Amino acid skeleton ionizable lipid as well as preparation method and application thereof

Abstract

The invention provides an amino acid skeleton ionizable lipid as well as a preparation method and application thereof, the amino acid skeleton ionizable lipid is modified by taking amino acid as a core, has more ester groups and peptide bonds, and can be quickly hydrolyzed by enzyme after RNA is effectively released in vivo; the transfection body is provided with four tail structures, the cross sectional area of the lipid tail can be increased, drugs such as RNA are helped to escape from an endosome, and then the transfection effect is enhanced; the charge capable of ionizing the lipid is electrically neutral under physiological conditions, so that the cytotoxicity caused by excessive positive charges is reduced, the stability of the lipid nanoparticles is further improved, the cycle time of the loaded nucleic acid medicine is prolonged, and the pharmacokinetic characteristics are improved. The LNP prepared from the ionizable lipid, auxiliary phospholipid, cholesterol and PEG lipid provided by the invention has more excellent nucleic acid carrier performance, and can effectively deliver nucleic acid drugs such as siRNA, mRNA, pDNA and the like into cells to play a role.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug carriers, and in particular relates to an amino acid skeleton ionizable lipid and a preparation method and application thereof. Background Art

[0002] Cancer vaccines are generally classified into four types: tumor or immune cell vaccines, peptide vaccines, viral vector vaccines, and nucleic acid vaccines. Nucleic acid-based vaccines are a very promising type of vaccine (e.g., DNA or RNA vaccines). RNA-based therapies primarily include antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), and circular RNA (circRNA). By manipulating different modes of action, they show great promise in treating a wide range of diseases. First, nucleic acid vaccines can simultaneously deliver multiple antigens, such as tumor-associated antigens (TAAs) or somatic tumor mutations, triggering humoral and cellular immunity and reducing vaccine resistance. Second, unlike peptide vaccines, nucleic acid vaccines allow APCs to simultaneously or cross-present multiple epitopes of class I and class II patient-specific human leukocyte antigens (HLAs), thus being less restricted by human HLA types and more likely to stimulate a broader T cell response. However, due to the inherent negative charge and instability of RNA molecules, RNA has difficulty breaking through biological barriers and reaching the cytoplasm. To overcome this problem, RNA requires a safe, effective, and stable delivery system to protect nucleic acids from degradation and accelerate cellular uptake and effective release of RNA. Currently, lipid nanoparticles (LNPs) are widely used in the field of drug delivery, but RNA vaccines still require more efficient delivery systems.

[0003] Nucleic acid delivery systems can be broadly divided into two categories: viral vectors and non-viral vectors. Viral vectors offer relatively high transfection efficiency, but they suffer from issues such as poor safety and targeting. Over the past few decades, liposomes, as a representative example of non-viral delivery systems, have rapidly developed. A new class of lipids, ionizable lipids, has been developed. These lipids can be protonated at weakly acidic pH, imparting a positive charge while remaining neutral at physiological pH. The pH sensitivity of ionizable lipids facilitates mRNA delivery in vivo, as neutral lipids interact less with the anionic membranes of blood cells, thereby improving the biocompatibility of nanoparticles. However, when lipid nanoparticles are placed in endosomes at weakly acidic pH, the ionizable lipids acquire a charge, promoting membrane destabilization and enhancing the nanoparticle's endosomal escape. However, ionizable lipids still have the following problems: (1) Toxicity issues: Ionizable lipids are the key components in lipid nanoparticles (LNPs) that trigger acute immune responses and long-term toxicity, but the lipid structure still needs to be further optimized; (2) Synthesis complexity: The current synthesis process of ionizable lipids is cumbersome and requires multi-step reactions and complex purification steps, which limits large-scale production and rapid screening; (3) Low delivery efficiency: Low endosomal escape is a common delivery efficiency problem of lipid nanoparticles, and more in-depth research is still needed to optimize the endosomal escape mechanism. Summary of the Invention

[0004] To overcome the problems of the above-mentioned prior art, one object of the present invention is to provide an ionizable amino acid backbone lipid. A second object of the present invention is to provide a method for preparing an ionizable amino acid backbone lipid. A third object of the present invention is to provide a lipid composition. A fourth object of the present invention is to provide the use of the above-mentioned ionizable amino acid backbone lipid and lipid composition in a delivery vehicle. A fifth object of the present invention is to provide a pharmaceutical composition. A sixth object of the present invention is to provide the use of the above-mentioned ionizable amino acid backbone lipid and pharmaceutical composition in the preparation of a medicament.

[0005] The present invention proposes an amino acid backbone ionizable lipid, the chemical structure of which contains three components: (i) an ionizable head group, (ii) a linker group, and (iii) a hydrophobic tail. The head group is a tertiary or secondary amine group, which can acquire protons at acidic pH, thereby carrying a positive charge. It can bind to negatively charged nucleic acid molecules or small molecule drugs through electrostatic interactions and then self-assemble with auxiliary lipids into lipid nanoparticles to deliver gene drugs. The hydrophobic tail structure has 2 to 4 more tail groups than the common double-tailed lipids in the prior art. Due to the increased cross-section of the tail region, this lipid produces a more tapered structure, giving it stronger endosome-destroying ability and enhancing delivery efficiency. Moreover, the tail is synthesized based on amino acids and has excellent biosafety. In response to a series of problems encountered in current gene drug delivery, such as low efficiency and high toxicity, this amino acid backbone ionizable lipid balances degradability in its chemical structure design, ensuring lipid safety while maintaining overall delivery efficiency. In addition, the preparation method of the amino acid skeleton ionized lipid of the present invention is different from the harsh and complex synthesis route of traditional cationic lipids. An ionizable lipid library can be obtained through Michael addition, which has the advantages of a simple synthesis route, a clear reaction mechanism, and is convenient for high-throughput screening.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides an amino acid backbone ionizable lipid,

[0008] Wherein, the R1 is selected from C2-C10 alkyl, C2-C10 heteroalkyl, aralkyl, or absent;

[0009] The R2 is selected from C3-C24 straight chain alkyl, C4-C24 straight chain alkenyl, n1=2~3, n2=3~24, n3=3~24;

[0010] Said X is O or NH;

[0011] The R3 is selected from

[0012] C1-C10 hydroxyalkyl; R6 and R7 are each independently selected from C1-C4 alkyl, C1-C4 hydroxyalkyl;

[0013] The R4 is selected from C1-C4 hydroxyalkyl; the R5 is C1-C4 alkyl, C1-C4 hydroxyalkyl, n4=2~3

[0014] The "*" is a connection site.

[0015] The term "isomer" refers to the fact that the compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are encompassed within the scope of the present invention.

[0016] Unless otherwise stated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of each other. Unless otherwise stated, the term "cis-trans isomer" or "geometric isomer" is caused by the inability to rotate freely around double bonds or single bonds of ring carbon atoms. Unless otherwise stated, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and are not mirror images of each other. Unless otherwise stated, "(D)" or "(+)" indicates dextrorotation, "(L)" or "(-)" indicates levorotation, and "(DL)" or "(±)" indicates racemic. Unless otherwise stated, bonds with a solid wedge are used. and dotted wedge key Indicates the absolute configuration of a stereocenter.

[0017] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, prepared by reacting the compounds of the present invention with relatively nontoxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, dihydrogen phosphate, dihydrogen phosphate, sulfuric acid, bisulfate, hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; and salts of amino acids (such as arginine) and organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and can be converted into either base or acid addition salts.

[0018] Preferably, the structural formula of the amino acid backbone ionizable lipid is selected from the structures shown in any one of the following formulas (1) to (104):

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027] The second aspect of the present invention provides a method for preparing the amino acid backbone ionizable lipid described in the first aspect, comprising the following steps: reacting a tail compound containing an amino acid structure or a stereoisomer or tautomer thereof with an organic amine compound through a Michael addition reaction to obtain the amino acid backbone ionizable lipid;

[0028] The structural formula of the tail compound containing an amino acid structure is shown in formula (a): The R1, R2, and X are as described in the first aspect.

[0029] Preferably, the reaction temperature of the Michael addition reaction is 70-110°C.

[0030] More preferably, the reaction temperature of the Michael addition reaction is 80-100°C.

[0031] More preferably, the reaction temperature of the Michael addition reaction is 85-95°C.

[0032] Preferably, the reaction time of the Michael addition reaction is 36 to 60 hours.

[0033] More preferably, the reaction time of the Michael addition reaction is 40 to 56 hours.

[0034] More preferably, the reaction time of the Michael addition reaction is 44 to 52 hours.

[0035] Preferably, the organic amine compound is selected from one of the following compounds:

[0036]

[0037]

[0038] Preferably, the preparation method of the tail compound containing an amino acid skeleton comprises the following steps: reacting acrylic acid chloride with compound a1

[0039] The tail compound containing an amino acid skeleton is prepared through an esterification reaction.

