Cationic lipid compound as well as preparation method and application thereof

Abstract

The invention belongs to the field of lipid delivery carriers, and particularly relates to a cationic lipid compound as well as a preparation method and application thereof. The cationic lipid compound is designed and synthesized, the lipid nanoparticle composition formed by the cationic lipid compound has the advantages in at least one aspect of the aspects of encapsulation efficiency, biological safety, transfection efficiency protein expression in cells and organisms and the like, and the problems existing in the prior art are solved. The method has non-important significance on development of gene therapy products. In order to improve the drug delivery effect of a gene vector and simplify the synthesis steps of the gene vector, the invention provides a cationic lipid compound as well as a preparation method and application thereof. The cationic lipid compound has positive electricity and can enter cells more easily, so that drugs are better delivered, a better treatment effect is generated, and the cationic lipid compound is simple to synthesize and easy to prepare and has practical application value.

Description

Technical Field

[0001] The invention belongs to the field of lipid delivery carriers, and specifically relates to a cationic lipid compound and a preparation method and application thereof. Background Art

[0002] Gene therapy is to introduce exogenous genes into target cells or tissues to replace, compensate, block or correct specific genes in order to achieve the purpose of treating and preventing diseases. However, exogenous genes have a short circulation time in the body and are easily degraded, making it difficult for them to enter target cells or target tissues. Therefore, finding a good gene delivery vector and improving the in vivo delivery efficiency of gene drugs is one of the key directions to improve the effectiveness of gene therapy products.

[0003] Gene therapy is a new technology born from the combination of modern medicine and molecular biology. It is a new means of disease treatment. It has been successfully applied in some cases, and the scientific breakthroughs achieved will continue to promote gene therapy to the mainstream medical field.

[0004] At present, the most widely used gene delivery vector is lipid nanoparticles, which can protect nucleic acids from rapid degradation in the body, prolong circulation time, and enhance targeted delivery. Lipid nanoparticles are composed of different lipid components, including cationic lipid compounds, structural lipids, phospholipids and PEG lipids. Among them, cationic lipid compounds play a key role in nucleic acid encapsulation and release, so it is very important to develop new, efficient and low-toxic cationic lipid compounds. In order to overcome the shortcomings of the prior art, there is still a need for a new cationic lipid compound that meets the delivery requirements of gene therapy products for the development of gene therapy products.

[0005] Gene vectors have to go through multiple complex processes when transporting genes: reaching target cells through blood circulation, cell uptake, endosome escape, intracellular movement, and vector release of gene material, etc. The main obstacles are extracellular obstacles of the complex blood environment and intracellular obstacles of lysosomal enzyme degradation. Therefore, how to find a good gene vector so that the target gene can reach the target site and take effect is a problem to be solved by gene vector researchers.

[0006] Commonly used non-viral vector systems are mainly lipid carriers. Lipid carriers usually contain positive charges of cationic lipids, which bind to negatively charged gene drugs through electrostatic interaction, thereby concentrating and packaging genetic materials into particles with smaller particle sizes to form lipid nanoparticles (LNPs). LNPs have shown unparalleled advantages over other types of liposomes in the preparation of gene delivery vectors and cell transfection. The smaller particle size of the complex reduces the chance of being recognized, phagocytosed, and cleared by macrophages in the body, thereby improving the in vivo bioavailability of the drug. At the same time, for brain tissue, the smaller particle size of the complex is more likely to penetrate into the brain through the gaps between vascular endothelial cells through penetration and retention effects, increasing drug aggregation in brain tissue. In terms of transfection, since the cell surface is slightly negatively charged, positively charged liposomes are more likely to adsorb to the cell surface and enter the cell through mechanisms such as endocytosis, greatly increasing the transfection ability of liposomes.

[0007] Currently, LNP has become the most widely used drug carrier due to its simple structure, easy operation and high biosafety. Summary of the invention

[0008] The cationic lipid compound (EGPLPD-3) designed and synthesized in the present application, and the lipid nanoparticle composition composed of the cationic lipid compound (EGPLPD-3) has advantages in at least one aspect of encapsulation efficiency, biosafety, cell and in vivo transfection efficiency, protein expression, etc., and solves the problems existing in the prior art. It is of great significance to the development of gene therapy products.

