group containing at least one carbon-carbon double bond. In some embodiments, alkenyl groups have 6-24 carbons, also referred to as C _6-24 alkenyl. Alkenyl groups include, for example, vinyl, propenyl, n-butenyl, isobutenyl, and the like. An alkenyl group may be unsubstituted or substituted with one or more substituents (eg 1, 2, 3 or 4) selected from those defined above for substituted alkyl substituents.

Unless otherwise stated, the term "alkynyl" or "alkyne" as used herein refers to a straight or branched chain hydrocarbon group containing at least one carbon-carbon triple bond. In some embodiments, an alkynyl group has 6-24 carbons, also referred to as C _6-24 alkynyl. Alkynyl groups include, for example, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like. An alkynyl group may be unsubstituted or substituted with one or more substituents (eg 1, 2, 3 or 4) selected from those defined above for substituted alkyl substituents.

"Substituted" means that one or more hydrogen atoms in a group are replaced independently of each other by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and the person skilled in the art is able to determine (either experimentally or theoretically) the possible substitutions without undue effort.

In the context of the present invention, alkyl, alkene and alkyne moieties as defined herein may further comprise one or more heteroatoms, e.g. a carbon in the alkyl, alkene or alkyne chain is selected such as from nitrogen, oxygen or sulfur and other heteroatom substitutions.

The present invention will be further described in detail below with reference to specific embodiments. It should be pointed out that the specific embodiments are for explanation rather than limitation of the present invention.

Example 1

Synthesis of intermediate 4-N,N’ didecyl aminobutyric acid tert-butyl ester (GA10a):

To a solution of 4-aminobutyric 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 ,1mmol). The reaction mixture was stirred and refluxed at 85°C for 48 hours. 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 then concentrated under reduced pressure. After being concentrated to dryness, the oil residue was purified by column chromatography (200-300 mesh silica gel, eluent: petroleum ether/ethyl acetate = 50:1-20:1) to obtain 4-N,N'didecylaminobutyl Tert-butyl acid ester (1.32g, yield 60.03%).

Synthesis of intermediate 4-N,N’ didecyl aminobutyric acid (GA10b):

Trifluoroacetic acid (2.5 mL) was added to a solution of GA10a (439.77 mg, 1 mmol) in glacial dichloromethane (6 mL) and stirred for 6 hours. Saturated sodium bicarbonate solution (5 mL) was added to the reaction mixture. Separate the organic layer, wash with saturated sodium bicarbonate (3 × 10 mL) and saturated sodium chloride solution (3 × 10 mL), dry the organic phase with anhydrous sodium sulfate, and remove the solvent on a rotary evaporator to obtain intermediate GA10b.

Example 2

Synthesis of intermediates 4-[di(dodecyl)amino]butyric acid-2-methylprop-2-yl ester (GA12a) and 4-[di(dodecyl)amino]butyric acid (GA12b) As described in Example 1, the difference is that 1-bromodecane is replaced by 1-bromododecane (11 mol); other steps and conditions are consistent with Example 1. The single-step yield of intermediate GA12a was 38.9%.

Example 3

The synthesis of intermediate (GA14a) and the synthesis of 4-[bis(tetradecyl)amino]butyric acid (GA14b) are as described in Example 1, except that 1-bromodecane is replaced by 1-bromotetradecane. Alkane (11 mol); other steps and conditions are consistent with Example 1. The single-step yield of intermediate GA14a was 64.73%.

Example 4

N-(11-Decyl-3-methyl-7-oxyylidene-3,6,11-triazanodecan-1-yl)-4-(didecylamino)butanamide (GAE10 )synthesis

Weigh GA10b (314.6 mg, 0.84 mmol) and dissolve it in 4 mL of methylene chloride. N-methyl-diaminoethylamine (46.9 mg, 0.4 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (184.03 mg, 0.96mmol), 1-hydroxybenzotriazole (HOBT) (129.72mg, 0.96mmol), N,N-diisopropylethylamine (DIPEA) (115.12μl, 1.2mmol) were added to the above solution, and stirred at room temperature for 24h. . The reaction was monitored using thin layer chromatography. The reaction mixture was washed with saturated brine (3×10 mL), and the organic phase was dried over anhydrous sodium sulfate. After concentration with a rotary evaporator, the oil residue was purified by column chromatography (column: 200-300 mesh silica gel; eluent: DCM/MeOH=100:0-10:1) to obtain GAE10 (111.3 mg, yield 43.76%). ¹ H NMR (400MHz, CDCl ₃ ) δ8.37 (s, 2H), 3.33 (s, 4H), 3.07 (s, 4H), 3.00 (s, 8H), 2.58 (d, J = 17.71Hz, 8H) , 2.26(s,3H), 2.04(s,4H), 1.69(s,8H), 1.22(d,J＝27.68,56H), 0.80(t,J＝5.12Hz,12H).

