Ionizable cationic lipid as well as preparation method and application thereof

Abstract

The invention provides ionizable cationic lipid as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The invention discloses an ionizable cationic lipid with a novel structure. Examples of the ionizable cationic lipid are as follows. Compared with the most efficient lipid nanoparticles DLin-MC3-DMA at present, the lipid nanoparticles disclosed by the invention not only are simpler in preparation process, but also are higher in delivery efficiency. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to an ionizable cationic lipid and a preparation method and application thereof. Background Art

[0002] Nucleic acid drugs include DNA drugs, mRNA drugs and small nucleic acid drugs. They mainly face the problems of how to efficiently enter target cells, release into the cytoplasm, and exert their effects efficiently and for a long time. Efficient and safe, precise targeting and improved stability are the key to technological breakthroughs. At present, nucleic acid in vivo delivery vectors are mainly divided into two types: viral vectors and non-viral vectors. Viral vectors have very high delivery efficiency and can ensure long-term gene expression, but their complex preparation process, high cost, easy to cause immune response in the body, involving biosafety issues, and limited size of target gene fragments limit the application of viral vectors. Compared with viral vectors, non-viral lipid-based delivery systems (such as lipid nanoparticles, LNP) have gradually become key tools for nucleic acid vaccines and treatments due to their advantages in safety, tolerance, repeated administration ability and large-scale gene cargo carrying capacity.

[0003] LNPs are usually composed of four components: ionizable cationic lipids, phospholipids, cholesterol, and PEGylated lipids. Each component plays a crucial role in the stability, transfection effect, and safety of LNPs. For the formation of mRNA-LNPs, different lipids and mRNA are usually dissolved in ethanol and acidic aqueous phases, respectively. Then, the ethanol and aqueous phases are mixed with a microfluidic device at a volume ratio of 1:3 to complete the self-assembly process. During the formation process, the ionizable cationic lipids are protonated and positively charged, and then bind to the negatively charged mRNA through electrostatic interactions, thereby encapsulating the mRNA in the LNP. At the same time, other auxiliary lipids, including phospholipids, cholesterol, and PEGylated lipids, self-assemble on them to stabilize the formed mRNA-LNPs. Subsequently, the mRNA-LNP solution is adjusted to a neutral pH by buffer replacement, during which the ionizable lipids become uncharged, making them stable and less toxic at physiological pH. LNPs have the advantages of definite structures of each component, good reproducibility, and good quality control, as well as long in vivo circulation time and good biocompatibility. After entering the cell, nanoparticles need to escape from the endosome / lysosome to release RNA in the cytoplasm so that it can be expressed to produce the target protein. Therefore, endosomal / lysosomal escape is a key step affecting nucleic acid delivery.

[0004] Chinese patent CN118388370A discloses a quaternary ammonium salt cationic lipid analog, its composition and application, by chemically modifying or modifying an ionizable cationic lipid analog to obtain a class of quaternary ammonium salt cationic lipid analogs, and using quaternary ammonium salt cationic lipid analogs to replace the auxiliary phospholipid component in the traditional four-component lipid nanoparticles to form a new LNP delivery system for organ / tissue targeted mRNA delivery. However, this patent only studies the performance of the new LNP delivery system for organ / tissue targeted mRNA delivery, and does not focus on the endosomal / lysosomal escape rate of the new LNP delivery system.

[0005] DLin-MC3-DMA (1,2dilinoleyloxy3dimethylaminopropane) is a synthetic cationic lipid that has been widely studied for gene therapy and mRNA vaccines due to its efficient nucleic acid delivery ability. DLin-MC3-DMA has a unique pH-dependent charge variable property: it is positively charged under acidic conditions and electrically neutral under physiological pH conditions. Therefore, nucleic acids are encapsulated under acidic conditions, and the formed liposomes have a very small positive charge density in the blood, that is, very low cytotoxicity. Although DLin-MC3-DMA cationic liposomes can increase the lysosomal escape of particles in the body due to their positive charge, only 1%-4% of RNA escapes the endosomes / lysosomes.