[0009] In order to improve the drug delivery effect of gene vectors and simplify their synthesis steps, the present application provides a cationic lipid compound (EGPLPD-3) and its preparation method and application. This cationic lipid compound (EGPLPD-3) is positively charged and can more easily enter cells, thereby better delivering drugs and producing better therapeutic effects. It is simple to synthesize and easy to prepare, and has practical application value.

[0010] In order to solve the above-mentioned technical problems, this application provides the following technical solutions:

[0011] The present invention provides a cationic lipid compound, the structure of which is as follows:

[0012]

[0013] Wherein, R1 is selected from a heterocyclic, alkane or olefin group; said R1 is a substituted or unsubstituted group;

[0014] The structure of R1 is selected from the following:

[0015]

[0016] Wherein, R2 is selected from a linear or branched alkane or olefin group; said R2 is a substituted or unsubstituted group;

[0017] The structure of R2 is selected from the following:

[0018]

[0019] Wherein, n1, n2, n3, n4, and n5 are independently selected from any integer between 1 and 10;

[0020] Wherein, R3 is selected from a linear or branched alkane or olefin group; said R3 is a substituted or unsubstituted group;

[0021] The structure of R3 is selected from the following:

[0022]

[0023] Wherein, n6 to n18 are independently selected from any integer between 1 and 10.

[0024] Preferably, R1:

[0025] R2: when n5 = 2;

[0026]

[0027] R3: n6 = 7; n7 = 8;

[0028]

[0029] The cationic lipid compound structure is as follows:

[0030]

[0031] The present invention also provides a method for preparing the above cationic lipid compound, comprising the following steps:

[0032] S11: Dissolve oleic acid in dichloromethane (CH2Cl2), add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP) and N,N-diisopropylethylamine (DIPEA), and mix for 8-12 minutes;

[0033] S12: Add 1,4-bis(2-hydroxyethyl)piperazine to the mixture obtained in step S11, and stir at room temperature (25±5° C.) for 20-28 hours to obtain the cationic lipid compound.

[0034] In the present application, the raw material in step S11 is a fatty acid compound, and in the actual production process, it can be specifically selected according to the structure of the specific cationic lipid compound to be synthesized.

[0035] Preferably, the fatty acid compound includes oleic acid.

[0036] Furthermore, in step S11, the molar ratio of the raw materials is oleic acid: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI): 4-dimethylaminopyridine (DMAP): N,N-diisopropylethylamine (DIPEA) = 1:1:0.1:1.

[0037] Furthermore, in step S12, the molar ratio of raw materials is oleic acid:1,4-bis(2-hydroxyethyl)piperazine=2:1.

[0038] Preferably, in step S11, 4-dimethylaminopyridine (DMAP) and N,N-diisopropylethylamine (DIPEA) are used as catalysts for the reaction.

[0039] Preferably, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) is used as the water absorbent for the reaction.

[0040] Preferably, in step S11, the reaction solvent includes dichloromethane (CH2Cl2).

[0041] Preferably, in step S11, the reaction is carried out at room temperature (28°C).

[0042] Preferably, in step S12, the reaction solvent includes DCM (dichloromethane).

[0043] Preferably, in step S12, the reaction is carried out at 28°C.

[0044] The present invention also provides a post-processing purification method for a cationic lipid compound, comprising the following steps:

[0045] (1) After the reaction in step S12 is completed, the reaction system is distilled under reduced pressure to remove the solvent;

[0046] (2) extracting with ethyl acetate and combining the extracted organic layers;

[0047] (3) washing the combined organic layers with saturated brine;

[0048] (4) washing the washed organic layer with saturated sodium bicarbonate;

[0049] (5) The organic phases were combined and dried by spin drying, and then purified by column separation to obtain the final product.

[0050] In the above post-treatment purification method, the solvent in step (2) is an organic reagent, which can be specifically selected according to the properties of the post-treatment product in the actual post-treatment process. The column in step (5) is a silica gel column, which can be specifically selected according to the polarity of the post-treatment product in the actual post-treatment process.

[0051] Preferably, in step (1), the temperature of the reduced pressure distillation is 35° C. and the rotation speed is 80 r / min.

[0052] Preferably, in step (1), the post-treatment temperature condition is 35°C.

[0053] Preferably, in step (2), ethyl acetate is used as the extractant for post-treatment.

