A branched cationic lipid compound, and a preparation method and application thereof

Abstract

The application discloses a branched cationic lipid compound and a preparation method and application thereof, and has the following structural characteristics: one, an ionizable tertiary amine head ensures high nucleic acid delivery capacity; two, an ether bond branched structure promotes the formation of a conical structure when mRNA is delivered and improves endosome escape capacity; three, a functional connecting bond connects a saturated or unsaturated tail chain, improves mRNA encapsulation efficiency and delivery safety. In summary, the polyamino amidine cationic lipid molecule has the characteristics of high delivery efficiency, good cell biological compatibility and excellent storage stability, provides more choices for nucleic acid drug delivery, and has important significance for the development and application of nucleic acid drugs.

Description

Technical Field

[0001] This invention belongs to the field of lipid materials, specifically relating to a branched cationic lipid compound, its preparation method, and its application. Background Technology

[0002] Nucleic acid drugs are a class of novel biopharmaceuticals that use DNA, RNA, or their chemically modified analogs as active ingredients to achieve therapeutic effects by regulating gene expression. Currently, commonly used nucleic acid drugs mainly include small interfering RNA (siRNA), messenger RNA (mRNA), antisense oligonucleotides (ASO), and microRNA (miRNA). Their main advantages are short design cycles, rapid response to novel pathogens, high specificity, and reduced off-target toxicity. However, due to the strong negative charge of nucleic acid drugs, they have difficulty crossing the lipid bilayer and are easily degraded by serum nucleases in vivo. Therefore, developing a nucleic acid drug carrier with high delivery efficiency and low toxicity is crucial.

[0003] Lipid nanoparticles (LNPs) are currently the most mature and clinically validated non-viral nucleic acid delivery carriers. A typical LNP is formed by the self-assembly of four core lipid components: ionizable cationic lipids, phospholipids, cholesterol, and PEGylated lipids. Studies have shown that the nucleic acid drug delivery efficiency of LNPs is mainly affected by the structure of the ionizable lipids. Ionizable lipids exert their effects by protonating in acidic endosomes, interacting with anionic endosome membranes, inducing non-bilayer phase transitions (such as hexagonal phases), disrupting the endosome membrane, and releasing nucleic acids into the cytoplasm. Currently, clinical LNPs mostly rely on a few lipids (such as MC3 and SM-102), lacking a diverse library to support rational design. Summary of the Invention

[0004] To overcome the problems existing in the prior art, one objective of the present invention is to provide a branched-chain cationic lipid compound. A second objective of the present invention is to provide a method for preparing the aforementioned branched-chain cationic lipid compound. A third objective of the present invention is to provide a lipid composition. A fourth objective of the present invention is to provide applications of the aforementioned branched-chain cationic lipid compound and lipid composition.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a branched cationic lipid compound, the structural formula of which is shown in Formula I: Formula I; Wherein, m and n are independently selected from integers 0-3 and 0-3, respectively; R1 and R2 are independently H, substituted or unsubstituted (C1-C2) compounds, respectively. 18 )alkyl, substituted or unsubstituted (C2-C 18 Alkenyl, substituted or unsubstituted phenyl or 4-7 membered heterocycles; Alternatively, R1 and R2, together with the nitrogen atoms they are attached to, form a heterocycle, which is a substituted or unsubstituted 4-7 membered nitrogen heterocycle; the heterocycle also contains 0-3 additional heteroatoms selected from N, O, and S; the substitution refers to the presence of a substituent at one or more substituted positions of the heterocycle, the substituent being selected from (C1-C8)alkyl, halo(C1-C8)alkyl, -O-(C1-C8)alkyl, -O-haloC1-C8alkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C6alkyl), and -N(C1-C6alkyl)2 substituents; R3, R4, R5, and R6 are independently H, substituted, or unsubstituted (C1-C2) compounds. 30 )alkyl, substituted or unsubstituted (C2-C 30 alkenyl; Wherein, the substitution of R1, R2, R3, R4, R5 or R6 is independently selected from at least one of deuterium, (C1-C8)alkyl, halo(C1-C8)alkyl, -O-(C1-C8)alkyl, -O-haloC1-C8alkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C6alkyl)2 and -N(C1-C6alkyl)2, wherein the number of substitutions is from monosubstituted to the maximum number of substitutions; The A is (C1-C6) alkenyl or single bond.