[0040] More preferably, the reaction temperature of the esterification reaction is 15-40°C.

[0041] More preferably, the reaction temperature of the esterification reaction is 20-35°C.

[0042] More preferably, the reaction time of the esterification reaction is 12 to 24 hours.

[0043] More preferably, the reaction temperature of the esterification reaction is 12 to 16 hours.

[0044] More preferably, the reaction system of the esterification reaction further comprises an organic base catalyst.

[0045] More preferably, the organic base catalyst is triethylamine.

[0046] More preferably, the molar ratio of acrylic acid chloride to compound a1 is (1-2):1.

[0047] More preferably, the molar ratio of acrylic acid chloride to compound a1 is (1-1.5):1.

[0048] More preferably, the preparation method of the compound a1 comprises the following steps: reacting a Boc-amino acid with an alkyl alcohol or an alkenyl alcohol, and subjecting the reaction product to a deprotection reaction with trifluoroacetic acid to obtain the compound a1.

[0049] More preferably, the reaction system further comprises an active agent and an acid promoter.

[0050] More preferably, the active agent comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI).

[0051] More preferably, the acid promoter comprises 4-dimethylaminopyridine (DMAP).

[0052] More preferably, the reaction temperature is 15-40°C.

[0053] More preferably, the reaction temperature of the deprotection reaction is 15-40°C.

[0054] The third aspect of the present invention provides a lipid composition comprising the amino acid backbone ionizable lipid described in the first aspect, a sterol, and a PEGylated lipid.

[0055] Preferably, a neutral helper lipid is also included.

[0056] More preferably, the neutral helper lipid includes at least one of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dilinoleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), and palmitoyloleylphosphatidylcholine (POPC).

[0057] Preferably, the polyethylene glycol (PEG)-ylated lipid includes at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0058] Preferably, the sterol includes at least one of cholesterol and β-sitosterol.

[0059] More preferably, when the lipid composition further comprises cholesterol, auxiliary phospholipid and PEGylated lipid, the molar ratio of the amino acid backbone ionizable lipid: cholesterol: auxiliary phospholipid: PEGylated lipid is (30-50): (35-60): (5-20): (1-5).

[0060] The fourth aspect of the present invention provides the use of the amino acid backbone ionizable lipid described in the first aspect, or the lipid composition described in the third aspect in the preparation of a bioactive substance delivery system, wherein the delivery system is a microparticle, nanoparticle, liposome, lipid nanoparticle or microbubble.

[0061] The fifth aspect of the present invention provides a pharmaceutical composition comprising the lipid composition described in the third aspect and an active substance; the active substance comprises at least one of a nucleic acid molecule, a small molecule compound, a polypeptide, and a protein.

[0062] Preferably, it comprises a carrier and an active substance, wherein the carrier comprises the lipid composition described in the third aspect; and the active substance is encapsulated in the carrier or combined with the carrier.

[0063] More preferably, the preparation method of the pharmaceutical composition comprises the following steps:

[0064] The amino acid backbone ionizable lipid, sterol, neutral auxiliary lipid, and PEGylated lipid are mixed in an ethanol solvent to prepare a lipid mixed solution; the active substance is mixed with an acidic buffer, and then mixed with the lipid mixed solution and incubated to prepare a drug carrier, i.e., the pharmaceutical composition;

[0065] Alternatively, the amino acid skeleton ionizable lipid, sterol, and neutral auxiliary lipid are dissolved in chloroform, blown dry with nitrogen to evaporate the solvent, and then an acidic or neutral buffer is added and mixed to prepare liposome nanoparticles for later use; the cationic polypeptide is mixed with the active substance, and then mixed with the liposome nanoparticles, and then pegylated lipid is added to prepare a drug carrier.

[0066] Further preferably, the cationic polypeptide is protamine.

[0067] More preferably, the acidic buffer solution has a pH of 3 to 7; the acidic buffer solution is sodium acetate or sodium citrate buffer solution.

[0068] Preferably, when the pharmaceutically active ingredient includes a nucleic acid molecule, the nitrogen-to-phosphorus ratio of the amino acid backbone ionizable lipid to the nucleic acid molecule is (1-100):1.

[0069] More preferably, when the pharmaceutically active ingredient includes a nucleic acid molecule, the nitrogen-to-phosphorus ratio of the amino acid backbone ionizable lipid to the nucleic acid molecule is (1-30):1.

[0070] Further preferably, when the pharmaceutically active ingredient includes a nucleic acid molecule, the nitrogen-to-phosphorus ratio of the amino acid backbone ionizable lipid to the nucleic acid molecule is (1-10):1.

[0071] Preferably, the nucleic acid molecule includes at least one of siRNA, miRNA, mRNA, circRNA, antisense RNA, CRISPR guide RNAs, replicable RNA, circular dinucleotide, poly IC, CpG ODN, plasmid DNA, and minicircular DNA.

[0072] Preferably, the protein includes at least one of colony stimulating factor, interleukins, lymphotoxins, interferon proteins, tumor necrosis factor, antibodies, and protein antigens.

[0073] The sixth aspect of the present invention provides the use of the amino acid backbone ionizable lipid described in the first aspect, or the pharmaceutical composition described in the fifth aspect in the preparation of nucleic acid drugs, gene vaccines, polypeptide or protein drugs, and small molecule drugs.

[0074] The beneficial effects of the present invention are:

[0075] (1) The present invention proposes an amino acid skeleton ionizable lipid, which is modified with amino acids as the core and has a large number of ester groups and peptide bonds. After effectively releasing RNA in the body, it can be quickly hydrolyzed by enzymes, is easy to be metabolized and eliminated in the body, and is biodegradable; it has a four-tail structure, which can increase the cross-sectional area of ​​the lipid tail, help RNA and other drugs escape from the endosome, and thus enhance the transfection effect; the charge of the ionizable lipid can change with the change of the pH of the environment, and is electrically neutral under physiological conditions, reducing the cytotoxicity caused by excessive positive charge, thereby increasing the stability of lipid nanoparticles, and helping to prolong the circulation time of the loaded nucleic acid drug and improve the pharmacokinetic characteristics.

[0076] (2) The LNPs provided by the present invention, which are made of ionizable lipids and auxiliary phospholipids, cholesterol and PEG lipids, have more excellent nucleic acid carrier properties and can effectively deliver nucleic acid drugs such as siRNA, mRNA, and pDNA into cells to exert their effects.

[0077] (3) The preparation method of the amino acid backbone ionized lipid of the present invention is different from the harsh and complex synthesis route of traditional cationic lipids. An ionizable lipid library can be obtained through Michael addition, which has the advantages of a simple synthesis route, a clear reaction mechanism, and is convenient for high-throughput screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 The relative luciferase activity results of cells transfected with different lipid nanoparticles are shown;

[0079] Figure 2(a) The relative luciferase activity results of cell transfection with lipid nanoparticles containing different neutral phospholipids; (b) The relative luciferase activity results of cell transfection with lipid nanoparticles containing different component ratios;

[0080] Figure 3 (c) is a graph showing the relative luciferase activity of cells transfected with lipid nanoparticles containing different nitrogen-phosphorus ratios; (d) is a graph showing the relative luciferase activity of cells transfected with lipid nanoparticles containing different buffers;

[0081] Figure 4 Fluorescence microscopy of Jurkat T cell lines transfected with different lipid nanoparticles;

[0082] Figure 5 Images of in vivo transfection of mice with different lipid nanoparticles;

[0083] Figure 6 The relative luciferase activity results of different lipid nanoparticles transfected in mice;

[0084] Figure 7 The relative luciferase activity results of the spleen and liver of mice transfected with different lipid nanoparticles in vivo;

[0085] Figure 8 This is the relative luciferase activity ratio of the spleen and liver in mice transfected with different lipid nanoparticles. DETAILED DESCRIPTION

[0086] The present invention is further described in detail below through specific examples. Unless otherwise specified, the raw materials used in the following examples can be obtained from conventional commercial sources or prepared and isolated by simple synthesis; unless otherwise specified, the processes used are conventional processes in the art.

[0087] The general preparation method of the partial amino acid backbone ionizable lipid of the present invention has the following specific steps:

[0088] (1) Synthesize the hydrophobic tail of the amino acid backbone

[0089] The specific steps are as follows: 10 mmol Boc-amino acid, 150 mL dichloromethane (DCM) and a magnet, 22 mmol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 20 mmol 4-dimethylaminopyridine (DMAP) were added to a 250 mL reaction flask in sequence, stirred at room temperature for 15 minutes, and then 20 mmol alkyl alcohol was added. The reaction mixture was reacted for 24 hours until the reaction was complete. The reaction mixture was transferred to a separatory funnel, DCM (2 x 100 mL) was added, and 1M HCl (2 x 100 mL) was used for washing, and saturated brine (2 x 200 mL) was extracted. The collected organic layer was dried over anhydrous magnesium sulfate and filtered, and then the organic solvent was removed using a reduced pressure rotary evaporator. The product was separated by thin layer chromatography.