[0054] Preferably, in step (2), the post-treatment temperature condition is carried out at room temperature (28°C).

[0055] Preferably, in step (3), saturated brine is used as a post-treatment detergent.

[0056] Preferably, in step (3), the post-treatment temperature condition is carried out at room temperature (28°C).

[0057] Preferably, in step (4), saturated sodium bicarbonate is used as a post-treatment detergent.

[0058] Preferably, in step (4), the post-treatment temperature condition is carried out at room temperature (28°C).

[0059] Preferably, in step (5), the eluent system of column chromatography is a system of dichloromethane and methanol.

[0060] Preferably, in step (5), the post-treatment temperature condition is carried out at room temperature (28°C).

[0061] Preferably, in step (5), the ratio of methyl chloride to methanol is 50:1.

[0062] In the present application, by optimizing the molar ratio of the reaction raw materials of the cationic lipid compound (EGPLPD-3), the yield of the cationic lipid compound (EGPLPD-3) is increased, and the purity of the cationic lipid compound (EGPLPD-3) is also increased.

[0063] The present invention also provides a lipid nanoparticle LNP carrier, wherein the lipid nanoparticle LNP carrier contains the cationic lipid compound.

[0064] Preferably, the lipid nanoparticle LNP carrier comprises 15-70% of the cationic lipid compound in terms of molar fraction.

[0065] Preferably, the lipid nanoparticle LNP carrier comprises 50% of a cationic lipid compound, 10% of a phospholipid, 38.5% of cholesterol and 1.5% of a polyethylene glycol lipid, by molar fraction.

[0066] In the present application, by optimizing the components of the LNP carrier and their respective addition amounts, a technical effect of synergistic gain is produced, the total amount of loaded drug is greater, and the adsorption effect on cells is better, thereby exerting a better drug loading effect, improving the utilization rate of the drug, and achieving a better therapeutic effect.

[0067] The present invention also provides a gene medicine preparation, which comprises the lipid nanoparticle LNP carrier.

[0068] Preferably, the gene drug preparation further comprises a nucleic acid drug; and the mass ratio of the nitrogen content of the cationic lipid compound in the lipid nanoparticle LNP carrier to the phosphorus content in the nucleic acid drug is 2-12:1.

[0069] Furthermore, the preparation method of the gene medicine preparation comprises the following steps:

[0070] S21: preparing lipid oil phase and nucleic acid (siRNA) aqueous phase respectively;

[0071] The lipid oil phase is obtained by mixing and dissolving a cationic lipid compound (EGPLPD-3), phospholipids, cholesterol and polyethylene glycol lipids;

[0072] The nucleic acid (siRNA) aqueous phase is obtained by diluting the nucleic acid drug with water;

[0073] S22: The lipid oil phase and the nucleic acid aqueous phase are mixed at a certain flow rate through a split injection pump to obtain the gene drug preparation.

[0074] Specifically, in the lipid oil phase, when phospholipids, cholesterol and polyethylene glycol lipids are dissolved, the solvent used includes anhydrous ethanol.

[0075] Specifically, in the nucleic acid (siRNA) aqueous phase, when diluting the nucleic acid drug, an acidic buffered sodium acetate solution is used for dilution.

[0076] The technical solution of the present invention has the following advantages over the prior art:

[0077] 1. This application optimizes the synthesis process of the cationic lipid compound (EGPLPD-3), so that the yield and purity of the cationic lipid compound (EGPLPD-3) are higher. The raw materials for the production of the cationic lipid compound (EGPLPD-3) are easily available, the preparation method is simple, and the synthesis efficiency is high, which creates conditions for large-scale production;

[0078] 2. The LNP carrier prepared using the above cationic lipid compound (EGPLPD-3) has a positive charge, so it is easier to adsorb to the cell surface and enter the cell, with higher efficiency in drug delivery and better therapeutic effect. After being prepared with nucleic acid molecules, it can accurately and efficiently enter the receptor cells and express related proteins, and induce the body's immune response, thereby playing a role in treating diseases as a therapeutic drug, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 It is a schematic diagram of the synthesis route of the cationic lipid compound (EGPLPD-3) in this application.

[0080] Figure 2 This is a picture of the mass spectrometry detection results of the cationic lipid compound (EGPLPD-3) in this application.