[0006] In view of this, there is an urgent need for an LNP delivery system that has both good nucleic acid encapsulation capacity and high endosomal / lysosomal escape ability. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies in previous studies and provide an ionizable cationic lipid and a preparation method and application thereof. The LNP delivery system composed of the ionizable cationic lipid prepared by the present invention has both good nucleic acid encapsulation capacity and high endosomal / lysosomal escape capacity, and the preparation method is simple, which has great research significance and practical needs.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] First, the present invention provides an ionizable cationic lipid, wherein the structure of the ionizable cationic lipid is selected from any one of formula (1), formula (2), formula (3), formula (4), and formula (5),

[0010]

[0011]

[0012] In formula (1),

[0013] a=1-5,b=1-6,

[0014] R1, R2, R3, R4, and R5 are each independently selected from hydrogen, (C1-C6)alkyl, (C1-C6)alkyl-COO-(C5-C 12 )alkyl;

[0015] In formula (2),

[0016] c = 1-6,

[0017] R6, R7, R8, R9, R 10 , R 11 Each independently selected from (C1-C6)alkyl, (C1-C6)alkyl-COO-(C5-C 12 )alkyl;

[0018] In formula (3),

[0019] d=1-5, e=1-6, f=1-6,

[0020] R 12 is (C1-C6)alkyl-COO-(C5-C 12 )alkyl;

[0021] In formula (4),

[0022] R 13 is (C1-C6)alkyl-COO-(C5-C 12 )alkyl;

[0023] In formula (5),

[0024] R 14 is (C1-C6)alkyl-COO-(C5-C 12 )alkyl.

[0025] Preferably,

[0026] In the formula (1),

[0027] a=2-4,b=2-5,

[0028] R1, R2, R3, R4, and R5 are each independently selected from hydrogen, (C1-C4)alkyl, (C2-C4)alkyl-COO-(C7-C 10 )alkyl;

[0029] In the formula (2),

[0030] c = 1-5,

[0031] R6, R7, R8, R9, R 10 , R 11Each independently selected from (C1-C4)alkyl, (C2-C4)alkyl-COO-(C7-C 10 )alkyl;

[0032] In the formula (3),

[0033] d=2-4, e=2-5, f=2-5,

[0034] R 12 is (C2-C4)alkyl-COO-(C7-C 10 )alkyl;

[0035] In the formula (4),

[0036] R 13 is (C2-C4)alkyl-COO-(C7-C 10 )alkyl;

[0037] In the formula (5),

[0038] R 14 is (C2-C4)alkyl-COO-(C7-C 10 )alkyl.

[0039] More preferably,

[0040] In the formula (1),

[0041] a=2-3,b=2-4,

[0042] R1, R2, R3, R4, R5 are each independently selected from hydrogen, (C1-C2) alkyl, (C2-C3) alkyl-COO-(C8-C9) alkyl;

[0043] In the formula (2),

[0044] c=1-4,

[0045] R6, R7, R8, R9, R 10 , R 11 Each independently selected from (C1-C2)alkyl, (C2-C3)alkyl-COO-(C8-C9)alkyl;

[0046] In the formula (3),

[0047] d=2-3, e=2-4, f=2-4,

[0048] R 12 is (C2-C3)alkyl-COO-(C8-C9)alkyl;

[0049] In the formula (4),

[0050] R13 is (C2-C3)alkyl-COO-(C8-C9)alkyl;

[0051] In the formula (5),

[0052] R 14 It is (C2-C3)alkyl-COO-(C8-C9)alkyl.

[0053] More preferably,

[0054] In the formula (1),

[0055] a=2-3,b=2-4,

[0056] R1, R2, R3, R4, and R5 are each independently selected from -H, -CH3,

[0057] -CH2CH2COOCH2CH(CH2CH3)CH2CH2CH2CH3,

[0058] -CH2CH2COOCH2CH2CH2CH2CH2CH(CH3)2,

[0059] -CH2CH2COOCH2CH2CH2CH2CH2CH2CH(CH3)2;

[0060] In the formula (2),

[0061] c = 1-3,

[0062] R6, R7, R8, R9, R 10 , R 11 Each independently selected from -CH3,

[0063] -CH2CH2COOCH2CH(CH2CH3)CH2CH2CH2CH3,

[0064] -CH2CH2COOCH2CH2CH2CH2CH2CH(CH3)2,

[0065] -CH2CH2COOCH2CH2CH2CH2CH2CH2CH(CH3)2;

[0066] In the formula (3),

[0067] d=2-3, e=2-4, f=2-4,

[0068] R 12 Selected from -CH2CH2COOCH2CH(CH2CH3)CH2CH2CH2CH3,

[0069] -CH2CH2COOCH2CH2CH2CH2CH2CH(CH3)2,

[0070] -CH2CH2COOCH2CH2CH2CH2CH2CH2CH(CH3)2;