[0090] Removal of amino protecting groups: Dissolve the product in 30 mL of DCM, add 20 mL of trifluoroacetic acid (TFA), and stir at room temperature for 4 h. Remove the organic solvent using a rotary evaporator under reduced pressure, then redissolve in 150 mL of DCM, wash with saturated sodium bicarbonate (2 x 100 mL), and extract with saturated brine (2 x 100 mL). The collected organic layer is dried over anhydrous magnesium sulfate and filtered. The organic solvent is then removed using a rotary evaporator under reduced pressure. The product is then carried on to the next step without further purification.

[0091] (2) Synthesis of linking groups

[0092] To a three-necked flask equipped with a magnetic separator, 5 mmol of the amino acid backbone alkyl tail product synthesized in the previous step, 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 minutes. Using a constant pressure funnel, 6.25 mmol of acryloyl chloride (premixed in 10 mL of dichloromethane) was slowly added dropwise. After the addition of acryloyl chloride was complete, the ice bath was removed. The reaction was allowed to react overnight at room temperature, then diluted with DCM (30 mL) and washed with 1 M HCl (50 mL). The organic layer was dried over anhydrous magnesium sulfate and filtered. The product was then isolated by flash chromatography.

[0093] (3) Reaction between head group and tail group

[0094] The chemically equivalent alkyl tail synthesized in step (2) and 100 mg of amine were added sequentially into a 3 mL reaction bottle lined with tetrafluoroethylene. The reaction was heated at 90°C for 48 h. After the reaction, the product could be directly used for cell transfection experiments or separated by rapid chromatography.

[0095] The Boc-amino acid synthesized by the present invention adopts L-type or D-type amino acid, thereby preparing an amino acid skeleton ionizable lipid containing L-type or D-type amino acid groups.

[0096] Example 1

[0097] Example 1 provides an amino acid backbone ionizable lipid, the preparation method of which is as follows:

[0098] (1) Synthesize the hydrophobic tail of the amino acid backbone

[0099]

[0100] To a 250 mL reaction flask, 10 mmol of BOC-L-glutamic acid, 150 mL of dichloromethane (DCM), a magnetic atom, 22 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 20 mmol of 4-dimethylaminopyridine (DMAP) were added sequentially. The mixture was stirred at room temperature for 15 minutes, followed by the addition of 20 mmol of oleyl alcohol. The reaction was allowed to react for 24 hours, and the reaction progress was monitored by thin-layer chromatography (TLC). Upon completion, the reaction mixture was transferred to a separatory funnel, washed with DCM (2 x 100 mL), washed with 1 M HCl (2 x 100 mL), and extracted with saturated brine (2 x 200 mL). The organic layer was dried over anhydrous magnesium sulfate and filtered, and the organic solvent was removed by rotary evaporation under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 20:1) to obtain the target product A in a yield of 73%.

[0101]

[0102] Removal of amino protecting groups: Dissolve intermediate product A in 30 mL of DCM, add 20 mL of trifluoroacetic acid (TFA), and stir at room temperature for 4 hours. Remove the organic solvent using a vacuum rotary evaporator, then redissolve in 150 mL of DCM, wash with saturated sodium bicarbonate (2 x 100 mL), and extract with saturated brine (2 x 100 mL). The collected organic layer is dried over anhydrous magnesium sulfate and filtered. The organic solvent is then removed using a vacuum rotary evaporator to obtain product B, which can be carried on to the next step without further purification.

[0103] (2) Synthesis of linking groups

[0104]

[0105] To a three-necked flask equipped with a magnetic separator, 5 mmol of the amino acid backbone alkyl tail of intermediate B, 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 minutes. Using a constant pressure funnel, 6.25 mmol of acryloyl chloride (premixed in 10 mL of dichloromethane) was slowly added dropwise. Once the addition of acryloyl chloride was complete, the ice bath was removed. The reaction was allowed to proceed overnight at room temperature, and progress was monitored by TLC. Upon completion, the reaction was diluted with DCM (30 mL) and washed with 1 M HCl (50 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, filtered, and the organic solvent removed using a reduced-pressure rotary evaporator. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 5:1) to obtain the desired product C in a 95% yield.

[0106] The hydrogen spectrum data of the obtained product are as follows:

[0107] 1 H NMR (400MHz, CDCl3): 6.37 (d, J = 3.6, 1H), 6.30 (d, J = 16Hz, 1H), 5.79 (m, 1H), 5.6 (d, J = 10.4Hz, 1H), 5.38-5.29 (m, 4H), 4.72-4.67 (m, 1H), 4.15- 4.02(dt,J=6.8Hz,4H),2.43-2.32(m,2H),2.27-2.19(m,1H),2.08-1.9 5(m,7H),1.29-1.25(m,42H),1.55-1.36(m,8H),0.87(t,J=13.6Hz,6H).

[0108] (3) Reaction between head group and tail group

[0109]

[0110] To a 5 mL reaction vial (with a Teflon-lined cap) equipped with a magnetic rod, add 100 mg of 3-aminopropanol and two chemical equivalents of intermediate C. The mixture is allowed to react at 90°C for 48 hours. After completion, the product is separated by thin-layer chromatography (DCM:methanol = 20:1) to yield the ionizable lipid 29GluOC18U.

[0111] The hydrogen spectrum data of the obtained product are as follows:

[0112] 1H NMR(400MHz, CDCl3):4.78-4.65(m,4H),4.08-4.03(m,4H),3.19-3.02(m,8H),2.81-2.77(m,2H), 2.63-2.42(m,10H),1.80-1.48(m,20H),1.39-1.25(m,20H),0.87(dd,J1=6.8Hz,J2=5.2Hz,12H).

[0113] Example 2

[0114] Example 2 provides an amino acid backbone ionizable lipid, the preparation method of which is as follows:

[0115]

[0116] To a 5 mL reaction vial (with a Teflon-lined cap) equipped with a magnetic rod, add 100 mg of 5-aminopentanol and two chemical equivalents of intermediate C. The mixture is allowed to react at 90°C for 48 hours. After completion, the product is separated by thin-layer chromatography (DCM:methanol = 20:1) to yield the ionizable lipid 34GluOC18U.

[0117] The hydrogen spectrum data of the obtained product are as follows:

[0118] 1 H NMR(400MHz, CDCl3):7.73-7.38(m,2H),5.41-5.35(m,8H),4.58-4.53(m,2H),4.14-4.06(m,8H),3.69-3.66(m,2H),3.16-3.10(m,8H),2 .81-2.77(m,2H),2.47-2.41(m,4H),2.24-1.98(m,20H),1.67-1.59(m,10H),1.45-1.41(m,4H),1.36-1.27(m,88H),0.91-0.88(m,12H).

[0119] Example 3

[0120] Example 3 provides an amino acid backbone ionizable lipid, the preparation method of which is as follows:

[0121] (1) Synthesis of linking groups

[0122]

[0123] In a three-necked flask equipped with a magnetic rod, 10 mmol of L-aspartic acid, 30 mmol of TEA, and 120 mL of DCM were added sequentially. The three-necked flask was pre-cooled in an ice bath for 30 minutes. 11 mmol of acryloyl chloride (premixed in 20 mL of dichloromethane) was slowly added dropwise using a constant pressure funnel. After the addition of acryloyl chloride was complete, the ice bath was removed. The reaction was allowed to proceed overnight at room temperature. The reaction progress was monitored by TLC until the reaction was complete. The mixture was diluted with DCM (50 mL) and washed with 1 M HCl (2 × 100 mL). The collected organic layer was dried over anhydrous magnesium sulfate and filtered, and then the organic solvent was removed using a reduced pressure rotary evaporator. Product D was used directly without purification.

[0124] (2) Synthesize the hydrophobic tail of the amino acid backbone

[0125]

[0126] To a 100 mL reaction flask, 5 mmol of intermediate D, 50 mL of dichloromethane (DCM), a magnetic atom, 11 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 5 mmol of 4-dimethylaminopyridine (DMAP) were added sequentially. The mixture was stirred at room temperature for 15 minutes, followed by the addition of 11 mmol of n-octanol. The reaction was allowed to react for 24 hours, and the reaction progress was monitored by thin-layer chromatography (TLC). Upon completion, the reaction mixture was transferred to a separatory funnel, washed with DCM (2 x 100 mL), washed with 1 M HCl (2 x 100 mL), and extracted with saturated brine (2 x 200 mL). The organic layer was dried over anhydrous magnesium sulfate and filtered, and the organic solvent was removed using a reduced-pressure rotary evaporator. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 20:1) to obtain the target product E in a 43% yield.