[0006] Preferably, the halogen refers to fluorine (F), chlorine (Cl), or bromine (Br).

[0007] Preferably, R1 and R2 are independently H, substituted or unsubstituted (C1-C2) compounds. 10 Alkyl group; or, R1 and R2 together with the nitrogen atom to which they are attached form a heterocycle, which is a 4-7 membered nitrogen heterocycle.

[0008] More preferably, R1 and R2 are independently H and (C1-C5) alkyl, respectively; or, R1 and R2 together with the nitrogen atoms they are attached to form a heterocycle, wherein the heterocycle is a 4-7 member nitrogen heterocycle.

[0009] More preferably, when R1 and R2 do not form heterocyclic rings, the definitions of R1 and R2 are the same.

[0010] More preferably, the structure of the nitrogen heterocycle is as follows: .

[0011] Preferably, R3 is H, substituted or unsubstituted (C1-C)10 )alkyl; The R4 is H, substituted or unsubstituted (C1-C2). 10 )alkyl; Wherein, the substitution of R3 or R4 is independently selected from at least one of deuterium, (C1-C5)alkyl, halo(C1-C5)alkyl, -O-(C1-C5)alkyl, -O-haloC1-C3alkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C5alkyl)2 and -N(C1-C5alkyl)2, wherein the number of substitutions is from monosubstituted to the maximum number of substitutions.

[0012] More preferably, R3 is H, or a substituted or unsubstituted (C1-C8) alkyl group; Wherein, the substitution of R3 is selected from at least one of deuterium, (C1-C3)alkyl, halo(C1-C3)alkyl, -O-(C1-C3)alkyl, -O-haloC1-C3alkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C3alkyl)2 and -N(C1-C3alkyl)2, wherein the number of substitutions is from monosubstituted to the maximum number of substitutions.

[0013] More preferably, R4 is H, or a substituted or unsubstituted (C1-C8) alkyl group; Wherein, the substitution of R3 is selected from at least one of deuterium, (C1-C3)alkyl, halo(C1-C3)alkyl, -O-(C1-C3)alkyl, -O-haloC1-C3alkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C3alkyl)2 and -N(C1-C3alkyl)2, wherein the number of substitutions is from monosubstituted to the maximum number of substitutions.

[0014] Preferably, R5 is H, substituted or unsubstituted (C1-C) 24 )alkyl; The R6 is H, substituted or unsubstituted (C1-C2). 24 )alkyl; Wherein, the substitution of R3 or R4 is independently selected from at least one of deuterium, (C1-C3)alkyl, halo(C1-C3)alkyl, -O-(C1-C3)alkyl, -O-haloC1-C3alkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C3alkyl)2 and -N(C1-C3alkyl)2, wherein the number of substitutions is from monosubstituted to the maximum number of substitutions.

[0015] More preferably, R5 is H, substituted or unsubstituted (C1-C) 20 )alkyl; The R6 is H, substituted or unsubstituted (C1-C2). 20 )alkyl; Wherein, the substitution of R5 or R6 is independently selected from at least one of deuterium, (C1-C3)alkyl, halo(C1-C3)alkyl, -O-(C1-C3)alkyl, -O-haloC1-C3alkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C3alkyl)2 and -N(C1-C3alkyl)2, wherein the number of substitutions is from monosubstituted to the maximum number of substitutions.

[0016] Preferably, L1 and L2 are each independently... , , , ; The linking site of the representative group, This indicates the connection site with R5 and R6.

[0017] Preferably, R3 and R4 have the same definition.

[0018] Preferably, R5 and R6 have the same definition.

[0019] More preferably, the branched cationic lipid compound has the structural formula shown in Formula II: Formula II.

[0020] Preferably, A is a (C2-C4) alkylene or a single bond.