[0081] Figure 3 This is a picture of the hydrogen spectrum detection results of the cationic lipid compound (EGPLPD-3) in this application.

[0082] Figure 4 This is a picture of the carbon spectrum detection results of the cationic lipid compound (EGPLPD-3) in this application. DETAILED DESCRIPTION

[0083] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0084] The present application provides a cationic lipid compound (EGPLPD-3), wherein the cationic lipid compound includes the group structures shown by R1, R2, and R3;

[0085] R1:

[0086] R2: When n5 = 2,

[0087]

[0088] R3: When n6=7, n7=8,

[0089]

[0090] Specifically, the cationic lipid compound (EGPLPD-3) includes the R1, R2, and R3 group structures shown in Formula I.

[0091] Specifically, this application takes EGPLPD-3 as an example to illustrate the preparation method of the cationic lipid compound, and its synthesis route is as follows: Figure 1As shown, the specific process is as follows;

[0092] Oleic acid reacts with 1,4-bis(2-hydroxyethyl)piperazine in dichloromethane solution under room temperature conditions to generate a cationic lipid compound (EGPLPD-3) using 4-dimethylaminopyridine (DMAP) and N,N-diisopropylethylamine (DIPEA) as catalysts and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) as a water absorbent.

[0093] Oleic acid (3.8 mL, 12.05 mmol) was dissolved in 15 mL of anhydrous dichloromethane, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.53 g, 13.20 mmol), 4-dimethylaminopyridine (0.14 g, 1.15 mmol), N,N-diisopropylethylamine (1.78 g, 13.77 mmol) were added, and stirred at 28°C for 10 min. After 10 min, 1,4-bis(2-hydroxyethyl)piperazine (1 g, 5.74 mmol) was added, and the mixture was reacted at room temperature (28°C) for 24 h.

[0094] The progress of the reaction was monitored by TLC. When the reaction was complete, the solvent was removed by vacuum distillation using a rotary evaporator. 100 mL of ethyl acetate was added for extraction three times, and the mixture was washed three times with an equal volume of saturated sodium chloride solution and twice with an equal volume of saturated sodium bicarbonate solution. The organic phases after washing were combined. The solvent was removed by vacuum distillation using a rotary evaporator and column separation and purification (silica gel column, eluent DCM: MeOH = 50: 1 (volume ratio)) was performed to purify the cationic lipid compound (EGPLPD-3).

[0095] The present application also provides an LNP carrier, which comprises, by mole fraction, 50% of a cationic lipid compound (EGPLPD-3), 10% of phospholipids, 38.5% of cholesterol and 1.5% of polyethylene glycol lipids.

[0096] The present application also provides a preparation, which includes an LNP carrier and a nucleic acid drug, wherein the molar ratio of the nitrogen content of the cationic lipid compound in the LNP carrier to the phosphorus content in the nucleic acid drug in the preparation is 2-12:1.

[0097] The preparation is prepared by the following method:

[0098] Weigh 5.4 mg of phospholipids, 10.17 mg of cholesterol, and 2.5722 mg of polyethylene glycol lipids and dissolve them in 40 mL of anhydrous ethanol at room temperature to prepare a stock solution;

[0099] At room temperature, 24 mg of the cationic lipid compound was added to the stock solution, and the mixture was mixed to obtain a lipid oil phase;

[0100] Use acidic buffered saline solution to dilute the nucleic acid drug (sodium acetate solution) to a final concentration of 33ug / mL and mix well to prepare a nucleic acid aqueous phase;

[0101] The lipid oil phase and the nucleic acid aqueous phase are mixed at a volume ratio of 1:3 at room temperature through a split syringe pump to prepare a drug intermediate, and ultrafiltration is performed through an ultrafiltration device to remove the solvent to obtain the preparation.

[0102] Combination Figures 1 to 4 The present application is further described in detail with reference to Examples 1 to 3.

[0103] Example 1

[0104] This embodiment provides a cationic lipid compound EGPLPD-3, and the structural formula of the cationic lipid compound EGPLPD-3 is shown in Formula I.

