Ionizable cationic lipids, methods of making and using the same

Abstract

The application provides an ionizable cationic lipid and a preparation method and application thereof, and relates to the technical field of biological medicines.A new structure of ionizable cationic lipid is disclosed, and examples of the ionizable cationic lipid are as follows.Compared with the current most efficient lipid nanoparticle, DLin-MC3-DMA, the lipid nanoparticle disclosed by the application is not only simpler in the preparation process, but also higher in the delivery efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an ionizable cationic lipid, its preparation method, and its application. Background Technology

[0002] Nucleic acid drugs, including DNA drugs, mRNA drugs, and small nucleic acid drugs, mainly face challenges such as how to efficiently enter target cells, release them into the cytoplasm, and exert their effects efficiently and for a long time. High efficiency, safety, precise targeting, and improved stability are key to technological breakthroughs. Currently, nucleic acid delivery vectors in vivo are mainly divided into viral vectors and non-viral vectors. Viral vectors have very high delivery efficiency and can ensure long-term gene expression, but their application is limited by drawbacks such as complex preparation processes, high cost, susceptibility to immune responses, biosafety concerns, and limitations on the size of target gene fragments. Compared to viral vectors, non-viral lipid-based delivery systems (such as lipid nanoparticles, LNPs) are gradually becoming key tools for nucleic acid vaccines and treatments due to their advantages in safety, tolerability, repeat dosing capability, and ability to carry large amounts of gene cargo.

[0003] LNPs typically consist of four components: ionizable cationic lipids, phospholipids, cholesterol, and PEGylated lipids. Each component plays a crucial role in the stability, transfection efficiency, and safety of the LNP. 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 1:3 volume ratio to complete the self-assembly process. During formation, the ionizable cationic lipids are protonated and become positively charged, then bind to the negatively charged mRNA through electrostatic interactions, thereby encapsulating the mRNA within the LNP. Simultaneously, other auxiliary lipids, including phospholipids, cholesterol, and PEGylated lipids, self-assemble on top of these components to stabilize the formed mRNA-LNP. Subsequently, the mRNA-LNP solution is adjusted to a neutral pH by buffer replacement. During this process, the ionizable lipids become uncharged, making it stable at physiological pH and less toxic. LNPs offer advantages such as well-defined component structures, good reproducibility, ease of quality control, long in vivo circulation time, and good biocompatibility. After entering the cell, nanoparticles must escape from endosomes / lysosomes to release RNA in the cytoplasm, enabling it to be expressed and generate the target protein. Therefore, endosome / lysosome escape is a key step affecting nucleic acid delivery.

[0004] Chinese patent CN118388370A discloses a quaternary ammonium salt-type cationic lipid analog, its composition, and its application. It describes obtaining a class of quaternary ammonium salt-type cationic lipid analogs through chemical modification or alteration of ionizable cationic lipid analogs, and using these analogs to replace the auxiliary phospholipid components in traditional four-component lipid nanoparticles to construct a novel LNP delivery system for targeted mRNA delivery to organs / tissues. However, this patent only studies the performance of the novel LNP delivery system for targeted mRNA delivery to organs / tissues, and does not focus on the endosome / lysosome escape rate of the novel LNP delivery system.

[0005] DLin-MC3-DMA (1,2-dilinoleyloxy-3-dimethylaminopropane) is a synthetic cationic lipid widely studied for gene therapy and mRNA vaccines due to its highly efficient nucleic acid delivery capabilities. DLin-MC3-DMA possesses 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, resulting in liposomes with a very low positive charge density in the blood, i.e., very low cytotoxicity. Although the positive charge of DLin-MC3-DMA cationic liposomes can increase lysosomal escape of particles in vivo, only 1%–4% of the RNA escapes from the endosomes / lysosomes.

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

[0007] The purpose of this invention is to overcome the shortcomings of previous research and provide an ionizable cationic lipid, its preparation method, and its applications. The LNP delivery system composed of the ionizable cationic lipid prepared by this invention possesses both excellent nucleic acid loading capacity and high endosome / lysosome escape capability. Furthermore, the preparation method is simple, making it of significant research value and meeting practical needs.

[0008] To achieve the above objectives, 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 formulas (1), (2), (3), (4), and (5).