[0021] A second aspect of the present invention provides a method for preparing the branched cationic lipid compound described in the first aspect, comprising the following steps: An amino compound and an epoxy compound are reacted 1 to obtain an intermediate product; the intermediate product is reacted 2 with a tail-chain compound to obtain the branched cationic lipid compound. The structural formulas of the amino compound, epoxide compound, and intermediate product are shown in formulas A through C, respectively; the structural formula of the tail-chain compound is shown in formula D1 or D2. Formula A Formula B Formula C; Formula D1 Formula D2; Wherein, m, n, R3, R5, and A are as described in the first aspect; R7 is a halogen.

[0022] Preferably, the reaction temperature of reaction 1 is 15-40°C.

[0023] Preferably, the reaction time of reaction 1 is 12-48 hours.

[0024] More preferably, the reaction time of reaction 1 is 16-24 hours.

[0025] Preferably, in reaction 1, the molar ratio of the amino compound to the epoxide compound is 1:(2-5).

[0026] More preferably, in reaction 1, the molar ratio of the amino compound to the epoxy compound is 1:(2.2-3).

[0027] Preferably, the reaction temperature of reaction 2 is 15-40°C.

[0028] Preferably, the reaction time of reaction 2 is 2h-10h.

[0029] More preferably, the reaction time of reaction 2 is 4h-6h.

[0030] Preferably, in reaction 2, the molar ratio of the intermediate product to the tail-chain compound is 1:(1-2).

[0031] More preferably, in reaction 2, the molar ratio of the intermediate product to the tail-chain compound is 1:(1-1.2).

[0032] Preferably, the reaction system of reaction 2 further includes 4-dimethylaminopyridine (DMAP) and triethylamine.

[0033] More preferably, in reaction 2, the molar ratio of intermediate product C to 4-dimethylaminopyridine is 1:(0.05-0.2).

[0034] More preferably, in reaction 2, the molar ratio of intermediate product C to 4-dimethylaminopyridine is 1:(0.1-0.15).

[0035] More preferably, in reaction 2, the molar ratio of intermediate product C to triethylamine is 1:(1-2).

[0036] More preferably, in reaction 2, the molar ratio of intermediate product C to triethylamine is 1:(1-1.2).

[0037] A third aspect of the present invention provides a lipid composition comprising the branched cationic lipid compound described in the first aspect, and further comprising at least one of the following lipids: (i) one or more non-cationic lipids; (ii) One or more cholesterol-based lipids; (iii) One or more PEG-modified lipids.

[0038] Preferably, the lipid composition is microparticles, nanoparticles, liposomes, lipid nanoparticles, or microbubbles.

[0039] The fourth aspect of the present invention provides the use of the branched cationic lipid compound described in the first aspect, or the lipid composition described in the third aspect, in the delivery of bioactive substances.

[0040] Preferably, the bioactive substance is a small molecule compound, nucleic acid, protein, peptide, metal, isotope-labeled compound, or vaccine.

[0041] More preferably, the nucleic acid is DNA, mRNA, siRNA, or circular RNA.

[0042] Preferably, the application is a drug delivery system, and the delivered drug may be a therapeutic agent, a diagnostic agent, or a preventive agent.

[0043] The drug delivery system may be in particle form. In some embodiments, the particle diameter is in the range of 1 μm to 1000 μm. In some embodiments, the particle diameter is in the range of 1 nm to 1000 nm. When the particle size range is in the range of 1 nm to 1000 nm, it is a nanoparticle as commonly known in the art. The particles may be prepared using any method known in the art. These methods include (but are not limited to) spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, nanoprecipitation, microfluidics, simple and complex agglomeration, and other methods well known to those skilled in the art.

[0044] More preferably, the application is in the form of lipid nanoparticles, which comprise an organic phase and an aqueous phase. The organic phase comprises branched-chain cationic lipid molecules, phospholipids, cholesterol, and PEG lipids, wherein the molar ratio of branched-chain cationic lipid molecules is 20%-80%, the molar ratio of phospholipids is 5%-50%, the molar ratio of cholesterol is 10%-60%, and the molar ratio of PEG lipids is 0%-5%. The aqueous phase comprises nucleic acid molecules. More preferably, the phospholipid is dioleoylphosphatidylethanolamine (DOPE) or distearate phosphatidylcholine (DSPC). More preferably, the nucleic acid is mRNA. More preferably, the solvent of the organic phase is an alcohol solvent.