[0127] The hydrogen spectrum data of the obtained product are as follows:

[0128] 1 H NMR (400MHz, CDCl3): 6.69 (d, J = 8Hz, 1H), 6.32 (d, J = 8Hz, 1H), 6.2-6.13 (m, 1H), 5.7 (d, J = 10.4Hz, 1H), 4.93-4 .89(m,1H),4.19-4.06(m,4H),3.10-2.88(m,2H),1.65-1.59(m,4H),1.34-1.21(m,20H),0.89(t,J=6Hz,6H).

[0129] (2) Reaction between head group and tail group

[0130]

[0131] To a 5 mL reaction vial (with a Teflon-lined cap) equipped with a magnetic rod, add 100 mg of N,N-dimethylethylenediamine and two chemical equivalents of intermediate E. The mixture was reacted at 90°C for 48 hours. After completion of the reaction, the product was separated by thin-layer chromatography (DCM:methanol = 20:1) to obtain the ionizable lipid 1AspOC8.

[0132] The hydrogen spectrum data of the obtained product are as follows:

[0133] 1 H NMR(400MHz, CDCl3):4.78-4.65(m,4H),4.08-4.03(m,4H),3.19-3.02(m,8H),2.81-2.77(m,2H), 2.63-2.42(m,10H),1.80-1.48(m,20H),1.39-1.25(m,20H),0.87(dd,J1=6.8Hz,J2=5.2Hz,12H).

[0134] Example 4

[0135] Example 4 provides an amino acid backbone ionizable lipid, the preparation method of which is as follows:

[0136] (1) Synthesize the hydrophobic tail of the amino acid backbone

[0137]

[0138] To a 250 mL reaction flask, 10 mmol of BOC-L-aspartic acid, 150 mL of dichloromethane (DCM), a magnetic atom, 22 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 20 mmol of 4-dimethylaminopyridine (DMAP) were added sequentially. The mixture was stirred at room temperature for 15 minutes, followed by the addition of 20 mmol of 9-nonadecanol. The reaction was allowed to react for 24 hours, and the reaction progress was monitored by thin-layer chromatography (TLC). Upon completion, the reaction mixture was transferred to a separatory funnel, washed with DCM (2 x 100 mL), washed with 1 M HCl (2 x 100 mL), and extracted with saturated brine (2 x 200 mL). The collected organic layer was dried over anhydrous magnesium sulfate and filtered, and the organic solvent was removed using a reduced-pressure rotary evaporator. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 30:1) to obtain the target product F in a yield of 68%.

[0139]

[0140] Removal of amino protecting groups: Dissolve intermediate F in 30 mL of DCM, add 20 mL of trifluoroacetic acid (TFA), and stir at room temperature for 4 h. Remove the organic solvent using a vacuum rotary evaporator, then redissolve in 150 mL of DCM, wash with saturated sodium bicarbonate (2 x 100 mL), and extract with saturated brine (2 x 100 mL). The collected organic layer is dried over anhydrous magnesium sulfate and filtered. The organic solvent is then removed using a vacuum rotary evaporator to obtain product G, which can be carried on to the next step without further purification.

[0141] (2) Synthesis of linking groups

[0142]

[0143] To a three-necked flask equipped with a magnetic rod, 5 mmol of the amino acid backbone alkyl tail of intermediate G, 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 minutes. Using a constant pressure funnel, 6.25 mmol of acryloyl chloride (premixed in 10 mL of dichloromethane) was slowly added dropwise. Once the addition of acryloyl chloride was complete, the ice bath was removed. The reaction was allowed to proceed overnight at room temperature, and progress was monitored by TLC. Upon completion, the reaction was diluted with DCM (30 mL) and washed with 1 M HCl (50 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, filtered, and the organic solvent removed using a reduced-pressure rotary evaporator. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 8:1) to obtain the desired product H in an 88% yield.

[0144] The hydrogen spectrum data of the obtained product are as follows:

[0145] 1 H NMR (400MHz, CDCl3): 6.69 (m, 1H), 6.32 (d, J = 16.8Hz, 1H), 6.19-6.12 (m, 1H), 5.68 (d, J = 10Hz, 1H), 4.93-4.89 (m,1H),4.08-3.93(m,4H),3.10-2.89(m,2H),1.63-1.59(m,2H),1.43-1.26(m,66H),0.88(t,J=6.4Hz,12H).

[0146] (3) Reaction between head group and tail group

[0147]

[0148] To a 5 mL reaction vial (with a Teflon-lined cap) equipped with a magnetic rod, 100 mg of 1-(2-aminoethyl)piperidine and two chemical equivalents of intermediate H were added and allowed to react at 90°C for 48 hours. Upon completion, the product was separated by thin-layer chromatography (DCM:methanol = 20:1) to yield the ionizable lipid 13AspOC8C10.

[0149] The hydrogen spectrum data of the obtained product are as follows:

[0150] 1 H NMR(400MHz, CDCl3):4.78-4.65(m,4H),4.08-4.03(m,4H),3.19-3.02(m,8H),2.81-2.77(m,2H), 2.63-2.42(m,10H),1.80-1.48(m,20H),1.39-1.25(m,20H),0.87(dd,J1=6.8Hz,J2=5.2Hz,12H).

[0151] Example 5

[0152] Example 5 provides an amino acid backbone ionizable lipid, the preparation method of which is as follows:

[0153] (1) Synthesize the hydrophobic tail of the amino acid backbone

[0154]

[0155] To a 250 mL reaction flask, 10 mmol of BOC-L-aspartic acid, 150 mL of dichloromethane (DCM), a magnetic atom, 22 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 20 mmol of 4-dimethylaminopyridine (DMAP) were added sequentially. The mixture was stirred at room temperature for 15 minutes, followed by the addition of 20 mmol of oleylamine. The reaction was allowed to react for 24 hours, and the reaction progress was monitored by thin-layer chromatography (TLC). Upon completion, the reaction mixture was transferred to a separatory funnel, washed with DCM (2 x 100 mL), washed with 1 M HCl (2 x 100 mL), and extracted with saturated brine (2 x 200 mL). The organic layer was dried over anhydrous magnesium sulfate and filtered, and the organic solvent was removed using a reduced-pressure rotary evaporator. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 4:1) to obtain the target product I in a yield of 73%.

[0156]

[0157] Removal of amino protecting groups: Dissolve intermediate I in 30 mL of DCM, add 20 mL of trifluoroacetic acid (TFA), and stir at room temperature for 4 h. Remove the organic solvent using a vacuum rotary evaporator, then redissolve in 150 mL of DCM, wash with saturated sodium bicarbonate (2 x 100 mL), and extract with saturated brine (2 x 100 mL). The collected organic layer is dried over anhydrous magnesium sulfate and filtered. The organic solvent is then removed using a vacuum rotary evaporator to obtain product J, which can be carried on to the next step without further purification.

[0158] (2) Synthesis of linking groups

[0159]

[0160] To a three-necked flask equipped with a magnetic separator, 5 mmol of the amino acid backbone alkyl tail of intermediate J, 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 minutes. Using a constant pressure funnel, 6.25 mmol of acryloyl chloride (premixed in 10 mL of dichloromethane) was slowly added dropwise. Once the addition of acryloyl chloride was complete, the ice bath was removed. The reaction was allowed to proceed overnight at room temperature, and progress was monitored by TLC. Upon completion, the reaction was diluted with DCM (30 mL) and washed with 1 M HCl (50 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, filtered, and the organic solvent removed using a reduced-pressure rotary evaporator. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 2:1) to obtain the target product K in an 86% yield.

[0161] The hydrogen spectrum data of the obtained product are as follows:

[0162] 1 H NMR (400MHz, CDCl3): 7.88(d,J=7.2Hz,1H),7.49(t,J=5.6Hz,1H),6.76(t,J=5.2Hz,1H),6.33-6.24(m,1H),5.67(d,J=9.6Hz,1H),5.40-5.33(m ,4H),4.82-4.79(m,1H),3.23-3.17(m,4H),2.86-2.81(m,1H),2.57-2. 52(m,1H),2.03-2.0(m,8H),1.37-1.18(m,48H),0.88(dd,J=6.4Hz,6H).

[0163] (3) Reaction between head group and tail group

[0164]

[0165] To a 5 mL reaction vial (with a Teflon-lined cap) equipped with a magnetic rod, 100 mg of 2-(2-methyl-1H-imidazol-1-yl)ethanamine and two chemical equivalents of intermediate K were added and reacted at 90°C for 48 hours. After completion of the reaction, the product was separated by thin-layer chromatography (DCM:methanol = 20:1) to obtain the ionizable lipid 1AspNC18U.

[0166] The hydrogen spectrum data of the obtained product are as follows:

[0167] 1 H NMR(400MHz, CDCl3):4.78-4.65(m,4H),4.08-4.03(m,4H),3.19-3.02(m,8H),2.81-2.77(m,2H), 2.63-2.42(m,10H),1.80-1.48(m,20H),1.39-1.25(m,20H),0.87(dd,J1=6.8Hz,J2=5.2Hz,12H).