[0010]

[0011]

[0012] In equation (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 equation (2),

[0016] c = 1 - 6,

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

[0018] In equation (3),

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

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

[0021] In equation (4),

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

[0023] In equation (5),

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

[0025] Preferably,

[0026] In the above 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 above formula (2),

[0030] c = 1 - 5,

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

[0032] In the above formula (3),

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

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

[0035] In the above formula (4),

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

[0037] In the above formula (5),

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

[0039] More preferably,

[0040] In the above formula (1),

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

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

[0043] In the above formula (2),

[0044] c = 1 - 4,

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

[0046] In the above formula (3),

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

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

[0049] In the above formula (4),

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

[0051] In the above formula (5),

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

[0053] More preferably,

[0054] In the above 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 above formula (2),

[0061] c = 1 - 3,

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

[0063] -CH2CH2COOCH2CH(CH2CH3)CH2CH2CH2CH3,

[0064] -CH2CH2COOCH2CH2CH2CH2CH2CH(CH3)2,

[0065] -CH2CH2COOCH2CH2CH2CH2CH2CH2CH(CH3)2;

[0066] In the above 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 above formula (4),

[0072] R 13 Selected from -CH2CH2COOCH2CH2CH2CH2CH2CH(CH3)2,

[0073] -CH2CH2COOCH2CH2CH2CH2CH2CH2CH(CH3)2;

[0074] In the above 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 formulas (6), (7), (8), (9), (10), (11), (12), and (13).

[0079]

[0080]

[0081] More preferably, the structure of the ionizable cationic lipid is selected from any one of formulas (6), (7), (12), and (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] Ultimately, preferably, the structure of the ionizable cationic lipid is as shown in 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 them at 70-90°C;

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

[0086]

[0087]

[0088] In equation (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 and (C1-C6) alkyl groups;

[0091] In equation (15),

[0092] j = 1-6,

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

[0094] In equation (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 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 includes a purification step, which is as follows: the product is purified by chromatography, and the eluent is a solution of dichloromethane, methanol and ammonia.

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

[0101] More preferably, the ammonia water contains 27% ammonia.

[0102] Furthermore, the present invention provides a lipid nanoparticle comprising the above-mentioned ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids.

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

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

[0105] Preferably, the volume ratio of the ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids is 10:5:10:3.

[0106] Finally, the present invention provides the use of the above-mentioned ionizable cationic lipids or lipid nanoparticles as targeted mRNA delivery carriers.

[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 this invention is simpler and the cost is lower.

[0109] (2) Compared with the most efficient lipid nanoparticles currently available, DLin-MC3-DMA, the lipid nanoparticles disclosed in this invention have higher delivery efficiency. The delivery efficiency of the lipid nanoparticles with the optimal structure is 8.2 times that of DLin-MC3-DMA. Detailed Implementation

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

[0111] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all pharmaceuticals used in the embodiments of the present invention are obtained through conventional commercial channels.

[0112] Raw materials of Examples 1-48

[0113] Table 1

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122] Preparation methods of Examples 1-48

[0123] Table 2

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] Example 49

[0130] The reaction temperature in Example 1 was changed from 80°C to 70°C, and the rest of the preparation process was the same as in 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 (containing 27% ammonia) in a mass ratio of 75:22:3 as the eluent. The desired fraction was collected, and the final product obtained was the one described in Example 49.

[0132] Example 50

[0133] The reaction temperature in Example 1 was changed from 80°C to 90°C, and the rest of the preparation process was the same as in 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 (containing 27% ammonia) in a mass ratio of 75:22:3 as the eluent. The desired fraction was collected, and the final product obtained was the product of Example 50.

[0135] Example 51

[0136] Preparation of mRNA / LNP:

[0137] (1) Prepare a 300 μL 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 mRNA in 10 mM citrate buffer (pH 3.0) to prepare a 900 μL mRNA solution;

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

[0140] (4) Sample concentration: The collected sample was concentrated in a 100 kDa concentrated acid tube, centrifuged at 3000 g for 10 min, and then PBS with 2 times the volume of the remaining solution was added and centrifuged again. The concentrated solution was collected and kept for later use.