[0071] In the formula (4),

[0072] R 13 Selected from -CH2CH2COOCH2CH2CH2CH2CH2CH2CH(CH3)2,

[0073] -CH2CH2COOCH2CH2CH2CH2CH2CH2CH(CH3)2;

[0074] In the formula (5),

[0075] R 14 Selected from -CH2CH2COOCH2CH(CH2CH3)CH2CH2CH2CH3,

[0076] -CH2CH2COOCH2CH2CH2CH2CH2CH(CH3)2,

[0077] -CH2CH2COOCH2CH2CH2CH2CH2CH2CH(CH3)2.

[0078] More preferably, the structure of the ionizable cationic lipid is selected from any one of formula (6), formula (7), formula (8), formula (9), formula (10), formula (11), formula (12), and formula (13),

[0079]

[0080]

[0081] More preferably, the structure of the ionizable cationic lipid is selected from any one of formula (6), formula (7), formula (12) and formula (13).

[0082] More preferably, the structure of the ionizable cationic lipid is selected from any one of formula (6), formula (7) and formula (12).

[0083] Finally, preferably, the structure of the ionizable cationic lipid is formula (7).

[0084] Secondly, the present invention provides a method for preparing the above-mentioned ionizable cationic lipid, comprising the following steps: mixing raw material 1 and raw material 2, and then reacting at 70-90° C.;

[0085] The structure of the raw material 1 is selected from any one of formula (14), formula (15), formula (16), formula (17), and formula (18),

[0086]

[0087]

[0088] In formula (14),

[0089] g = 1-5, h = 1-6,

[0090] R 15 , R 16 , R 17 , R 18 , R 19 Each is independently selected from hydrogen, (C1-C6) alkyl;

[0091] In formula (15),

[0092] j = 1-6,

[0093] R 20 , R 21 , R 22 , R 23 , R 24 , R 25 Each is independently selected from hydrogen, (C1-C6) alkyl;

[0094] In formula (16),

[0095] k=1-5, m=1-6, n=1-6;

[0096] The raw material 2 is (C5-C 12 )alkyl-COO-(C1-C6)olefin.

[0097] Preferably, the raw material 1 and the raw material 2 are reacted at 80°C.

[0098] Preferably, the molar ratio of raw material 1 to raw material 2 is 1:5-9.

[0099] Preferably, the preparation method further comprises a purification step, which is as follows: purifying the product by chromatography, wherein the eluent is a mixed solution of dichloromethane, methanol and aqueous ammonia.

[0100] Further preferably, the mass ratio of dichloromethane, methanol and aqueous ammonia is 75:22:3.

[0101] More preferably, the ammonia content of the ammonia water is 27%.

[0102] Furthermore, the present invention provides a lipid nanoparticle, which comprises the above-mentioned ionizable cationic lipid, phospholipid, cholesterol and pegylated lipid.

[0103] Preferably, the phospholipid is diphosphocholine (DSPC) or dioleoylphosphatidylethanolamine (DOPE).

[0104] Preferably, the PEGylated lipid is dimyristoylglycerol-polyethylene glycol (DMG-PEG).

[0105] Preferably, the volume ratio of the ionizable cationic lipid, phospholipid, cholesterol and pegylated lipid is 10:5:10:3.

[0106] Finally, the present invention provides the use of the above-mentioned ionizable cationic lipid or the above-mentioned lipid nanoparticle as a targeted mRNA delivery carrier.

[0107] Compared with the prior art, the present invention has the following beneficial effects:

[0108] (1) Compared with the most efficient lipid nanoparticles currently available, DLin-MC3-DMA, the preparation process of the lipid nanoparticles disclosed in the present invention is simpler and has lower cost.

[0109] (2) Compared with the most efficient lipid nanoparticle currently available, DLin-MC3-DMA, the lipid nanoparticles disclosed in the present invention have a higher delivery efficiency, and the delivery efficiency of the lipid nanoparticles with the best structure is 8.2 times that of DLin-MC3-DMA. DETAILED DESCRIPTION

[0110] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0111] The present invention is further described below by way of specific examples. Unless otherwise specified, the various drugs used in the examples of the present invention are obtained through conventional commercial channels.