[0168] Example 6

[0169] Example 6 provides an amino acid backbone ionizable lipid, the preparation method of which is as follows:

[0170] (1) Synthesize the hydrophobic tail of the amino acid backbone

[0171]

[0172] To a 250 mL reaction flask, 10 mmol of BOC-L-valine, 150 mL of dichloromethane (DCM), a magnetic atom, 11 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 10 mmol of 4-dimethylaminopyridine (DMAP) were added sequentially. The mixture was stirred at room temperature for 15 minutes, followed by the addition of 10 mmol of oleyl alcohol. The reaction was allowed to react for 24 hours, and the reaction progress was monitored by thin-layer chromatography (TLC). Upon completion, the reaction mixture was transferred to a separatory funnel, washed with DCM (2 x 100 mL), washed with 1 M HCl (2 x 100 mL), and extracted with saturated brine (2 x 200 mL). The organic layer was dried over anhydrous magnesium sulfate and filtered, and the organic solvent was removed by rotary evaporation under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 20:1) to obtain the target product L in a yield of 77%.

[0173]

[0174] Removal of amino protecting groups: Dissolve intermediate product L in 30 mL of DCM, add 15 mL of trifluoroacetic acid (TFA), and stir at room temperature for 4 hours. Remove the organic solvent using a vacuum rotary evaporator, then redissolve in 150 mL of DCM, wash with saturated sodium bicarbonate (2 x 100 mL), and extract with saturated brine (2 x 100 mL). The collected organic layer is dried over anhydrous magnesium sulfate and filtered. The organic solvent is then removed using a vacuum rotary evaporator to obtain product M, which can be carried on to the next step without further purification.

[0175] (2) Synthesis of linking groups

[0176]

[0177] To a three-necked flask equipped with a magnetic rod, 5 mmol of the amino acid backbone alkyl tail of intermediate M, 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 minutes. Using a constant pressure funnel, 6.25 mmol of acryloyl chloride (premixed in 10 mL of dichloromethane) was slowly added dropwise. Once the addition of acryloyl chloride was complete, the ice bath was removed. The reaction was allowed to proceed overnight at room temperature, and progress was monitored by TLC. Upon completion, the reaction was diluted with DCM (30 mL) and washed with 1 M HCl (50 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, filtered, and the organic solvent removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 10:1) to obtain the desired product N in a 98% yield.

[0178] The hydrogen spectrum data of the obtained product are as follows:

[0179] 1 H NMR(400MHz, CDCl3):6.41-6.30(m,1H),6.22-6.15(m,1H),5.68-5.66(m,1H),5.41-5.30(m,2H),4.69-4. 66(m,1H),4.17-4.10(m,1H),2.23-1.95(m,5H),1.68-1.61(m,2H),1.33-1.26(m,22H),0.97-0.85(m,9H).

[0180] (3) Reaction between head group and tail group

[0181]

[0182] To a 5 mL reaction vial (with a Teflon-lined cap) equipped with a magnetic rod, add 100 mg of 3-aminopropanol and two chemical equivalents of intermediate N. The mixture is reacted at 90°C for 48 hours. After completion, the product is separated by thin-layer chromatography (DCM:methanol = 20:1) to yield the ionizable lipid 17ValOC18U.

[0183] The hydrogen spectrum data of the obtained product are as follows:

[0184] 1 H NMR(400MHz, CDCl3):8.05-7.83(m,1H),7.39-7.37(m,1H),7.21-7.14(m,1 H),7.09-7.01(m,1H),5.40-5.34(m,4H),4.58-4.50(m,2H),4.16-4.01(m,6 H),3.15-3.10(q,J=8.0Hz,4H),2.96-2.67(m,4H),2.53-2.40(m,4H),2.23 -1.95(m,10H),1.66-1.61(m,4H),1.42-1.26(m,44H),0.97-0.87(m,18H)).

[0185] Example 7

[0186] Example 7 provides an amino acid backbone ionizable lipid, the preparation method of which is as follows:

[0187] (1) Synthesize the hydrophobic tail of the amino acid backbone

[0188]

[0189] To a 250 mL reaction flask, 10 mmol of BOC-L-methionine, 150 mL of dichloromethane (DCM), a magnetic atom, 11 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 10 mmol of 4-dimethylaminopyridine (DMAP) were added sequentially. The mixture was stirred at room temperature for 15 minutes, followed by the addition of 10 mmol of oleyl alcohol. The reaction was allowed to react for 24 hours, and the reaction progress was monitored by thin-layer chromatography (TLC). Upon completion, the reaction mixture was transferred to a separatory funnel, washed with DCM (2 x 100 mL), washed with 1 M HCl (2 x 100 mL), and extracted with saturated brine (2 x 200 mL). The organic layer was dried over anhydrous magnesium sulfate and filtered, and the organic solvent was removed by rotary evaporation under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 20:1) to obtain the target product O in a 64% yield.

[0190]

[0191] Removal of amino protecting groups: Dissolve intermediate O in 30 mL of DCM, add 15 mL of trifluoroacetic acid (TFA), and stir at room temperature for 4 h. Remove the organic solvent using a rotary evaporator under reduced pressure, then redissolve in 150 mL of DCM, wash with saturated sodium bicarbonate (2 x 100 mL), and extract with saturated brine (2 x 100 mL). The collected organic layer is dried over anhydrous magnesium sulfate and filtered. The organic solvent is then removed using a rotary evaporator under reduced pressure to obtain product P, which can be carried on to the next step without further purification.

[0192] (2) Synthesis of linking groups

[0193]

[0194] To a three-necked flask equipped with a magnetic rod, 5 mmol of the amino acid backbone alkyl tail of intermediate M, 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 minutes. Using a constant pressure funnel, 6.25 mmol of acryloyl chloride (premixed in 10 mL of dichloromethane) was slowly added dropwise. Once the addition of acryloyl chloride was complete, the ice bath was removed. The reaction was allowed to proceed overnight at room temperature, and progress was monitored by TLC. Upon completion, the reaction was diluted with DCM (30 mL) and washed with 1 M HCl (50 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, filtered, and the organic solvent removed using a reduced-pressure rotary evaporator. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 10:1) to obtain the target product Q in an 88% yield.

[0195] The hydrogen spectrum data of the obtained product are as follows:

[0196] 1 H NMR(400MHz, CDCl3):6.34-6.30(m,1H),6.20-6.13(m,1H),5.70-5.67(m,1H),5.40-5.30(m,2H),4.82-4.77(m,2H) ,4.16-4.11(m,1H),2.59-2.47(m,2H),2.26-1.94(m,9H),1.68-1.61(m,2H),1.34-1.23(m,22H),0.89-0.85(m,3H).

[0197] (3) Reaction between head group and tail group

[0198]

[0199] To a 5 mL reaction vial (with a Teflon-lined cap) equipped with a magnetic rod, add 100 mg of 3-aminopropanol and two chemical equivalents of intermediate Q. The mixture is reacted at 90°C for 48 hours. After completion, the product is separated by thin-layer chromatography (DCM:methanol = 20:1) to yield the ionizable lipid 17ValOC18U.

[0200] The hydrogen spectrum data of the obtained product are as follows:

[0201] 1 H NMR(400MHz, CDCl3):7.77-7.69(m,2H),5.41-5.35(m,4H),4.69-4.62(m,2H),4.15-4.11(m,4H),3.80-3.76(m,2H),3.18-3.1 0(m,4H),2.84-2.71(m,4H),2.62-2.57(m,4H),2.14-1.97(m,20H),1.68-1.64(m,4H),1.45-1.27(m,44H),0.91-0.88(m,6H).

[0202] Example 8

[0203] Example 8 provides an amino acid backbone ionizable lipid, the preparation method of which is as follows:

[0204] (1) Synthesize the hydrophobic tail of the amino acid backbone

[0205]

[0206] To a 250 mL reaction flask, 10 mmol of BOC-L-phenylalanine, 150 mL of dichloromethane (DCM), a magnetic atom, 11 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 10 mmol of 4-dimethylaminopyridine (DMAP) were added sequentially. The mixture was stirred at room temperature for 15 minutes, followed by the addition of 10 mmol of oleyl alcohol. The reaction was allowed to react for 24 hours, and the reaction progress was monitored by thin-layer chromatography (TLC). Upon completion, the reaction mixture was transferred to a separatory funnel, washed with DCM (2 x 100 mL), washed with 1 M HCl (2 x 100 mL), and extracted with saturated brine (2 x 200 mL). The organic layer was dried over anhydrous magnesium sulfate and filtered, and the organic solvent was removed by rotary evaporation under reduced pressure. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 20:1) to obtain the target product R in a 97% yield.

