Preparation and application of cationic lipid material

Abstract

The invention relates to the field of biological medicine, in particular to a cationic lipid compound capable of being used for nucleic acid delivery and a preparation method and application thereof. The LNP prepared from the cationic lipid compound with the brand new structure has the advantages of high transfection efficiency and the like, and is particularly only distributed at an injection part.

Description

Preparation and application of a cationic lipid material Technical Field

[0001] The present invention relates to the field of biomedicine, in particular to a compound that can be used for nucleic acid delivery, and a preparation method and use thereof. Background Art

[0002] Recently, with the outbreak of the global COVID-19 pandemic, COVID-19 mRNA vaccines have achieved remarkable success. Currently, the three major mRNA vaccine giants Moderna, CureVac, and BioNTech all use lipid nanoparticles (LNPs) delivery technology for their COVID-19 vaccines. Among them, the cationic lipid material in LNP delivery technology plays a very critical role. The nucleic acid gene drug to be delivered is encapsulated or wrapped through specific microfluidic technology to form nanoparticles, which protects mRNA and delivers it across the cell membrane, promoting endosome escape, and the release of mRNA drugs. LNP preparations play a vital role in the field of nucleic acid drug delivery.

[0003] The cationic lipid materials commonly used at present include non-ionizable cationic materials and ionizable cationic materials.

[0004] As for non-ionized cationic materials, LNPs currently prepared using this type of lipid material utilize their positive charge to make their formulations relatively stable and can improve transfection efficiency at the cellular level and enhance the expression of nucleic acid drugs. However, they also face many challenges: (1) LNPs prepared from non-ionized cationic materials generally have systemic toxicity problems in vivo, which affects their dosage and corresponding effects; (2) LNPs prepared from non-ionized cationic materials generally have low delivery efficiency in vivo, which limits their actual clinical application.

[0005] In addition, with regard to ionizable cationic materials, there are currently a number of COVID-19 vaccine products using LNP delivery technology that are in the market sales stage, but there are also many challenges: (1) The cationic material itself is blocked by many leading foreign companies, making structural innovation difficult; (2) The LNP component itself is also subject to patent protection and blockade, and the LNP formulation prepared according to the proportion of commercially available products has the risk of infringement; (3) As a vaccine product, the commercially available product has a strong accumulation in the liver and poses certain safety risks.

[0006] In summary, in view of the above problems and challenges that currently exist, in order to overcome problems such as in vivo toxicity and delivery efficiency, it is still necessary to independently design and synthesize new cationic lipid materials and use LNP preparation technology to achieve efficient drug delivery.

[0007] Summary of the Invention

[0008] To meet the delivery needs of nucleic acid drugs for diverse diseases and clinical settings, while reducing systemic toxicity, minimizing the risk of liver accumulation, and improving delivery efficiency, the present invention provides a cationic lipid compound, its preparation method, and its use. The LNPs prepared from this novel cationic lipid compound exhibit advantages such as high transfection efficiency. For example, non-ionizable cationic lipid nanoparticles (LNPs) containing compound 3 primarily distribute at the injection site, rarely entering organs through the bloodstream. Ionizable cationic lipid nanoparticles (LNPs) containing compounds 1 and 2, at a 25 mol% ratio, accumulate primarily at the injection site compared to MC3. At a 50 mol% ratio, the in vivo transfection efficiency of the lipid nanoparticles containing compound 2 is comparable to that of MC3, and at a 50 mol% ratio, the in vivo transfection efficiency of the lipid nanoparticles containing compound 1 is 1.668 times that of MC3. Furthermore, ionizable cationic lipid nanoparticles containing compounds 17 and 18 achieve localized transfection equivalent to MC3 LNPs while completely avoiding liver toxicity.

[0009] The present invention provides a cationic lipid compound represented by formula (I) or a pharmaceutically acceptable salt thereof:

[0010] in,

[0011] R 1 and R 2 Each independently is C6-C 20 Alkyl or C6-C 20 Alkenyl, or substituted C2-C5 alkyl, the substituent is C 16 -C 20 Alkyl-substituted -OC(=O)-, -C(=O)O-, -SC(=O)-, or -C(=O)S-;

[0012] G1 and G2 are each independently C1-C4 alkylene;

[0013] L1 and L2 are each independently -OC(=O)-, -C(=O)O-, -SC(=O)- or -C(=O)S-;

[0014] R 3 is independently a 5-6 membered saturated heterocyclic group containing 1 or 2 ring heteroatoms independently selected from N, O and S, which is optionally replaced by R 6 replace;

[0015] R 4 are independently C1-C4 alkyl, -OH or -SH, wherein the alkyl is optionally substituted by -OH or -SH;

[0016] R 5 are independently absent or C1-C4 alkyl;

[0017] R 6 are independently C1-C6 alkyl, which is optionally substituted with -OH or -SH.

[0018] In one embodiment of the present invention, in the lipid compound represented by the above formula (I), R 1 and R 2 Each independently is C6-C 20 Alkyl or C6-C 20 Alkenyl, preferably C 10 -C 20 Alkyl or C 10 -C 20 Alkenyl, more preferably C 15 -C 20 Alkyl or C 15 -C 20 Alkenyl.

[0019] In one embodiment of the present invention, in the lipid compound represented by the above formula (I), R 1 and R 2 Each independently

[0020] In one embodiment of the present invention, in the lipid compound represented by the above formula (I), R 1 and R 2 are each independently a substituted C2-C5 alkyl group, preferably a substituted C4 alkyl group, wherein the substituent is a C 16 -C 20 Alkyl-substituted -OC(=O)-, -C(=O)O-, -SC(=O)-, or -C(=O)S-.