[0112] Raw materials of Example 1 to Example 48

[0113] Table 1

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122] Preparation methods of Examples 1 to 48

[0123] Table 2

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] Embodiment 49

[0130] The reaction temperature of Example 1 was replaced from 80° C. to 70° C., and the rest of the preparation process was the same as that of Example 1.

[0131] In a 50 mL round-bottom flask, raw material 1 (145.25 mg, 1 mmol) and raw material 2 (921.4 mg, 5 mmol) were added and reacted at 70°C for 48 h to obtain a crude product. The product was then purified by column chromatography, using a solution of dichloromethane, methanol and ammonia water (ammonia content of 27%) in a mass ratio of 75:22:3 as the eluent, and the desired fractions were collected to obtain the final product of Example 49.

[0132] Embodiment 50

[0133] The reaction temperature of Example 1 was replaced from 80° C. to 90° C., and the rest of the preparation process was the same as that of Example 1.

[0134] In a 50 mL round-bottom flask, raw material 1 (145.25 mg, 1 mmol) and raw material 2 (921.4 mg, 5 mmol) were added and reacted at 90°C for 48 h to obtain a crude product. The product was then purified by column chromatography, using a solution of dichloromethane, methanol and ammonia water (ammonia content of 27%) in a mass ratio of 75:22:3 as the eluent, and the desired fractions were collected to obtain the final product of Example 50.

[0135] Embodiment 51

[0136] Preparation of mRNA / LNP:

[0137] (1) Prepare 300 μL of solution in a 1.5 mL EP tube: first add 216 μL of ethanol, then add 9 μL of DMG-PEG, 30 μL of cholesterol, 15 μL of DSPC and 30 μL of ionizable cationic lipid (prepared in Example 4) in a volume ratio of (3:10:5:10).

[0138] (2) In another 1.5 mL EP, dissolve 60 μg of mRNA in 10 mM citrate buffer (pH 3.0) to prepare 900 μL of mRNA solution;

[0139] (3) The above two solutions were aspirated into a threaded syringe, synthesized at a rate of 12 mL / min using microfluidics, and the samples were collected.

[0140] (4) Concentrated samples: The collected samples were concentrated in a 100Kd concentrated acid tube, centrifuged at 3000 g for 10 min, and then PBS with a volume twice that of the remaining solution was added and centrifuged again to collect the concentrated solution for later use.

[0141] Embodiment 52

[0142] The 30 μL ionizable cationic lipid (prepared in Example 4) in Example 51 was replaced with 30 μL ionizable cationic lipid (prepared in Example 7), and the rest of the raw materials and processes were the same as in Example 51.

[0143] Embodiment 53

[0144] The 30 μL ionizable cationic lipid (prepared in Example 4) in Example 51 was replaced with 30 μL ionizable cationic lipid (prepared in Example 10), and the rest of the raw materials and processes were the same as in Example 51.

[0145] Embodiment 54

[0146] The 30 μL ionizable cationic lipid (prepared in Example 4) in Example 51 was replaced with 30 μL ionizable cationic lipid (prepared in Example 11), and the rest of the raw materials and processes were the same as in Example 51.

[0147] Embodiment 55

[0148] The 30 μL ionizable cationic lipid (prepared in Example 4) in Example 51 was replaced with 30 μL ionizable cationic lipid (prepared in Example 13), DSPC was replaced with DOPE, and the remaining raw materials and processes were the same as in Example 51.

[0149] Embodiment 56

[0150] The 30 μL ionizable cationic lipid (prepared in Example 4) in Example 51 was replaced with 30 μL ionizable cationic lipid (prepared in Example 19), DSPC was replaced with DOPE, and the remaining raw materials and processes were the same as in Example 51.

[0151] Embodiment 57

[0152] The 30 μL ionizable cationic lipid (prepared in Example 4) in Example 51 was replaced with 30 μL ionizable cationic lipid (prepared in Example 20), DSPC was replaced with DOPE, and the remaining raw materials and processes were the same as in Example 51.

[0153] Embodiment 58

[0154] The 30 μL ionizable cationic lipid (prepared in Example 4) in Example 51 was replaced with 30 μL ionizable cationic lipid (prepared in Example 37), DSPC was replaced with DOPE, and the remaining raw materials and processes were the same as in Example 51.