[0207]

[0208] Removal of amino protecting groups: Dissolve intermediate product R in 30 mL of DCM, add 15 mL of trifluoroacetic acid (TFA), and stir at room temperature for 4 hours. Remove the organic solvent using a vacuum rotary evaporator, then redissolve in 150 mL of DCM, wash with saturated sodium bicarbonate (2 x 100 mL), and extract with saturated brine (2 x 100 mL). The collected organic layer is dried over anhydrous magnesium sulfate and filtered. The organic solvent is then removed using a vacuum rotary evaporator to obtain product S, which can be carried on to the next step without further purification.

[0209] (2) Synthesis of linking groups

[0210]

[0211] To a three-necked flask equipped with a magnetic rod, 5 mmol of the intermediate S amino acid backbone alkyl tail, 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 minutes. Using a constant pressure funnel, 6.25 mmol of acryloyl chloride (premixed in 10 mL of dichloromethane) was slowly added dropwise. Once the addition of acryloyl chloride was complete, the ice bath was removed. The reaction was allowed to proceed overnight at room temperature, and progress was monitored by TLC. Upon completion, the mixture was diluted with DCM (30 mL) and washed with 1 M HCl (50 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, filtered, and the organic solvent removed using a reduced-pressure rotary evaporator. The product was separated by thin-layer chromatography (petroleum ether:ethyl acetate = 10:1) to obtain the desired product, T, in a 69% yield.

[0212] The hydrogen spectrum data of the obtained product are as follows:

[0213] 1 H NMR(400MHz, CDCl3):7.31-7.11(m,5H),6.33-6.27(m,1H),6.16-6.09(m,1H),5.68-5.65(m,1H),5.41-5.35(m,2H),4.99-4. 95(m,1H),4.15-4.08(m,2H),3.19-3.16(m,2H),2.06-2.03(m,4H),1.63-1.59(m,2H),1.37-1.28(m,22H),0.91-0.87(m,3H).

[0214] (3) Reaction between head group and tail group

[0215]

[0216] To a 5 mL reaction vial (with a Teflon-lined cap) equipped with a magnetic rod, add 100 mg of 3-aminopropanol and two chemical equivalents of intermediate T. The reaction is allowed to proceed at 90°C for 48 hours. After completion, the product is separated by thin-layer chromatography (DCM:methanol = 20:1) to yield the ionizable lipid 34PheOC18U.

[0217] The hydrogen spectrum data of the obtained product are as follows:

[0218] 1 H NMR(400MHz, CDCl3):7.31-7.16(m,10H),5.41-5.35(m,4H),4.84-4.80(m,2H),4.10-4.06(m,4H),3.66-3.63(m,4H),3.66-3. 05(m,6H),2.95-2.94(m,2H),2.65-2.52(m,4H),2.08-1.96(m,8H),1.62-1.53(m,6H),1.45-1.27(m,48H),0.91-0.88(m,6H).

[0219] The amino acid backbone ionizable lipids synthesized by the present invention are (the synthesis methods of amino acid backbone ionizable lipids with other structures refer to the above general preparation method and Examples 1-8): 1GluOC8, 1AspOC8, 1GluOC10, 1AspOC10, 1GluOC8C10, 1AspOC8C10, 1GluOC10, 1AspOC10, 1GluOC12, 1AspOC12, 1GluOC16, 1AspOC16, 1GluOC18, 1AspOC18, 1GluOC18U, 1AspOC18U, 1GluOC18U2, 1AspOC18U2, 1GluNC8, 1AspNC8, 1G luNC18U, 1AspNC18U, 3GluOC18U, 3AspOC18U, 4GluOC18U, 4AspOC18U, 1LeuOC18U, 1LeuNC18U, 6AspOC18U, 6GluOC18U, 8GluOC18, 8AspOC18, 8GluOC18 U, 9GluOC8, 8AspOC18U, 9AspOC8, 9GluOC10, 9AspOC10, 9GluOC18U, 9AspOC18U, 9GluOC18U2, 9AspOC18U2, 11GluOC8, 11AspOC8, 11GluOC10, 11AspOC10 , 12GluOC18U, 12AspOC18U, 13AspOC8, 13AspOC10, 13GluOC8C10, 13AspOC8C10, 16AspOC10, 16GluOC18U2, 16GluOC18U, 16AspOC18U, 17AspOC10, 17Gl uOC18U2, 17GluOC18U, 17AspOC18U, 18GluOC18U, 18AspOC18U, 19GluOC8C10, 19AspOC8C10, 19GluOC18U, 19AspOC18U, 20GluOC18U, 20AspOC18U, 21Gl uOC18U, 21GluNC18U, 22GluOC18U, 22AspOC18U, 24GluOC18U, 24AspOC18U, 25GluOC18U, 25AspOC18U, 26GluOC18U, 26AspOC18U, 27GluOC18U, 27AspOC 18U, 28GluOC18U, 28AspOC18U, 29GluOC18U, 29AspOC18U, 30GluOC18U, 30AspOC18U, 31GluOC18U, 31AspOC18U, 32GluOC18U, 32GluNC18U, 33GluOC18U,33AspOC18U, 34GluOC18U, 34AspOC18U, 17PheOC18, 17MetOC18U, 34ValOC18U, 17GayOC18U, 29PheO C18U, 29MetOC18U, 29ValOC18U, 29GayOC18U, 34PheOC18U, 34MetOC18U, 34ValOC18U, 34GayOC18U. ,

[0220] Lipid experimental characterization

[0221] 1. Characterization of enzyme delivery efficiency of fluorescent proteins and luciferin

[0222] The efficiency of LNPs containing ionizable lipids in delivering plasmids encoding green fluorescent protein (GFP) and firefly luciferase (Luc) (DNA-GFP-Luc) was verified in 293T cell lines. The ionizable lipids 1GluOC18, 8GluOC18, 3GluOC18U, 4GluOC18U, 12GluOC18U, 13GluOC18U, 17GluO18U, 1AspOC18U, 3AspOC18U, 1AspOC8, 1AspOC10, 8GluOC18U, 9AspOC8, 9AspOC10, 9GluOC18U, 13AspOC8, 13AspOC10 were used. , 13AspOC8C10, 11AspOC8, 11AspOC10, 16AspOC10, 16AspOC18U, 17AspOC10, 17AspOC18U, 19AspOC18U , 19AspOC8C10, 22AspOC18U, 21G; uNC18U, 16GluOC18U, 20GluOC18U, 21GluOC18U, 1GluOC18U2, 9GluOC 18U2, 16GluOC18U2, 17GluOC18U2, 9GluOC18U, 16GluOC18U, 19GluOC18U, 25GluOC18U, 26GluOC18U, 2 7GluOC18U, 29GluOC18U, 30GluOC18U, 31GluOC18U, 32GluOC18U, 33GluOC18U, 34GluOC18U, 17GayOC18 U, 29GayOC18U, 34GayOC18U, 17PheOC18U, 29PheOC18U, 34PheOC18U, 17ValOC18U, 29ValOC18U, 34ValOC18U, 17MetOC18U, 29MetOC18U, 34MetOC18U and commercial materials ALC-0315 and SM-102 were used as delivery materials to express DNA-GFP-Luc in cells.

[0223] Specific steps:

[0224] (1) Cell culture: The day before the experiment, cultured 293T cells were seeded in a 96-well cell culture plate. After the cell density grew to about 70-80%, the cell transfection experiment was performed.

[0225] (2) Preparation of lipid nanoparticles LNP-DNA-GFP-Luc for cell transfection

[0226] The aforementioned ionizable lipids were dissolved in anhydrous ethanol with DSPC, Cholesterol, and DSPE-PEG at concentrations of 10, 3, 6, and 1 mg / mL, respectively. The mixture was then uniformly mixed at a molar ratio of ionizable lipid: Cholesterol: DSPC: DSPE-PEG = 40:48:10:2. Simultaneously, an appropriate amount of DNA-GFP-Luc was dissolved in sodium citrate buffer (the volume of sodium citrate buffer was three times the total volume of the lipid mixture, pH = 4.0-4.5). The DNA buffer and lipid mixture solutions were then rapidly mixed and incubated at room temperature for 15 minutes to assemble stable LNPs (150 ng of DNA-GFP-Luc was transfected per well). The LNPs were diluted with two volumes of sterile PBS and added to 96-well cell culture plates for transfection. The nitrogen-phosphorus ratio of the ionizable lipid to the nucleic acid was 6:1, i.e., the molar ratio between the protonated amino group and the phosphate group on the nucleic acid (the same applies below).