[0105]

[0106] The cationic lipid compound EGPLPD-3 is prepared by the following method, and the synthetic route is as shown in FIG. Figure 1 As shown:

[0107] (1) Oleic acid is reacted with 1,4-bis(2-hydroxyethyl)piperazine in a dichloromethane (DCM) solution under room temperature conditions to produce a cationic lipid compound (EGPLPD-3) using 4-dimethylaminopyridine (DMAP) and N,N-diisopropylethylamine (DIPEA) as catalysts and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) as a water absorbent;

[0108] In a 100mL round-bottom flask, weigh 2.53g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.14g of 4-dimethylaminopyridine and dissolve them in 15mL of anhydrous dichloromethane (DCM). Then weigh 2.4mL of N,N-diisopropylethylamine and 3.8mL of oleic acid and add them to the round-bottom flask. After stirring at 28°C for 10min, weigh 1g of 1,4-bis(2-hydroxyethyl)piperazine and add it to the round-bottom flask. After all the substances are dissolved, stir at 28°C for 24h.

[0109] Monitor the progress of the reaction by TLC. After the reaction is complete, use a rotary evaporator to remove the solvent from the system in the round-bottom flask. Add 100 mL of ethyl acetate to extract three times, wash with an equal volume of saturated sodium chloride solution three times, and wash with an equal volume of saturated sodium bicarbonate solution twice, and combine the washed organic phases. Use a rotary evaporator to remove the solvent by reduced pressure rotary evaporation and perform column separation and purification (silica gel column, eluent DCM:MeOH=50:1 (volume ratio)) to purify the cationic lipid compound (EGPLPD-3). The hydrogen spectrum detection results of the cationic lipid compound EGPLPD-3 are shown as follows Figure 2 shown.

[0110] Example 2

[0111] This embodiment provides a cationic lipid compound EGPLPD-3, and the structural formula of the cationic lipid compound EGPLPD-3 is shown in Formula I.

[0112]

[0113] The cationic lipid compound EGPLPD-3 is prepared by the following method, and the synthetic route is as shown in FIG. Figure 1 As shown:

[0114] Oleic acid reacts with 1,4-bis(2-hydroxyethyl)piperazine in dichloromethane (DCM) solution under room temperature conditions to generate a cationic lipid compound (EGPLPD-3) using 4-dimethylaminopyridine (DMAP) and N,N-diisopropylethylamine (DIPEA) as catalysts and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) as a water absorbent.

[0115] In a 100mL round-bottom flask, weigh 0.4875g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.0610g of 4-dimethylaminopyridine and dissolve them in 12mL of anhydrous dichloromethane (DCM). Then weigh 0.35mL of N,N-diisopropylethylamine and 0.95mL of oleic acid and add them to the round-bottom flask. After stirring at 28°C for 10min, weigh 0.1742g of 1,4-bis(2-hydroxyethyl)piperazine and add it to the round-bottom flask. After all the substances are dissolved, stir at 28°C for 24h.

[0116] The progress of the reaction was monitored by TLC. After the reaction was complete, the system in the round-bottom flask was distilled under reduced pressure using a rotary evaporator to remove the solvent. 100 mL of ethyl acetate was added for extraction three times, and the solution was washed with an equal volume of saturated sodium chloride solution three times and an equal volume of saturated sodium bicarbonate solution twice, and the organic phases after washing were combined. The solvent was removed by rotary evaporation under reduced pressure using a rotary evaporator and column separation and purification (silica gel column, eluent DCM: MeOH = 50: 1 (volume ratio)) was performed to purify the cationic lipid compound (EGPLPD-3).

[0117] In the above examples, the statistical results of the quality and yield of the obtained products are shown in Table 1.

[0118] Table 1 Statistical results of the quality and yield of the products obtained in each embodiment

[0119]

[0120] Example 3

[0121] This embodiment provides a gene drug preparation, which is prepared by the following method:

[0122] 5.4 mg of phospholipids, 10.17 mg of cholesterol and 2.5722 mg of polyethylene glycol lipid were weighed and dissolved in 40 mL of anhydrous ethanol at room temperature to prepare a stock.

[0123] At room temperature, 24 mg of the cationic lipid compound EGPLPD-3 was added to the stock solution and mixed to obtain a lipid oil phase.

[0124] 1 OD of siRNA was dissolved in 1 mL of sodium acetate solution at pH = 4.0 and mixed evenly to prepare a nucleic acid aqueous phase.

[0125] 1 mL of lipid oil phase and 3 mL of nucleic acid aqueous phase were mixed at room temperature by a split syringe pump to prepare a drug intermediate sample.