[0045] More preferably, the drug is selected from any one of the following: (1) a drug for treating tumors; (2) a drug for treating autoimmune diseases; (3) a drug for treating infectious diseases; (4) a drug for treating infectious diseases; (5) a drug for treating cardiovascular diseases; (6) a drug for treating diabetes; (7) a drug for treating nervous system diseases; (8) a drug for treating respiratory system diseases; and (9) a drug for treating blood system diseases.

[0046] The beneficial effects of this invention are: This invention provides a branched-chain cationic lipid compound with the following structural features: 1. An ionizable tertiary amine head ensures efficient nucleic acid delivery; 2. The ether-bonded branched structure promotes the formation of a cone-shaped structure during mRNA delivery and enhances endosome escape capability; 3. Functional linkers connect saturated or unsaturated tail chains, improving mRNA encapsulation efficiency and delivery safety. In summary, the cationic lipid compound of this invention is a branched-chain cationic lipid molecule that combines high delivery efficiency, good cell biocompatibility, and excellent storage stability, providing more options for nucleic acid drug delivery and having significant implications for the development and application of nucleic acid drugs.

[0047] In addition, this invention has developed two-component and three-component LNP formulations based on branched cationic lipid molecules. These formulations are not only simpler than traditional four-component LNP formulations, achieving a simplified formulation, but also, since there are currently few reports of stable two-component LNP clinical or research formulations, the two-component LNP formulations of this invention are expected to promote the development of precision mRNA therapies for various diseases. Attached Figure Description

[0048] Figure 1 The results are the proton NMR spectra of the lipid molecule A5BT1; Figure 2 Mass spectrometry results for lipid molecule A5BT1; Figure 3 The 1H NMR spectrum of the lipid molecule A5BT2; Figure 4 The mass spectrometry results are for the lipid molecule A5BT2. Figure 5 The 1H NMR spectrum of the lipid molecule A5BT3; Figure 6 The mass spectrometry results are for the lipid molecule A5BT3. Figure 7 The 1H NMR spectrum of the lipid molecule A5BT4; Figure 8 Mass spectrometry results for lipid molecule A5BT4; Figure 9 The 1H NMR spectrum results are for the ether-free sample of Comparative Example 1. Figure 10 Mass spectrometry results for the ether-free sample of Comparative Example 1; Figure 11 The results of transfection of cationic lipid molecules with and without ether bonds in 293T cells; Figure 12 The relative transfection rates of the three samples; Figure 13 The results of transfection of cationic lipid molecules with different linkages in 293T cells. Detailed Implementation

[0049] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from conventional commercial channels or prepared and isolated through simple synthesis; unless otherwise specified, the processes employed are conventional processes in the art.

[0050] This invention utilizes combinatorial chemistry to construct a branched cationic lipid library, the composition of which is shown in Table 1 below. The chemical structures of each part are represented by A1-A6, B, C, and T1-T5, respectively. The general structural formula of the Head is as follows: The general structural formula of a Linker is: The structures of Tail are T1-T4 respectively: Table 1. Schematic diagram of the chemical structure of branched cationic lipid libraries

[0051] This lipid library contains 60 polyaminoamidinyl cationic lipid molecules. The experiment first involved preliminary screening of these 60 cationic lipids in HEK293T cells. The screening process is as follows: The synthesized branched cationic lipid molecules were prepared into a 10 mM solution. 75 μL of the 10 mM cationic lipid molecule solution and 75 μL of a 10 mM neutral phospholipid (DOPE) solution were thoroughly mixed in an EP tube. Then, 350 μL of a 200 mM sodium acetate buffer solution (pH 5) was added, and the mixture was immediately vortexed for 10 seconds to obtain a two-component lipid nanoparticle solution suitable for verifying delivery efficiency. Unless otherwise specified, the preparation steps for the two-component lipid nanoparticles are the same.