[0227] Positive control group: commercial lipids ALC-0315 and SM-102 were used to assemble into LNPs according to the published preparation method. The specific operation is as follows: ALC-0315 or SM-102, DSPC, Cholesterol, ALC-0159 or DMG-PEG 2000 Dissolved in anhydrous ethanol at concentrations of 5 mg / mL, 1.5 mg / mL, 3 mg / mL, and 1 mg / mL, and prepared according to the following formulas: ALC-0315: Cholesterol: DSPC: ALC-0159 = 46.3: 42.7: 9.4: 1.6 or SM-102: Cholesterol: DSPC: DMG-PEG 2000 =50:38.5:10:1.5 molar ratio was uniformly mixed. Meanwhile, an appropriate amount of DNA-GFP-Luc was dissolved in sodium citrate buffer (the volume of sodium citrate buffer was three times the total volume of the lipid mixture, pH = 4.0). The DNA buffer and lipid mixture solution were then rapidly mixed and incubated at room temperature for 15 minutes to assemble stable LNPs (150 ng of DNA-GFP-Luc LNPs were transfected per well). The LNPs were diluted with two volumes of sterile PBS and added to 96-well cell culture plates for transfection. The nitrogen-phosphorus ratio of ALC-0315 and SM-102 to nucleic acid was 6:1.

[0228] Negative control group: 293T cells were cultured normally and unencapsulated DNA-GFP-Luc was added.

[0229] (3) Analysis of cell transfection efficiency

[0230] 36 hours after cell transfection, the expression of green fluorescent protein was detected using a fluorescence microscope; the culture medium of the 96-well cell culture plate was aspirated, and the cells were lysed on ice for 30 minutes after adding cell lysis buffer. After centrifugation, the supernatant was transferred to a white 96-well detection plate, and the firefly luciferase substrate was added. The firefly luciferase content (chemiluminescence) was detected using a microplate reader. The relative luciferase activity results are shown in Figure 2. Figure 1 As shown. The results show that the ionizable lipids synthesized by the present invention can greatly enhance the transfection efficiency of nucleic acids. When the tail of the ionizable lipid is GluO18U or AspOC18U, the RNA expression efficiency is the highest. When the tail is GluO8 or AspOC8, the RNA expression efficiency is weak. The transfection efficiency of lipids represented by 29GluO18U, 34GluO18U, and 17AspOC18U is better than that of commercial lipids ALC-0315 and SM-102, with an efficiency increase of about 2 to 3 times, verifying the rationality and high efficiency of the overall chemical structure of the ionizable lipids designed by the present invention.

[0231] 2. LNP component optimization experiment

[0232] The efficiency of LNP delivery of saRNA-GFP-Luc containing ionizable lipids was verified in the 293T cell line. The composition of LNP was optimized using the ionizable lipid 17LGluO18U.

[0233] Specific steps:

[0234] (1) Referring to the above experimental method, the ionizable lipid used in the experimental group was: 17GluO18U. The ionizable lipids, DOPE, DSPC or DOPC, Cholesterol or β-sitosterol, DSPE-PEG or DMG-PEG, were dissolved in anhydrous ethanol at a concentration of 10 mg / mL, 3 mg / mL, 6 mg / mL, and 1 mg / mL, respectively. The ratio of ionizable lipid 17LGluO18U: (Cholesterol or β-sitosterol): (DOPE, DSPC or DOPC): (DSPE-PEG or DMG-PEG) = 40:48:10:2.

[0235] (2) Analysis of cell transfection efficiency

[0236] 36 hours after transfection, the expression of green fluorescent protein was detected using a fluorescence microscope; the culture medium of the 96-well cell culture plate was aspirated, and the cells were lysed on ice for 30 minutes after adding cell lysis buffer. After centrifugation, the supernatant was transferred to a white 96-well detection plate, and the firefly luciferase substrate was added. The firefly luciferase content (chemiluminescence) was detected using a microplate reader. The relative luciferase activity results are shown in Figure 2. Figure 2As shown in (a) in the figure. The results show that the chemical structure of the neutral co-phospholipid significantly affects RNA delivery efficiency. When DSPC is the neutral co-phospholipid, the delivery efficiency of the three ionizable lipids is significantly better than that of DOPE or DOPC, so DSPC is the preferred neutral co-phospholipid. Cholesterol is preferred over β-sitosterol. The chain length of PEG significantly affects RNA delivery efficiency, and the shorter DMG-PEG is preferred.

[0237] 3. LNP component ratio optimization experiment

[0238] The efficiency of LNP delivery of saRNA-GFP-Luc containing ionizable lipids was verified in the 293T cell line. The ratio of each component in LNP was optimized using the ionizable lipid 17LGluO18U.

[0239] Specific steps:

[0240] (1) Referring to the above experimental method, the ionizable lipid used in the experimental group was: 17LGluO18U. The ionizable lipids, DSPC, Cholesterol, and DSPE-PEG were dissolved in anhydrous ethanol at 10 mg / mL, 3 mg / mL, 6 mg / mL, and 1 mg / mL, respectively, and mixed using six different molar ratios: ratio A was ionizable lipid: Cholesterol: DSPC: DMG-PEG = 40:48:10:2; ratio B was ionizable lipid: Cholesterol: DSPC: DMG-PEG = 30:28.5:10:0.75; ratio C was ionizable lipid : Cholesterol: DSPC: DMG-PEG = 50:38.5:10:1.5; ratio D is ionizable lipid: Cholesterol: DSPC: DMG-PEG = 35:46:16:2.5; ratio E is ionizable lipid: Cholesterol: DSPC: DMG-PEG = 46.3:42.7:9.4:1.6; ratio F is ionizable lipid: Cholesterol: DSPC: DMG-PEG = 46.3:42.7:9.4:1.5.

[0241] (2) Analysis of cell transfection efficiency

[0242] 36 hours after transfection, the expression of green fluorescent protein was detected using a fluorescence microscope; the culture medium of the 96-well cell culture plate was aspirated, and the cells were lysed on ice for 30 minutes after adding cell lysis buffer. After centrifugation, the supernatant was transferred to a white 96-well detection plate, and the firefly luciferase substrate was added. The firefly luciferase content (chemiluminescence) was detected using a microplate reader. The relative luciferase activity results are shown in Figure 2. Figure 2The results show that the molar ratio of the components of LNP also affects the RNA delivery efficiency to a certain extent, and the optimal ratio is ionizable lipid: Cholesterol: DSPC: DMG-PEG = 40:48:10:1.5.

[0243] 4. LNP nitrogen-phosphorus ratio optimization experiment

[0244] The efficiency of LNPs containing ionizable lipids in delivering saRNA-GFP-Luc was verified in the 293T cell line. The nitrogen-phosphorus ratio of LNPs was optimized using the ionizable lipid 17GluO18U.

[0245] Specific steps:

[0246] (1) Referring to the above experimental method, the ionizable lipid used in the experimental group was: 17GluO18U. The ionizable lipids, DSPC, Cholesterol, and DMG-PEG were dissolved in anhydrous ethanol at concentrations of 10 mg / mL, 3 mg / mL, 6 mg / mL, and 1 mg / mL, respectively. The ratio of ionizable lipid compound 17GluO18U: Cholesterol: DSPC: DMG-PEG was 40:48:10:1.5. The nitrogen-phosphorus ratios of LNPs were 4:1, 6:1, 8:1, 12:1, and 16:1, respectively.

[0247] (2) Analysis of cell transfection efficiency

[0248] 36 hours after transfection, the expression of green fluorescent protein was detected using a fluorescence microscope; the culture medium of the 96-well cell culture plate was aspirated, and the cells were lysed on ice for 30 minutes after adding cell lysis buffer. After centrifugation, the supernatant was transferred to a white 96-well detection plate, and the firefly luciferase substrate was added. The firefly luciferase content (chemiluminescence) was detected using a microplate reader. The relative luciferase activity results are shown in Figure 2. Figure 3 The results show that the transfection efficiency of saRNA-GFP-Luc is optimal when the nitrogen-phosphorus ratio is 6:1.

[0249] 5. LNP buffer formulation optimization experiment

[0250] The efficiency of LNP delivery of saRNA-GFP-Luc containing ionizable lipids was verified in 293T cell lines. The buffer formulation of LNP was optimized using the ionizable lipid 17LGluO18U.

[0251] Specific steps:

[0252] (1) Referring to the above experimental method, the ionizable lipid used in the experimental group was: 17LGluO18U. The ionizable lipids, DSPC, Cholesterol, and DMG-PEG were dissolved in anhydrous ethanol at concentrations of 10 mg / mL, 3 mg / mL, 6 mg / mL, and 1 mg / mL, respectively. The ratio of ionizable lipid compound 17LGluO18U, 34LGluO18U, or 16AspOC18U: Cholesterol: DSPC: DMG-PEG was 40:48:10:1.5. The solution for premixing RNA was sodium acetate or sodium citrate buffer, and the nitrogen-phosphorus ratio for preparing LNPs was 6:1.