Example 5

4-[Di(dodecyl)amino]-N-(3-methyl-7-oxyylidene-11-dodecyl-3,6,11-triazatricosane-1- Synthesis of butylamide (GAE12)

GAE12 was prepared as described in Example 4, except that GA10b (0.84mmol) was replaced by GA12b (0.63mmol); other steps and conditions and the proportions of each reagent were consistent with Example 4. Obtain GAE12 (55.3mg, 19.2%). ¹ HNMR (400MHz, CDCl ₃ ) δ8.03 (s, 2H), 3.32 (t, 4H), 3.05 (s, 4H), 2.95 (s, 8H), 2.62 ( t,J＝5.82Hz,4H),2.52(d,J＝4.95Hz,4H),2.21(s,3H),2.08(m,4H),1.74(s,8H),1.29(m,72H), 0.88(t,J＝6.78Hz,12H).

Example 6

4-[Di(tetradecyl)amino]-N-(3-methyl-7-oxyylidene-11-tetradecyl-3,6,11-triazapentadecane-1- Synthesis of butylamide (GAE14)

GAE14 was prepared as described in Example 4, except that GAE10b (0.84mmol) was replaced by GAE14b (0.84mmol); other steps and conditions were consistent with Example 4. GAE14 (85.47 mg, yield 21.63%) was obtained. ¹ H NMR (400MHz, CDCl ₃ ) δ8.37 (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＝6.50Hz,12H ).

Example 7

4-[Di(dodecyl)amino]-N-(13-dodecyl-4-methyl-9-oxyylidene-4,8,13-triazapentadecane-1- Synthesis of butylamide (GAP12)

GAP12 was prepared as described in Example 4, except that GA10b (0.84mmol) was replaced by GA12b (0.84mmol), and N-methyl-diaminoethylamine was replaced by N,N-bis(3-amino Propyl)methylamine (0.4mmol); other steps and conditions were consistent with Example 4. GAP12 (58.3 mg, 14.75%) was obtained. ¹ H NMR (400MHz, CDCl3) δ7.43 (s, 2H), 3.29 (dd, J1 = 6.15Hz, J2 = 12.13Hz, 4H), 2.63 (d, J = 25.78Hz, 12H), 2.39 (t, J＝6.33Hz,4H),2.31(t,J＝6.83Hz,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＝6.64Hz,12H).

Example 8

N-(13-Decyl-4-methyl-9-oxyylidene-4,8,13-triazatricosan-1-yl)-4-(didecylamino)butanamide (GAP10 )synthesis.

GAP10 was prepared as described in Example 4, except that N-methyl-diaminoethylamine (0.4mmol) was replaced by N,N-bis(3-aminopropyl)methylamine (0.4mmol). ;Other steps, conditions and reagent molar ratios are consistent with Example 4. GAP10 (56.5 mg, yield 16.11%) was obtained. ¹ H NMR (400MHz, CDCl ₃ ) δ7.15 (s, 2H), 3.29 (dd, J1＝6.24Hz, J2＝6.25Hz, 4H), 2.44 (m, 8H), 2.37 (s, J＝6.46Hz ,4H),2.24(d,J＝7.06Hz,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＝6.65Hz,12H).

Example 9

Lipid GAE14 was selected as a representative compound to prepare lipid nanoparticles. First, the ionizable lipid (GAE14), cholesterol, DOPE, and DSPE-PEG2k used to prepare lipid nanoparticles were dissolved in ethanol at a molar ratio of 35:25:30:0.5 to prepare an ethanol phase solution. Then add EGFP (or Luciferase) mRNA to 10-50mM citrate buffer (pH=4) to obtain an mRNA aqueous phase solution, and prepare the mRNA lipid nanocomplex by rapidly mixing the ethanol phase solution and the aqueous phase solution evenly. . Among them, the weight ratio of ionizable lipid:mRNA is 10:1. Lipid nanoparticles encapsulating mRNA can be obtained through dialysis, ultrafiltration and other operations.

