Ionizable cationic lipid, and preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of biomedicine. Provided are an ionizable cationic lipid, and a preparation method therefor and the use thereof. Disclosed in the present invention is an ionizable cationic lipid with a new structure. An example of the ionizable cationic lipid is as follows. Compared to DLin-MC3-DMA, which is currently the most efficient lipid nanoparticle, the lipid nanoparticle disclosed in the present invention not only has a simpler preparation process, but also exhibits a higher delivery efficiency.

Description

An ionizable cationic lipid, its preparation method and application

[0001] This application claims priority to the patent application filed on November 5, 2024, application number 202411569986.4. Technical Field

[0002] This invention belongs to the field of biomedical technology and relates to an ionizable cationic lipid, its preparation method, and its application. Background Technology

[0003] 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.

[0004] 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.

[0005] 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.

[0006] 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.

[0007] 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

[0008] 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 compounds disclosed in this invention exhibits excellent nucleic acid loading capacity, showing significant advantages in fluorescence intensity, delivery efficiency, and expression duration. Furthermore, the LNPs of this invention demonstrate high safety at different concentrations, suggesting significant potential clinical application value. In addition, the preparation method of the ionizable cationic lipid compounds of this invention is simple and cost-effective.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] First, the present invention provides an ionizable cationic lipid, wherein the structure of the ionizable cationic lipid is selected from any one of the compounds of formula (1), formula (2), formula (3), formula (4) and formula (5).

[0011] In formula (1), a = 1-5, b = 1-6, and R1, R2, R3, R4 or R5 are each independently selected from hydrogen, (C1-C6)alkyl, (C1-C6)alkyl-COO-(C5-C 12 )alkyl;

[0012] In equation (2), c = 1 - 6, R6, R7, R8, R9, R 10 Or R 11 Each is independently selected from (C1-C6)alkyl, (C1-C6)alkyl-COO-(C5-C 12 )alkyl;

[0013] In equation (3), d = 1-5, e = 1-6, f = 1-6, R 12 (C1-C6)alkyl-COO-(C5-C 12 )alkyl;

[0014] In equation (4), R 13 (C1-C6)alkyl-COO-(C5-C 12 )alkyl;

[0015] In equation (5), R 14 (C1-C6)alkyl-COO-(C5-C 12 )alkyl.

[0016] Preferably, in formula (1), a = 2-4, b = 2-5, and R1, R2, R3, R4 or R5 are each independently selected from hydrogen, (C1-C4)alkyl, (C2-C4)alkyl-COO-(C7-C 10 )alkyl.

[0017] More preferably, in formula (1), a = 2-3, b = 2-4, and R1, R2, R3, R4 or R5 are each independently selected from hydrogen, (C1-C2)alkyl, (C2-C3)alkyl-COO-(C8-C9)alkyl.

[0018] More preferably, in formula (1), a = 2, b = 2, R1, R2, R3 or R4 are each independently selected from (C2-C3)alkyl-COO-(C8-C9)alkyl, and R5 is selected from hydrogen and (C1-C2)alkyl.

[0019] More preferably, in equation (1), a = 2, b = 2, and R1, R2, R3, or R4 are each independently selected from...

[0020] -CH2CH2COOCH2CH(CH2CH3)CH2CH2CH2CH3,

[0021] -CH2CH2COOCH2CH2CH2CH2CH2CH2CH(CH3)2 and

[0022] -CH2CH2COOCH2CH2CH2CH2CH2CH2CH(CH3)2.

[0023] More preferably, the structure of the ionizable cationic lipid is selected from any one of the compounds of formula (6), formula (7), formula (8), formula (9), formula (10), formula (11), formula (12), formula (13), and formula (19).

[0024] More preferably, the structure of the ionizable cationic lipid is selected from any one of the compounds of formula (6), formula (12) and formula (19).

[0025] 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;

[0026] The structure of the raw material 1 is selected from any one of the compounds of formula (14), formula (15), formula (16), formula (17) and formula (18).

[0027] In equation (14),

[0028] g = 1 - 5, h = 1 - 6, R 15 R 16 R 17 R 18 Or R 19 Each is independently selected from hydrogen and (C1-C6) alkyl groups;

[0029] In equation (15),

[0030] j = 1-6, R 20 R 21 R 22 R 23 R 24 Or R 25 Each is independently selected from hydrogen and (C1-C6) alkyl groups;

[0031] In equation (16),

[0032] k=1-5, m=1-6, n=1-6;

[0033] The raw material 2 is (C5-C) 12 )alkyl-COO-(C1-C6)olefin.

[0034] Preferably, the raw material 1 and raw material 2 are reacted at 80°C.

[0035] Preferably, the molar ratio of raw material 1 to raw material 2 is 1:5-9.

[0036] 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.

[0037] More preferably, the mass ratio of dichloromethane, methanol, and ammonia is 75:22:3.

[0038] More preferably, the ammonia water contains 27% ammonia.