[0052] The delivery efficiency of the lipid nanoparticle solution prepared above was verified in 293T cells. The specific steps are as follows: 1. Add 20 μL of PBS solution to a PCR tube, and then add 1.0 μL of the two-component lipid nanoparticle solution; 2. Dilute EGFP mRNA to a concentration of 220 ng / 10 μL, and add 20 μL of the solution to the PBS solution. 1. Prepare a 220 ng / 10 μL mRNA solution, pipette it evenly, and incubate at room temperature for approximately 30 minutes. 2. Digest 293T cells into a cell suspension, dilute the cells with culture medium to a concentration of 75,000 cells / 100 μL, and after incubation in step 2, add 200 μL of cell suspension to each PCR tube, pipette it evenly, and then add 120 μL of the cell suspension-lipid nanoparticle mixture to each well of a 96-well plate. 3. After 36 hours, add 20 μL of 10 mM Hoechst nuclear staining agent to each well of cells and stain in the dark at 37°C for approximately 30 minutes. After successful staining, use a high-content microscope to quantitatively analyze the cell transfection efficiency. The formula for calculating the transfection efficiency is: Unless otherwise specified, the procedures for subsequent cell delivery efficiency verification experiments are the same.

[0053] Based on the above verification of cell delivery efficiency, several samples with transfection efficiency superior to Lipo2000 transfection reagent were finally screened. Among them, A5BT2 has excellent transfection delivery efficiency. The following uses A5BT1 to illustrate this: Example 1 This embodiment provides an ionizable cationic lipid molecule, A5BT1, whose synthetic route, preparation method, and structural formula are as follows:

[0054] S1, 2.4 equivalents of n-butyl glycidyl ether B, and 1 equivalent of N,N-diethylethylenediamine A5 (2 mmol) were dissolved in 3 mL of methanol in a 25 mL flask. The mixture was stirred thoroughly and reacted at room temperature for 24 hours. The solvent was rotary evaporated to obtain the crude product. The crude product was purified by alumina column chromatography to obtain the intermediate product A5B. (Dichloromethane:methanol = 15:1, Rf = 0.2), the yield of this reaction step is 80%; S2, 1 equivalent of intermediate product A5B (1 mmol), and 0.12 equivalents of DMAP were thoroughly dissolved in 2 ml of dichloromethane, and then 1.2 equivalents of triethylamine (TEA) and 1.2 equivalents of octanoyl chloride T1 were added sequentially. The mixture was stirred thoroughly and reacted at room temperature for 4 hours. The reaction was stopped by rotary evaporation to obtain the crude product. Finally, the crude product was purified by alumina column chromatography to obtain the final product A5BT1. (Dichloromethane:methanol = 30:1, Rf = 0.2), the yield of this reaction step is 80%.

[0055] Figure 1 and 2 The results of the 1H NMR and mass spectrometry of the lipid molecule A5BT1 can verify the preparation of the ionizable cationic lipid molecule with the target structure.

[0056] Unless otherwise specified, the synthesis steps of other lipid molecules described in this invention are the same as or similar to those described herein.

[0057] Figure 3-8 The proton NMR and mass spectrometry results of lipid molecules A5BT2, A5BT3, and A5BT4 respectively confirm that ionizable cationic lipid molecules with the target structure were obtained in all cases.

[0058] Comparative Example 1 This comparative example provides a branched, ionizable cationic lipid molecule without ether bonds. Its preparation method and structural formula are as follows: S1, 2.4 equivalents of epoxy octane, and 1 equivalent of N,N-diethylethylenediamine A5 (2 mmol) were dissolved in 3 mL of methanol in a 25 mL flask. The mixture was stirred thoroughly and reacted at room temperature for 24 hours. The solvent was rotary evaporated to obtain the crude product. The crude product was purified by alumina column chromatography to obtain the intermediate product. (Dichloromethane:methanol = 15:1, Rf = 0.2), the yield of this reaction step is 80%; S2, 1 equivalent of the intermediate (1 mmol), and 0.12 equivalents of DMAP were dissolved thoroughly in 2 mL of dichloromethane, and then 1.2 equivalents of triethylamine (TEA) and 1.2 equivalents of octanoyl chloride (T1) were added sequentially. The mixture was stirred thoroughly and reacted at room temperature for 4 hours. The reaction was stopped by rotary evaporation to obtain the crude product. Finally, the crude product was purified by alumina column chromatography to obtain the final product. (Dichloromethane:methanol = 30:1, Rf = 0.2), the yield of this reaction step is 80%.