For further optimization of the prescription, in order to achieve good atomization stability of lipid nanoparticles and deep lung delivery, the natural lung surfactant component DPPC was added to a single phospholipid component, and further screening was performed to obtain the optimized prescription. Including but not limited to, ionizable lipids, cholesterol, DOPE, DPPC, DSPE-PEG2k molar ratio 35:35:6:4:1.5. According to the above operation and prescription ratio, lipid nanoparticles were prepared and labeled as NGAE14 for further characterization.

Experimental example 1

Characterization of Ionizable Lipid Nanoparticles:

The lipid nanoparticles prepared in Example 9 were further characterized. The morphology of the above-mentioned lipid nanoparticles was characterized by transmission electron microscopy. The dynamic light scattering laser particle size analyzer (Malvern Zetasizer Nano ZS) was used to characterize the nanometer size, polydispersity coefficient PDI and zeta potential. Quant-iT RiboGreen RNAAssay Kit RNA quantitative detection reagent Cartridge was used to measure the encapsulation efficiency, and the results are shown in Figures 2 to 5 (only the prescription: lipid prepared by ionizable lipid, cholesterol, DOPE, DPPC, DSPE-PEG2k molar ratio 35:35:6:4:1.5 Nanoparticles GAE14L). Lipid nanoparticles are spherical in shape, with uniform particle size (PDI<0.3), potential close to neutral, good encapsulation efficiency, and stable performance before and after atomization.

Experimental example 2

Investigation of in vitro transfection efficiency of ionizable lipid nanoparticles:

MLE12 cells in the logarithmic growth phase were seeded into a 96-well plate filled with DMEM/F12 medium at a plating density of 1*10 ⁵ cells per well. After the cells adhered, prepare for transfection (37°C, about 12 hours). Lipid nanoparticles containing 0.2 μg of mRNA were added to each well, and three duplicate wells were set in each group. 24 hours after transfection, use a fluorescence microscope or flow cytometer to detect the fluorescence ratio of cells in each group.

The overall experimental results show that the particle size of different prescription LNPs prepared with different ionizable lipids is basically maintained within 200nm, but there are large differences in delivery efficiency. We further optimize the prescription to prepare highly efficient transfection lipid nanoparticles to meet the needs of different disease treatments while reducing the risks caused by multiple administrations. Part of the flow cytometry experimental results are shown in Figure 6 (only GAE14L lipid nanoparticles are shown), in which the positive control is lipid nanoparticles prepared by commercially available DLin-MC3. The transfection efficiencies of LNP prepared with the same prescription for the remaining lipids GAE12, GAE10, GAP10, and GAP12 were 84.7%, 73.8%, 54.3%, and 67.5% respectively.

The above are only some embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. Biodegradable amino acid derived ionizable lipid, characterized in that it is a compound of formula (i), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof;

wherein R is ₁ 、R ₂ Are identical or different from each other and are each independently C ₆ -C ₂₄ Alkyl, C ₆ -C ₂₄ Alkenyl, C ₆ -C ₂₄ Alkynyl, substituent group-substituted C ₆ -C ₂₄ C substituted by alkyl, substituent groups ₆ -C ₂₄ Alkenyl, substituent group-substituted C ₆ -C ₂₄ With or without one or more heteroatoms;

R ₃ 、R ₄ are identical or different from each other and are each independently C ₆ -C ₂₄ Alkyl, C ₆ -C ₂₄ Alkenyl, C ₆ -C ₂₄ Alkynyl, substituent group-substituted C ₆ -C ₂₄ C substituted by alkyl, substituent groups ₆ -C ₂₄ Alkenyl, substituent group-substituted C ₆ -C ₂₄ With or without one or more heteroatoms;

R ₁ 、R ₂ and R is R ₃ 、R ₄ The same or different;

R ₅ independently selected from hydrogen, C ₁ -C ₁₀ C substituted by alkyl, substituent groups ₁ -C ₁₀ An alkyl group;

X ₁ 、X ₂ selected from oxygen or nitrogen or sulfur;

a is selected from positive integers from 1 to 3;

b is selected from positive integers from 1 to 3;

c is selected from integers from 0 to 2;

d is selected from integers from 0 to 2.

2. The biodegradable amino acid derived ionizable lipid of claim 1, wherein c = d = 0, said amino acid is α -aminoacetic acid;

or, c=d=1, the amino acid is β -alanine;

or, when c=d=2, the amino acid is gamma-aminobutyric acid;

or, R ₁ 、R ₂ 、R ₃ 、R ₄ Are identical to or different from each other and are independently selected from C ₆ -C ₂₄ An alkyl group; r is R ₁ ＝R ₂ ，R ₃ ＝R ₄ ；R ₅ Is hydrogen or methyl; x is nitrogen; a=b, c=d;

or, R ₁ 、R ₂ 、R ₃ 、R ₄ Independently selected from C ₈ -C ₁₈ Alkyl, R ₅ Methyl, c=d=2.