[0039] Furthermore, the present invention provides a lipid nanoparticle comprising the above-mentioned ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids.

[0040] Preferably, the phospholipid is phosphocholine (DSPC) or dioleoylphosphatidylethanolamine (DOPE).

[0041] Preferably, the PEGylated lipid is dimyristic glycerol-polyethylene glycol (DMG-PEG).

[0042] Preferably, the volume ratio of the ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids is 10:5:10:3.

[0043] Finally, the present invention provides the use of the above-mentioned ionizable cationic lipids or lipid nanoparticles as targeted mRNA delivery carriers.

[0044] Compared with the prior art, the present invention has the following beneficial technical effects:

[0045] (1) Compared with the most efficient lipid nanoparticles currently available, DLin-MC3-DMA, the lipid nanoparticles prepared by the lipid compounds disclosed in this invention have certain advantages in fluorescence intensity, delivery efficiency and expression duration. Furthermore, the LNPs of this invention exhibit high safety at different concentrations and are expected to have high potential clinical application value.

[0046] (2) 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. Detailed Implementation

[0047] 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.

[0048] 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.

[0049] Preparation Example: Preparation of Ionizable Cationic Lipids

[0050] The ionizable cationic lipids were prepared according to the preparation methods in Table 1 below.

[0051] Table 1: Preparation methods of each preparation example

[0052] The corresponding raw materials 1 and 2 used in the preparation of the above preparation examples are shown in Table 2 below.

[0053] Table 2: Corresponding raw materials for each preparation example

[0054] Preparation Example: Preparation of mRNA / LNP

[0055] Preparation Example 4: mRNA / LNP was prepared according to the following method:

[0056] (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 (the compound in formula (6) above) in a volume ratio of (3:10:5:10).

[0057] (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;

[0058] (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.

[0059] (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.

[0060] Preparation Example 5: mRNA / LNP was prepared according to the following method:

[0061] The ionizable cationic lipid in Preparation Example 4 was replaced with the compound of Formula (12) above, and the other raw materials and processes were the same as in Preparation Example 4.

[0062] Preparation Example 6: mRNA / LNP was prepared according to the following method:

[0063] The ionizable cationic lipid in Preparation Example 4 was replaced with compound of formula (19), and the other raw materials and processes were the same as in Preparation Example 4.

[0064] Comparative example (DLin-MC3-DMA): purchased from Xiamen Sinobond Biotechnology Co., Ltd. (06040008900). The lipid compound structure of DLin-MC3-DMA is shown in formula (20) (Reference: US8158601B2).

[0065] Test Implementation Example: Technical Effect Detection

[0066] Test Example 1: The NMR results of the lipid compounds obtained above are shown in Table 3 below.

[0067] Table 3: NMR results of lipid compounds from each preparation example

[0068] Test Example 2: Cell Experiment

[0069] 1. HeLa cell transfection

[0070] 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 5-8 was diluted in fresh culture medium (1.5 mL EP tube) 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.

[0071] 2. Fluorescence detection

[0072] (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.

[0073] (2) Dissolve the firefly luciferase detection reagent and bring it to room temperature;

[0074] (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.

[0075] (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. The results are shown in Table 4 below.

[0076] Table 4: Fluorescence detection results of each preparation example

[0077] As shown in Table 4, the fluorescence intensity of the representative compound of this invention is higher than that of DLin-MC3-DMA.

[0078] Test Example 3: Animal Experiment

[0079] Animal experiments were conducted using the mRNA / LNP obtained in the preparation examples 4-6 above to verify its delivery efficiency and expression duration:

[0080] Female Balb / c mice (6-8 weeks old) were used. A 0.5 mL insulin syringe, a tail vein injection fixator, cotton swabs, and other consumables were prepared. Six mice were used in each group. Three mice received a 100 μL (containing 2 μg Luc-mRNA) injection via tail vein (for delivery efficiency studies), and the other three received a 50 μL (containing 10 μg Luc-mRNA) injection via intramuscular injection (for expression duration studies). In vivo imaging preparation was performed 4 hours after intravenous injection, and intramuscular injections were performed at 0.25, 1, 3, 5, and 7 days prior. Before imaging, D-Luciferin sodium (3 mg / mouse, 200 μL PBS) was injected intraperitoneally into the mice. The mice were then anesthetized and imaging was performed 5 minutes later. The experimental results are shown in Tables 5-6 below.

[0081] Table 5: Chemiluminescence intensity of each preparation example and control example

[0082] Table 6: Chemiluminescence intensity of Preparation Example 4 over time

[0083] Animal experiments further validated the delivery efficiency of LNPs using the lipid compounds of this invention. As shown in Table 5 above, the chemiluminescence signal was significantly higher than that of the control MC3. In terms of expression duration, the longer the mRNA delivered by ionizable cationic lipids is expressed in animals, the longer it exerts its biological effect in vivo, which is more conducive to achieving a sustained immune effect in therapeutic vaccines. Table 6 also shows that the chemiluminescence signal of the preparation example 4 of this invention was 3.7 times that of MC3 on day 1, and still 6.6 times that of MC3 on day 7. This indicates that the overall luminescence intensity of the LNPs in this application is higher than that of the commercially available product MC3, demonstrating a greater advantage in expression duration.