[0141] Example 52

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

[0143] Example 53

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

[0145] Example 54

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

[0147] Example 55

[0148] The 30 μL of ionizable cationic lipid (prepared in Example 4) in Example 51 was replaced with 30 μL of 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] Example 56

[0150] The 30 μL of ionizable cationic lipid (prepared in Example 4) in Example 51 was replaced with 30 μL of 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] Example 57

[0152] The 30 μL of ionizable cationic lipid (prepared in Example 4) in Example 51 was replaced with 30 μL of 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] Example 58

[0154] The 30 μL of ionizable cationic lipid (prepared in Example 4) in Example 51 was replaced with 30 μL of 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] Result detection

[0156] Cell experiments

[0157] 1. HeLa cell transfection

[0158] 100 μL of freshly resuspended HeLa cell solution (1×10⁻⁶) 4 Cells were passaged into 96-well plates and incubated overnight. The next day, the mRNA / LNP prepared in Examples 51-58 was diluted in fresh culture medium (1.5 mL EP tubes) to a concentration of 0.2 μg / mL. The old culture medium was discarded, and 100 μL of fresh culture medium was added for 24 h. After incubation, the cell viability was initially observed under a microscope. The culture medium was discarded, and the cells were detected using a firefly luciferase reporter gene assay kit. DLin-MC3-DMA was used as a positive control for comparison.

[0159] 2. Fluorescence detection

[0160] (1) Cell lysis: Add 100 μL of reporter gene cell lysis buffer to each well. After complete lysis, centrifuge at 10,000-15,000 × g for 3-5 min, and collect the supernatant for assay.

[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 operation manual, 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, mix well by shaking the plate for 60 s with an ELISA reader, and then measure the fluorescence intensity.

[0164] Technical effect testing

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

[0166] Table 3

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176] 2. Cellular Experiment Results

[0177] Cell experiments were conducted on Examples 51-58, and the fluorescence intensity values ​​of each example were statistically analyzed, as shown in Table 4.

[0178] Table 4

[0179] Example number fluorescence intensity Example 51 27518 Example 52 48912 Example 53 40000 Example 54 160579 Example 55 185750 Example 56 24238 Example 57 24238 Example 58 29150 Control (DLin-MC3-DMA) 22514

[0180] As shown in Table 4, the fluorescence intensity of Examples 51-58 was higher than that of DLin-MC3-DMA. Example 52 had the highest fluorescence intensity, which was 8.2 times that of DLin-MC3-DMA, while Example 56 had the lowest, which was 1.07 times that of DLin-MC3-DMA. These results demonstrate that the ionizable cationic lipids (48 compounds in total) disclosed in this invention are highly efficient mRNA delivery vectors.

[0181] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart 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 cationic lipid is shown as formula (6):

2. A process for the preparation of an ionizable cationic lipid as claimed in claim 1, characterized in that, The preparation method comprises the following steps: mixing raw material 1 and raw material 2 shown in the following structure, and then reacting at 70-90 DEG C:

3. The production method according to claim 2, characterized by, The preparation method is: in a 50 mL round bottom flask, 1 mmol of raw material 1 and 5 mmol of raw material 2 are added, and the reaction is carried out at 80 DEG C for 48 h to obtain a crude product; then the product is purified by column chromatography, and the eluent is a solution mixed by dichloromethane, methanol and ammonia water containing 27% of ammonia in a mass ratio of 75:22:3, and the required fraction is collected to obtain the product.

4. A lipid nanoparticle characterized in that, The lipid nanoparticle comprises the ionizable cationic lipid, a phospholipid, cholesterol and a pegylated lipid.

5. The lipid nanoparticle of claim 4, wherein, The phospholipid is phosphatidylcholine or dioleoyl phosphatidyl ethanolamine; The pegylated lipid is dimyristyl glycerol-polyethylene glycol.

6. The lipid nanoparticle of claim 5, wherein, The volume ratio of the ionizable cationic lipid, the phospholipid, the cholesterol and the pegylated lipid is 10:5:10:

3.

7. Use of the ionizable cationic lipid of claim 1 or the lipid nanoparticle of any one of claims 4-6 in the preparation of a targeted mRNA delivery carrier.

Images