[0253] (2) Analysis of cell transfection efficiency

[0254] 36 hours after transfection, the expression of green fluorescent protein was detected using a fluorescence microscope; the culture medium of the 96-well cell culture plate was aspirated, and the cells were lysed on ice for 30 minutes after adding cell lysis buffer. After centrifugation, the supernatant was transferred to a white 96-well detection plate, and the firefly luciferase substrate was added. The firefly luciferase content (chemiluminescence) was detected using a microplate reader. The relative luciferase activity results are shown in Figure 2. Figure 3 The results show that the RNA delivery efficiency is better when the buffer used to prepare the nanoparticles is sodium citrate buffer than when using sodium acetate buffer. Therefore, sodium citrate buffer is preferred.

[0255] 6. Characterization of modRNA-GFP delivery efficiency

[0256] The efficiency of modRNA-GFP delivery using LNPs containing ionizable lipids was verified in the Jurkat T cell line. The transfection efficiency of LNPs was verified using the following ionizable lipids: 9LGluOC18U, 17LGluO18U, 25DGluO18U, and 29LGluO18U.

[0257] Specific steps:

[0258] (1) Referring to the above experimental method, the Juekat T cell line was used. The ionizable lipids used in the experimental group were: 9LGluOC18U, 17LGluO18U, 25DGluO18U, 29LGluO18U, and the ionizable lipids used in the control group were: SM-10 and ALC-0315. The other components of LNP were DSPC, Cholesterol, and DMG-PEG, which were dissolved in anhydrous ethanol at concentrations of 10 mg / mL, 3 mg / mL, 6 mg / mL, and 1 mg / mL, respectively. The ratio of ionizable lipid compound: Cholesterol: DSPC: DMG-PEG was 40:48:10:1.5. The solution for premixing modRNA was sodium citrate buffer, and the nitrogen-phosphorus ratio for preparing LNP was 6:1.

[0259] (2) Analysis of cell transfection efficiency

[0260] 24 hours after transfection, the expression of green fluorescent protein was detected using a fluorescence microscope. Figure 4 As shown, it is shown that the ionizable lipids of the present invention can efficiently deliver nucleic acids to immune cells and are superior to commercial lipids.

[0261] 7. In vivo imaging system characterization

[0262] LNPs containing ionizable lipids 9GluOC18U, 17GluOC18U, 25GluOC18U, 29GluOC18U, 33GluOC18U, 34GluOC18U and commercial lipids SM-102 and ALC-0315 were used to deliver chemically synthesized modified messenger ribonucleotides (modRNA-Luc) encoding firefly luciferase in Balb / c mice. The expression of the reporter gene luciferase was detected using an in vivo imaging system (IVIS) 6 hours after intravenous injection.

[0263] Specific steps:

[0264] (1) Referring to the above experimental method, the ionizable lipids used in the experimental group were: 9GluOC18U, 17GluOC18U, 25GluOC18U, 29GluOC18U, 33GluOC18U, and 34GluOC18U. The ionizable lipids, DSPC, Cholesterol, and DMG-PEG were dissolved in anhydrous ethanol at concentrations of 10 mg / mL, 6 mg / mL, 12 mg / mL, and 5 mg / mL, respectively. An appropriate amount of modRNA-Luc was dissolved in sodium acetate buffer (the volume of sodium acetate buffer was twice the total volume of the lipid mixture, pH = 4.0). The buffer containing modRNA-Luc was added to the ethanol solution of the lipid mixture and quickly mixed to assemble LNPs. The mixed solution was incubated at room temperature for 15 minutes, dialyzed in PBS for 1 hour using a dialysis bag (MWCO = 14000 MW), and then injected into the tail vein (each injection contained 5 μg of modRNA-Luc LNPs). The ratio of ionizable lipids 9GluOC18U, 17GluOC18U, 25GluOC18U, 29GluOC18U, 33GluOC18U, or 34GluOC18U: Cholesterol: DSPC: DMG-PEG was 40:48.5:10:1.5. The RNA premix solution was sodium citrate buffer, and the nitrogen-to-phosphorus ratio for LNP preparation was 6:1.

[0265] Positive control group: commercial lipids ALC-0315 and SM-102 were used to assemble the corresponding positive control LNPs according to the published preparation method. The specific operation is as follows: ALC-0315 or SM-102, DSPC, Cholesterol, ALC-0159 or DMG-PEG 2000 The mRNA was dissolved in anhydrous ethanol at concentrations of 5 mg / mL, 1.5 mg / mL, 3 mg / mL, and 1 mg / mL, and uniformly mixed according to a molar ratio of ALC-0315: Cholesterol: DSPC: ALC-0159 = 46.3: 42.7: 9.4: 1.6 or SM-102: Cholesterol: DSPC: DMG-PEG2000 = 50: 38.5: 10: 1.5. At the same time, an appropriate amount of modRNA-Luc was drawn into sodium citrate buffer (the volume of sodium citrate buffer was three times the total volume of the lipid mixture, pH = 4.0). The mRNA-containing buffer was then quickly mixed with the lipid mixture solution, incubated at room temperature for 15 min to assemble into LNPs, and then dialyzed in PBS for 1 h using a dialysis bag (MWCO = 14000 MW) and injected into the tail vein (5 μg of modRNA-Luc LNPs were injected per injection). The nitrogen-phosphorus ratio of ALC-0315, SM-102 and mRNA is 6:1.

[0266] (2) Analysis of in vivo imaging results

[0267] IVIS results showed that 6 h after tail vein injection (e.g. Figure 5 、 Figure 6 、 Figure 7 、 Figure 8 ), the expression intensity of the ionizable lipids 29GluOC18U and 34GluOC18U of the present invention is better than that of the commercial lipids SM-102 and ALC-0315, and has excellent organ targeting and can accurately target the spleen.

[0268] According to literature, most ionizable lipid-loaded nucleic acids are expressed in multiple organs throughout the body after intravenous injection, with a tropism for the liver. However, the ionizable lipids of the present invention can precisely target the spleen. As the spleen is the largest immune organ in the body, spleen-targeted LNPs could lead to more significant immunotherapeutic effects in nucleic acid therapy applications.

[0269] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

[0270] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. An amino acid backbone ionizable lipid, characterized in that It is a compound represented by formula (A) or formula (B), a pharmaceutically acceptable salt thereof, a stereoisomer thereof or a tautomer thereof; Wherein, the R1 is selected from C2-C10 alkyl, C2-C10 heteroalkyl, aralkyl, or absent; The R2 is selected from C3-C24 straight chain alkyl, C4-C24 straight chain alkenyl, n1=2~3, n2=3~24, n3=3~24; Said X is O or NH; The R3 is selected from C1-C10 hydroxyalkyl; R6 and R7 are each independently selected from C1-C4 alkyl, C1-C4 hydroxyalkyl; The R4 is selected from C1-C4 hydroxyalkyl; the R5 is C1-C4 alkyl, C1-C4 hydroxyalkyl, n4=2~3 The "*" is a connection site.

2. The amino acid backbone ionizable lipid according to claim 1, wherein The amino acid backbone ionizable lipid may have a structure represented by any one of formulas (1) to (104):

3. The method for preparing the amino acid backbone ionizable lipid according to claim 1 or 2, characterized in that: The method comprises the following steps: a tail compound containing an amino acid structure or a stereoisomer or tautomer thereof is subjected to a Michael addition reaction with an organic amine compound to prepare the amino acid skeleton ionizable lipid; The structural formula of the tail compound containing an amino acid structure is shown in formula (a): Said R1, R2 and X are as described in claim 1.

4. The method for preparing an ionizable lipid having an amino acid backbone according to claim 3, wherein: The organic amine compound is selected from one of the following compounds:

5. The method for preparing an ionizable lipid with an amino acid backbone according to claim 3, wherein The preparation method of the tail compound containing an amino acid skeleton comprises the following steps: reacting acryloyl chloride with compound a1 The tail compound containing an amino acid skeleton is prepared through an esterification reaction.

6. A lipid composition, characterized in that The invention comprises the amino acid skeleton ionizable lipid, sterol and PEGylated lipid according to claim 1 or 2.

7. Use of the amino acid backbone ionizable lipid according to claim 1 or 2, or the lipid composition according to claim 6, in preparing a bioactive substance delivery system, characterized in that: The delivery system is a microparticle, nanoparticle, liposome, lipid nanoparticle or microbubble.

8. A pharmaceutical composition, characterized in that It comprises the lipid composition according to claim 6 and an active substance; the active substance comprises at least one of a nucleic acid molecule, a small molecule compound, a polypeptide, and a protein.

9. The pharmaceutical composition according to claim 8, characterized in that When the pharmaceutically active ingredient includes a nucleic acid molecule, the nitrogen-phosphorus ratio of the amino acid backbone ionizable lipid to the nucleic acid molecule is (1-100):

1.

10. Use of the amino acid backbone ionizable lipid according to claim 1 or 2, or the pharmaceutical composition according to claim 8 or 9 in the preparation of nucleic acid drugs, gene vaccines, polypeptide or protein drugs, or small molecule drugs.

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