[0084] Test Example 4: Security Verification Experiment

[0085] The testing steps are as follows:

[0086] ① The mRNA-LNPs of Examples 4-6 were synthesized and prepared, with MC3 as a control.

[0087] ② HeLa cells were seeded at 5000 cells per well and cultured at 37°C, 5% CO2, and controlled humidity for 24 hours. The old culture medium was discarded, and 100 μL of fresh culture medium containing mRNA-LNP was added to each well to achieve mRNA concentrations of 5, 2.5, 1.25, and 0.75 μg / mL. Wells without mRNA-LNP were used as blank controls.

[0088] ③ After incubation in an incubator for 24 hours, add 10 μL of CCK8 solution to each well. Wells containing culture medium and CCK8 solution but without cells serve as a complete blank control. After incubation in an incubator for 0.5 hours, measure the absorbance at 450 nm using a microplate reader and record the data.

[0089] ④ Data Analysis

[0090] Cell viability = (A(drug-treated) - A(blank)) / (A(blank) - A(blank)) × 100%

[0091] The calculation formula is as above, where:

[0092] A (Drug Addition): Absorbance of the mRNA-LNP group

[0093] A (blank): Absorbance of the group without mRNA-LNP addition

[0094] A (Complete Blank): Contains no cells or mRNA-LNP, only absorbance values ​​of CCK8 solution and culture medium.

[0095] In the safety experiments, higher cell viability at higher concentrations indicated higher safety for LNP. Specific results are shown in Table 7 below.

[0096] Table 7: Safety test results for each preparation example

[0097] As can be seen, at low concentrations of 0.75-1.25 μg / mL, the cell viability of the representative preparation example 4 of this invention was above 90%, indicating that the LNP at low concentrations had high safety and no significant difference. However, at a high concentration of 5.00 μg / mL, MC3 decreased significantly, while the cell viability of the preparation example 4 of this application still remained above 90%, proving that it still has high safety at high concentrations. Considering that concentrations much higher than 5.00 μg / mL are often required in clinical treatment, this result indicates that the LNP prepared from the representative lipid compound of this application has high potential clinical application value.

[0098] 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 will not depart from the essence and scope of the present invention.

Claims

1. An ionizable cationic lipid, characterized in that, The structure of the ionizable cationic lipid is shown in formula (1). in, a＝1-5， b＝1-6， R1, R2, R3, R4, or R5 are each independently selected from hydrogen, (C1-C6)alkyl, and (C1-C6)alkyl-COO-(C5-C 12 )alkyl.

2. The ionizable cationic lipid according to claim 1, characterized in that, In the above formula (1), a = 2 - 3, b = 2 - 4, R1, R2, R3, R4, or R5 are each independently selected from hydrogen, (C1-C2)alkyl, and (C2-C3)alkyl-COO-(C8-C9)alkyl.

3. The ionizable cationic lipid according to claim 1 or 2, characterized in that, In the above formula (1), a = 2, b = 2, R1, R2, R3 or R4 are each independently (C2-C3)alkyl-COO-(C8-C9)alkyl, and R5 is selected from hydrogen and (C1-C2)alkyl.

4. The ionizable cationic lipid according to claim 3, characterized in that, In the above formula (1), a = 2, b = 2, and R1, R2, R3, or R4 are each independently selected. -CH2CH2COOCH2CH(CH2CH3)CH2CH2CH2CH3, -CH2CH2COOCH2CH2CH2CH2CH2CH2CH(CH3)2 and -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 formulas (6), (12), and (19).

6. A method for preparing an ionizable cationic lipid according to any one of claims 1-5, characterized in that, The process includes the following steps: mixing raw material 1 and raw material 2, and then reacting them at 70-90°C; The structure of raw material 1 is shown in formula (14). In equation (14), g＝1-5， h＝1-6， R 15 R 16 R 17 R 18 Or R 19 Each is independently selected from hydrogen and (C1-C6) alkyl groups; 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 raw material 1 to raw material 2 is 1:5-9.

8. A lipid nanoparticle, characterized in that, The lipid nanoparticles include ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids according to any one of claims 1-5.

9. The lipid nanoparticles according to claim 8, characterized in that, The phospholipid is phosphocholine or dioleoylphosphatidylethanolamine; The PEGylated lipid is dimyristic glycerol-polyethylene glycol.

10. The lipid nanoparticles according to claim 9, characterized in that, The volume ratio of the ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids is 10:5:10:

3.

11. The use of the ionizable cationic lipids according to any one of claims 1-5 or the lipid nanoparticles according to any one of claims 8-10 as targeted mRNA delivery carriers.

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