3. The biodegradable amino acid derived ionizable lipid of claim 1, wherein said biodegradable amino acid derived ionizable lipid is selected from one of the following compounds:

4. a method of preparing a biodegradable amino acid derived ionizable lipid according to any one of claims 1-3, comprising:

a step of obtaining a compound represented by formula (1) by an esterification reaction or an amidation reaction with a compound represented by formula (I1) and a compound represented by formula (III);

wherein in the formulas (II), (III), R ₁ 、R ₂ 、R ₅ 、X ₁ 、X ₂ A, b and c have the same meaning as in the compounds of the formula (I).

5. The method of preparing a biodegradable amino acid derived ionizable lipid of claim 4, comprising one or more of the following conditions:

i. the organic solvent is selected from one or more 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-dimethylformamide or triethanolamine;

ii. The catalyst is selected from one or more than two of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, N-hydroxysuccinimide, dicyclohexylcarbodiimide, diisopropylcarbodiimide, 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, O-benzotriazol-tetramethylurea hexafluorophosphate, 4-dimethylaminopyridine or O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroboric acid;

or the catalyst is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole;

the mol ratio of the compound shown in the formula (I1) to the compound shown in the formula (III) is 1:1-1.1;

iv, the reaction temperature is room temperature, and the reaction time is 10-30h.

6. Use of a biodegradable amino acid derived ionizable lipid according to any of claims 1-3 for the preparation of a drug delivery vehicle.

7. A lipid nanoparticle comprising the biodegradable amino acid-derived ionizable lipid of any one of claims 1-4, a helper lipid, a phospholipid, and a PEG-lipid.

8. The lipid nanoparticle of claim 7, wherein the helper lipid is selected from the group consisting of a steroid, a cholesterol hemisuccinate, a cholesterol, and an alkyl resorcinol;

or, the phospholipid is selected from one or more than two of distearoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylcholine, diethyl pyrocarbonate, dilauroyl phosphatidylcholine, egg yolk lecithin, hydrogenated soybean phosphatidylcholine, sphingomyelin or dimyristoyl phosphatidylcholine;

or, the PEG lipid is selected from one or more than two of DSPE-PEG, DMG-PEG, DPPE-PEG or DMA-PEG;

or, the molar ratio of the biodegradable amino acid derived ionizable lipid, the auxiliary lipid, the phospholipid and the PEG lipid is 20-50:20-60:10-40:0.5-10; the mass ratio of the ionizable lipid to the medicine is 1-100:1;

the medicine comprises one or more than two of biological medicine or chemical medicine; the biological medicine comprises one or more than two of nucleic acid medicine, protein medicine, polypeptide medicine or polysaccharide medicine;

or, the nucleic acid drug comprises one or more than two of small interfering RNA, messenger RNA, microRNA, circular mRNA, long-chain non-coding RNA, plasmid DNA, mini circle DNA, antisense oligonucleotide, small activating RNA or nucleic acid aptamer;

or, the chemical medicine comprises one or more than two of small molecule medicine, fluorescein or developer;

alternatively, the drug is an mRNA, including linear mRNA and circular mRNA.

9. A method of preparing lipid nanoparticles according to claim 7 or 8, comprising: liposome extrusion, thin film differentiation, nano precipitation, microfluidic and impact jet mixing;

or, the preparation method of the lipid nanoparticle comprises the following steps: dissolving biodegradable amino acid derived ionizable lipid, auxiliary lipid, phospholipid and PEG lipid in ethanol to obtain lipid mixed ethanol phase; dispersing the medicine in citric acid buffer solution with pH=4-4.5 to obtain medicine water phase; mixing the lipid mixed ethanol phase and the drug water phase by utilizing micro-flow control to prepare a solution containing lipid nanoparticles; then the lipid nanoparticle is prepared through dialysis and ultrafiltration.

10. Use of a biodegradable amino acid derived ionizable lipid according to any of claims 1-3, and a lipid nanoparticle according to claim 7 or 8, for the preparation of a genetic medicament, characterized in that said genetic medicament comprises: an active ingredient and a delivery carrier, wherein the active ingredient is a nucleic acid drug.