[0059] Figure 9-10 The structure of the ether-free sample of Comparative Example 1 and the corresponding proton NMR and mass spectrometry results are shown.

[0060] Application Example 1 This application example provides a comparative example of the ether-free branched chain of the branched-chain ionizable cationic lipid molecule A5BT2 and two-component lipid nanoparticles of Lipo2000, along with their preparation steps and transfection steps in 293T cells, as detailed below: Preparation steps of the two-component lipid nanoparticles: A 10 mM solution of cationic lipid molecules was prepared. 75 μL of the 10 mM cationic lipid molecule solution and 75 μL of a 10 mM neutral phospholipid (DOPE) solution were thoroughly mixed in an EP tube. Then, 350 μL of a 200 mM sodium acetate buffer solution (pH 5) was added and immediately vortexed for 10 seconds to obtain the two-component lipid nanoparticle solution suitable for verifying delivery efficiency. Unless otherwise specified, the preparation steps of the two-component lipid nanoparticles described in this invention are the same or similar.

[0061] The delivery efficiency of the two-component lipid nanoparticles prepared above was verified in 293T cells. The specific steps are as follows: 1. Add 20 μL of PBS solution to a PCR tube, then add 2.0 μL of the two-component lipid nanoparticle solution; 2. Dilute EGFP mRNA to a concentration of 220 ng / 10 μL, and add 20 μL of the solution to the PBS solution. 1. Prepare a 220 ng / 10 μL mRNA solution, pipette it evenly, and incubate at room temperature for approximately 30 minutes. 2. Digest 293T cells into a cell suspension, dilute the cells with culture medium to a concentration of 75,000 cells / 100 μL, and after the incubation step in step 2, add 200 μL of cell suspension to each PCR tube, gently pipette it evenly, and then add 120 μL of the cell suspension-lipid nanoparticle mixture to each well of a 96-well plate. 3. After 36 hours, add 20 μL of 10 mM Hoechst nuclear staining agent to each well of cells and stain in the dark at 37°C for approximately 30 minutes. After successful staining, use a high-content microscope to quantitatively analyze the cell transfection efficiency. The formula for calculating the transfection efficiency is: The results of 293T cell transfection with the three lipid molecules are shown in the figure. Figure 11 .

[0062] like Figure 11 As shown, Figure 11 In the figures, AF represent the cell transfection fluorescence results and corresponding bright-field fluorescence of the A5BT1 sample from Example 1, the sample from Comparative Example 1, and Lipo2000, respectively. As can be seen from the figures, compared to other samples, when the branched chains are pure carbon chains without ether bonds, the cell transfection efficiency is extremely low under the same tail chain length. Additionally... Figure 12 The relative transfection rates of the three samples are shown. The A5BT2 sample has a higher cell transfection efficiency than Lipo2000, indicating that the present invention designs and synthesizes a branched cationic lipid molecule that has high delivery efficiency, good cell biocompatibility and excellent storage stability.

[0063] Furthermore, based on the branched cationic lipid molecule library in Table 1, different ionizable cationic lipid molecules were prepared. The corresponding two-component lipid nanoparticles were then prepared using the method described above, and their delivery efficiency was verified. The results are as follows: Figure 13 As shown, different ionizable cationic lipid molecules all have certain delivery efficiencies, providing more options for nucleic acid drug delivery and having important significance for the development and application of nucleic acid drugs.