[0155] Results

[0156] Cell experiments

[0157] 1. Hela cell transfection

[0158] 100 μL of freshly resuspended HeLa cell solution (1×10 4 The mRNA / LNP prepared by Example 51-58 was diluted into fresh culture medium (1.5mL EP tube) and made into 0.2ug / mL the next day. The old culture medium was abandoned and 100uL fresh culture medium was added to process 24h. After incubation, the cell death state was observed under the microscope, culture fluid was abandoned, and firefly luciferase reporter gene detection kit was used to detect. DLin-MC3-DMA was used as a positive control for comparison.

[0159] 2. Fluorescence detection

[0160] (1) Lyse cells: Add 100 μL of reporter gene cell lysis buffer to each well. After sufficient lysis, centrifuge at 10,000-15,000 × g for 3-5 min and take the supernatant for determination;

[0161] (2) Dissolve the firefly luciferase detection reagent and bring it to room temperature;

[0162] (3) Turn on the chemiluminescence analyzer or multifunctional microplate reader with chemiluminescence detection function according to the instrument operating instructions, and set the interval time and measurement time according to the requirements of each instrument;

[0163] (4) Add 100 μL of sample and 100 μL of firefly luciferase detection reagent to each well, shake the plate for 60 seconds to mix, and then measure the fluorescence intensity.

[0164] Technical effect testing

[0165] 1. NMR results of the products of Examples 1 to 50

[0166] Table 3

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176] 2. Cell experiment results

[0177] Examples 51-58 were subjected to cell experiments, and the fluorescence intensity values ​​of each example were statistically analyzed, as shown in Table 4:

[0178] Table 4

[0179] Example No. Fluorescence intensity Embodiment 51 27518 Embodiment 52 48912 Embodiment 53 40000 Embodiment 54 160579 Embodiment 55 185750 Embodiment 56 24238 Embodiment 57 24238 Embodiment 58 29150 Control (DLin-MC3-DMA) 22514

[0180] As shown in Table 4, the fluorescence intensity of Examples 51-58 is higher than that of DLin-MC3-DMA, among which Example 52 with the highest fluorescence intensity is 8.2 times that of DLin-MC3-DMA, and Example 56 with the lowest fluorescence intensity is 1.07 times that of DLin-MC3-DMA. The above results show that the ionizable cationic lipid (48 compounds in total) disclosed in the present invention is an efficient mRNA delivery carrier.

[0181] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. An ionizable cationic lipid, characterized in that The structure of the ionizable cationic lipid is selected from any one of formula (1), formula (2), formula (3), formula (4), and formula (5), In formula (1), a＝1-5， b＝1-6， R1, R2, R3, R4, and R5 are each independently selected from hydrogen, (C1-C6)alkyl, (C1-C6)alkyl-COO-(C5-C 12 )alkyl; In formula (2), c＝1-6， R6, R7, R8, R9, R 10 , R 11 Each independently selected from (C1-C6)alkyl, (C1-C6)alkyl-COO-(C5-C 12 )alkyl; In formula (3), d＝1-5， e＝1-6， f＝1-6， R 12 is (C1-C6)alkyl-COO-(C5-C 12 )alkyl; In formula (4), R 13 is (C1-C6)alkyl-COO-(C5-C 12 )alkyl; In formula (5), R 14 is (C1-C6)alkyl-COO-(C5-C 12 )alkyl.

2. The ionizable cationic lipid according to claim 1, characterized in that In the formula (1), a＝2-4， b＝2-5， R1, R2, R3, R4, and R5 are each independently selected from hydrogen, (C1-C4)alkyl, (C2-C4)alkyl-COO-(C7-C 10 )alkyl; In the formula (2), c＝1-5， R6, R7, R8, R9, R 10 , R 11 Each independently selected from (C1-C4)alkyl, (C2-C4)alkyl-COO-(C7-C 10 )alkyl; In the formula (3), d＝2-4， e＝2-5， f＝2-5， R 12 is (C2-C4)alkyl-COO-(C7-C 10 )alkyl; In the formula (4), R 13 is (C2-C4)alkyl-COO-(C7-C 10 )alkyl; In the formula (5), R 14 is (C2-C4)alkyl-COO-(C7-C 10 )alkyl.