[0126] The intermediate sample is ultrafiltered through an ultrafiltration device to remove the solvent, thereby obtaining the cationic lipid gene drug preparation.

[0127] Effect evaluation 1

[0128] The pharmaceutical preparation prepared in Example 3 was subjected to performance tests, specifically including particle size detection, potential detection and dispersion degree detection.

[0129] Particle size, potential and dispersion detection

[0130] The particle size potential and dispersion degree (PDI) of nanoparticles were determined using a laser scattering particle size analyzer. A 671nm solid laser was used as the incident light. The dynamic light scattering test was carried out at 25°C, and the scattering light path was detected by 173° backscattering. The average value of three consecutive tests was used as the test data.

[0131] The test results of particle size, potential and dispersion degree are shown in Table 2.

[0132] Table 2 Test results of particle size, potential and dispersion of gene drug preparations

[0133] drug N / P Particle size Potential PDI Gene medicine 6 152.7±0.4359nm -1.73±0.312mV 0.17±0.031

[0134] It can be seen from Table 2 that the gene drug preparation prepared according to the formula and method of Example 3 has a suitable particle size and uniform particle size distribution, and also has a high potential, which creates conditions for subsequent practical applications.

[0135] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A cationic lipid compound, characterized in that The cationic lipid compound structure is as follows: Wherein, R1 is selected from a heterocyclic, alkane or olefin group; said R1 is a substituted or unsubstituted group; The structure of R1 is selected from the following: Wherein, R2 is selected from a linear or branched alkane or olefin group; said R2 is a substituted or unsubstituted group; The structure of R2 is selected from the following: Wherein, n1, n2, n3, n4, and n5 are independently selected from any integer between 1 and 10; Wherein, R3 is selected from a linear or branched alkane or olefin group; said R3 is a substituted or unsubstituted group; The structure of R3 is selected from the following: Wherein, n6 to n18 are independently selected from any integer between 1 and 10.

2. The cationic lipid compound according to claim 1, characterized in that The R1 is selected from R2 is selected from R3 is selected from The cationic lipid compound structure is as follows:

3. A method for preparing the cationic lipid compound according to claim 1 or 2, characterized in that: The steps include: S11: dissolving oleic acid in dichloromethane, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine and N,N-diisopropylethylamine, and mixing for 8-12 minutes; S12: Add 1,4-bis(2-hydroxyethyl)piperazine to the mixture obtained in step S11, and stir at room temperature for 20-28 hours to obtain the cationic lipid compound.

4. The preparation method according to claim 3, characterized in that: The molar ratio of oleic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, N,N-diisopropylethylamine and 1,4-bis(2-hydroxyethyl)piperazine is 1:1:0.1:1:0.

5.

5. A lipid nanoparticle LNP carrier, characterized in that: The lipid nanoparticle LNP carrier contains the cationic lipid compound according to claim 1 or 2.

6. The lipid nanoparticle LNP carrier according to claim 5, characterized in that, The lipid nanoparticle LNP carrier contains 15-70% of the cationic lipid compound in terms of molar fraction.

7. The lipid nanoparticle LNP carrier according to claim 6, characterized in that, The lipid nanoparticle LNP carrier further comprises 10% phospholipids, 38.5% cholesterol and 1.5% polyethylene glycol lipids by molar fraction.

8. A gene medicine preparation, characterized in that: The gene medicine preparation comprises the lipid nanoparticle LNP carrier according to any one of claims 5-7.

9. The gene medicine preparation according to claim 8, characterized in that: The gene medicine preparation also includes a nucleic acid drug; the mass ratio of the nitrogen content of the cationic lipid compound in the lipid nanoparticle LNP carrier to the phosphorus content in the nucleic acid drug is 2-12:

1.

10. The gene medicine preparation according to claim 8 or 9, characterized in that: The preparation method of the gene medicine preparation comprises the following steps: S21: preparing lipid oil phase and nucleic acid aqueous phase respectively; The lipid oil phase is obtained by mixing and dissolving a cationic lipid compound, phospholipids, cholesterol and polyethylene glycol lipids; The nucleic acid aqueous phase is obtained by diluting the nucleic acid drug with water; S22: The lipid oil phase and the nucleic acid aqueous phase are mixed at a certain flow rate through a split injection pump to obtain the gene drug preparation.

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