[0064] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A branched cationic lipid compound, characterized in that, Its structural formula is shown in Formula I: Formula I; Wherein, m and n are independently selected from integers 0-3 and 0-3, respectively; R1 and R2 are independently H, substituted or unsubstituted (C1-C2) compounds, respectively. 18 )alkyl, substituted or unsubstituted (C2-C 18 Alkenyl, substituted or unsubstituted phenyl or 4-7 membered heterocycles; Alternatively, R1 and R2, together with the nitrogen atoms they are attached to, form a heterocycle, which is a substituted or unsubstituted 4-7 membered nitrogen heterocycle; the heterocycle also contains 0-3 additional heteroatoms selected from N, O, and S; the substitution refers to the presence of a substituent at one or more substituted positions of the heterocycle, the substituent being selected from (C1-C8)alkyl, halo(C1-C8)alkyl, -O-(C1-C8)alkyl, -O-haloC1-C8alkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C6alkyl), and -N(C1-C6alkyl)2 substituents; R3, R4, R5, and R6 are independently H, substituted, or unsubstituted (C1-C2) compounds. 30 )alkyl, substituted or unsubstituted (C2-C 30 alkenyl; Wherein, the substitution of R1, R2, R3, R4, R5 or R6 is independently selected from at least one of deuterium, (C1-C8)alkyl, halo(C1-C8)alkyl, -O-(C1-C8)alkyl, -O-haloC1-C8alkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C6alkyl)2 and -N(C1-C6alkyl)2, wherein the number of substitutions is from monosubstituted to the maximum number of substitutions; L1 and L2 are each independently , , , ; The linking site of the representative group, Indicates the connection sites with R5 and R6; The A is a (C1-C6) alkylene or a single bond.

2. The branched cationic lipid compound according to claim 1, characterized in that, R1 and R2 are independently H, substituted or unsubstituted (C1-C2) compounds, respectively. 10 Alkyl group; or, R1 and R2 together with the nitrogen atom to which they are attached form a heterocycle, which is a 4-7 membered nitrogen heterocycle.

3. The branched cationic lipid compound according to claim 1, characterized in that, The R3 is H, substituted or unsubstituted (C1-C2). 10 )alkyl; The R4 is H, substituted or unsubstituted (C1-C2). 10 )alkyl; Wherein, the substitution of R3 or R4 is independently selected from at least one of deuterium, (C1-C5)alkyl, halo(C1-C5)alkyl, -O-(C1-C5)alkyl, -O-haloC1-C3alkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C5alkyl)2 and -N(C1-C5alkyl)2, wherein the number of substitutions is from monosubstituted to the maximum number of substitutions.

4. The branched cationic lipid compound according to claim 1, characterized in that, The R5 is H, substituted or unsubstituted (C1-C1) 24 )alkyl; The R6 is H, substituted or unsubstituted (C1-C2). 24 )alkyl; Wherein, the substitution of R5 or R6 is independently selected from at least one of deuterium, (C1-C5)alkyl, halo(C1-C5)alkyl, -O-(C1-C5)alkyl, -O-haloC1-C3alkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C5alkyl)2 and -N(C1-C5alkyl)2, wherein the number of substitutions is from monosubstituted to the maximum number of substitutions.

5. The branched cationic lipid compound according to claim 1, characterized in that, The definitions of R3 and R4 are the same; And / or, the definitions of R5 and R6 are the same.

6. The method for preparing the branched-chain cationic lipid compound according to claim 5, characterized in that, Includes the following steps: An amino compound and an epoxy compound are reacted 1 to obtain an intermediate product; the intermediate product is reacted 2 with a tail-chain compound to obtain the branched cationic lipid compound. The structural formulas of the amino compound, epoxide compound, and intermediate product are shown in formulas A through C, respectively; the structural formula of the tail-chain compound is shown in formula D1 or D2. Formula A Formula B Formula C; Formula D1 Formula D2; Wherein, m, n, R3, R5, and A are as described in claim 5; R7 is a halogen.

7. A lipid composition, characterized in that, It comprises the branched cationic lipid compound as described in any one of claims 1 to 5, and further comprises at least one of the following lipids: (i) one or more non-cationic lipids; (ii) One or more cholesterol-based lipids; (iii) One or more PEG-modified lipids.

8. The lipid composition according to claim 7, characterized in that, The lipid composition is microparticles, nanoparticles, liposomes, lipid nanoparticles, or microbubbles.

9. The use of the branched cationic lipid compound according to any one of claims 1 to 5, or the lipid composition according to claim 7 or 8, in the delivery of bioactive substances.

10. The application according to claim 9, characterized in that, The bioactive substances are small molecule compounds, nucleic acids, proteins, peptides, metals, isotopically labeled compounds, and vaccines.

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