3. The ionizable cationic lipid according to claim 2, characterized in that In the formula (1), a＝2-3， b＝2-4， R1, R2, R3, R4, R5 are each independently selected from hydrogen, (C1-C2) alkyl, (C2-C3) alkyl-COO-(C8-C9) alkyl; In the formula (2), c＝1-4， R6, R7, R8, R9, R 10 , R 11 Each independently selected from (C1-C2)alkyl, (C2-C3)alkyl-COO-(C8-C9)alkyl; In the formula (3), d＝2-3， e＝2-4， f＝2-4， R 12 is (C2-C3)alkyl-COO-(C8-C9)alkyl; In the formula (4), R 13 is (C2-C3)alkyl-COO-(C8-C9)alkyl; In the formula (5), R 14 It is (C2-C3)alkyl-COO-(C8-C9)alkyl.

4. The ionizable cationic lipid according to claim 3, characterized in that In the formula (1), a＝2-3， b＝2-4， R1, R2, R3, R4, and R5 are each independently selected from -H, -CH3, -CH2CH2COOCH2CH(CH2CH3)CH2CH2CH2CH3, -CH2CH2COOCH2CH2CH2CH2CH2CH(CH3)2, -CH2CH2COOCH2CH2CH2CH2CH2CH2CH(CH3)2; In the formula (2), c＝1-3， R6, R7, R8, R9, R 10 , R 11 Each is independently selected from -CH3, -CH2CH2COOCH2CH(CH2CH3)CH2CH2CH2CH3, -CH2CH2COOCH2CH2CH2CH2CH2CH(CH3)2, -CH2CH2COOCH2CH2CH2CH2CH2CH2CH(CH3)2; In the formula (3), d＝2-3， e＝2-4， f＝2-4， R 12 Choose -CH2CH2COOCH2CH(CH2CH3)CH2CH2CH2CH3, -CH2CH2COOCH2CH2CH2CH2CH2CH(CH3)2, -CH2CH2COOCH2CH2CH2CH2CH2CH2CH(CH3)2; In the formula (4), R 13 Select self -CH2CH2COOCH2CH2CH2CH2CH2CH(CH3)2, -CH2CH2COOCH2CH2CH2CH2CH2CH2CH(CH3)2; In the formula (5), R 14 Select -CH2CH2COOCH2CH(CH2CH3)CH2CH2CH2CH3, -CH2CH2COOCH2CH2CH2CH2CH2CH(CH3)2, -CH2CH2COOCH2CH2CH2CH2CH2CH2CH(CH3)2.

5. The ionizable cationic lipid according to claim 4, characterized in that The structure of the ionizable cationic lipid is selected from any one of formula (6), formula (7), formula (8), formula (9), formula (10), formula (11), formula (12), and formula (13), Preferably, the structure of the ionizable cationic lipid is selected from any one of formula (6), formula (7), formula (12), and formula (13); More preferably, the structure of the ionizable cationic lipid is selected from any one of formula (6), formula (7), and formula (12); Finally, preferably, the structure of the ionizable cationic lipid is formula (7).

6. The method for preparing the ionizable cationic lipid according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: mixing raw material 1 and raw material 2, and then reacting at 70-90° C.; The structure of the raw material 1 is selected from any one of formula (14), formula (15), formula (16), formula (17), and formula (18), In formula (14), g＝1-5， h＝1-6， R 15 , R 16 , R 17 , R 18 , R 19 Each is independently selected from hydrogen, (C1-C6) alkyl; In formula (15), j＝1-6， R 20 , R 21 , R 22 , R 23 , R 24 , R 25 Each is independently selected from hydrogen, (C1-C6) alkyl; In formula (16), k＝1-5， m＝1-6， n＝1-6； The raw material 2 is (C5-C 12 )alkyl-COO-(C1-C6)olefin.

7. The preparation method according to claim 6, characterized in that: The molar ratio of the raw material 1 to the raw material 2 is 1:5-9.

8. A lipid nanoparticle, characterized in that: The lipid nanoparticles include the ionizable cationic lipids, phospholipids, cholesterol and PEGylated lipids according to any one of claims 1 to 5.

9. The lipid nanoparticle according to claim 8, characterized in that The phospholipid is phosphorylcholine or dioleoylphosphatidylethanolamine; The PEGylated lipid is dimyristoylglycerol-polyethylene glycol; The volume ratio of the ionizable cationic lipid, phospholipid, cholesterol and PEGylated lipid is 10:5:10:

3.

10. Use of the ionizable cationic lipid according to any one of claims 1 to 5 or the lipid nanoparticle according to any one of claims 8 to 9 as a targeted mRNA delivery vector.

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