[0021] In one embodiment of the present invention, in the lipid compound represented by the above formula (I), R 1 and R 2 Each independently

[0022] In one embodiment of the present invention, in the lipid compound represented by the above formula (I), G1 is independently a methylene group, and G2 is independently a methylene group or an ethylene group.

[0023] In one embodiment of the present invention, in the lipid compound represented by the above formula (I), R 3 are independently pyrrolidinyl or piperazinyl, which are optionally replaced by R 6 Replace, and R 6is independently methyl, ethyl, propyl or butyl, which is optionally substituted with -OH or -SH.

[0024] In one embodiment of the present invention, in the lipid compound represented by the above formula (I), R 4 are independently methyl, hydroxymethyl, hydroxyethyl or -OH, and R 5 Does not exist.

[0025] In one embodiment of the present invention, in the lipid compound represented by the above formula (I), R 4 are independently methyl, and R 5 are independently methyl.

[0026] The present invention provides a cationic lipid compound represented by formula (I) or a pharmaceutically acceptable salt thereof:

[0027] in,

[0028] R 1 and R 2 Each independently is C6-C 20 Alkyl or C6-C 20 alkenyl;

[0029] G1 and G2 are each independently C1-C4 alkylene;

[0030] L1 and L2 are each independently -OC(=O)-, -C(=O)O-, -SC(=O)- or -C(=O)S-;

[0031] R 3 is independently a 5-6 membered saturated heterocyclic group containing 1 or 2 ring heteroatoms independently selected from N, O and S, which is optionally replaced by R 6 replace;

[0032] R 4 are independently C1-C4 alkyl, -OH or -SH, wherein the alkyl is optionally substituted by -OH or -SH;

[0033] R 5 are independently absent or C1-C4 alkyl;

[0034] R 6 are independently C1-C6 alkyl, which is optionally substituted with -OH or -SH.

[0035] In one embodiment of the present invention, in the lipid compound represented by the above formula (I), R 1 and R 2 Each independently is C 10 -C 20 Alkyl or C 10 -C20 Alkenyl, preferably C 15 -C 20 Alkyl or C 15 -C 20 Alkenyl.

[0036] In one embodiment of the present invention, in the lipid compound represented by the above formula (I), R 1 and R 2 Each independently

[0037] In one embodiment of the present invention, in the lipid compound represented by the above formula (I), G1 is independently a methylene group, and G2 is independently a methylene group or an ethylene group.

[0038] In one embodiment of the present invention, in the lipid compound represented by the above formula (I), R 3 are independently pyrrolidinyl or piperazinyl, which are optionally replaced by R 6 Replace, and R 6 is independently methyl, ethyl, propyl or butyl, which is optionally substituted with -OH or -SH.

[0039] In one embodiment of the present invention, in the lipid compound represented by the above formula (I), R 4 are independently methyl, hydroxymethyl, hydroxyethyl or -OH, and R 5 Does not exist.

[0040] In one embodiment of the present invention, in the lipid compound represented by the above formula (I), R 4 are independently methyl, and R 5 are independently methyl.

[0041] The present invention provides an ionizable cationic lipid compound represented by formula (Ia) or a pharmaceutically acceptable salt thereof:

[0042] in,

[0043] R 1 and R 2 Each independently is C6-C 20 Alkyl or C6-C 20 Alkenyl, or substituted C2-C5 alkyl, the substituent is C 16 -C 20 Alkyl-substituted -OC(=O)-, -C(=O)O-, -SC(=O)-, or -C(=O)S-;

[0044] G1 and G2 are each independently C1-C4 alkylene;

[0045] L1 and L2 are each independently -OC(=O)-, -C(=O)O-, -SC(=O)- or -C(=O)S-;

[0046] R 3 is independently a 5-6 membered saturated heterocyclic group containing 1 or 2 ring heteroatoms independently selected from N, O and S, which is optionally replaced by R 6 replace;

[0047] R 4 are independently C1-C4 alkyl, -OH or -SH, wherein the alkyl group is optionally substituted with -OH or -SH;

[0048] R 6 are independently C1-C6 alkyl, which is optionally substituted with -OH or -SH.

[0049] In one embodiment of the present invention, in the lipid compound represented by the above formula (Ia), R 1 and R 2 Each independently is C6-C 20 Alkyl or C6-C 20 Alkenyl, preferably C 10 -C 20 Alkyl or C 10 -C 20 Alkenyl, more preferably C 15 -C 20 Alkyl or C 15 -C 20 Alkenyl.

[0050] In one embodiment of the present invention, in the lipid compound represented by the above formula (Ia), R 1 and R 2 Each independently

[0051] In one embodiment of the present invention, in the lipid compound represented by the above formula (Ia), R 1 and R 2 are each independently a substituted C2-C5 alkyl group, preferably a substituted C4 alkyl group, wherein the substituent is a C 16 -C 20 Alkyl-substituted -OC(=O)-, -C(=O)O-, -SC(=O)-, or -C(=O)S-.

[0052] In one embodiment of the present invention, in the lipid compound represented by the above formula (Ia), R 1 and R 2 Each independently

[0053] In one embodiment of the present invention, in the lipid compound represented by the above formula (Ia), G1 is independently a methylene group, and G2 is independently a methylene group or an ethylene group.

[0054] In one embodiment of the present invention, in the lipid compound represented by the above formula (Ia), R 3 are independently pyrrolidinyl or piperazinyl, which are optionally replaced by R 6 Replace, and R 6 is independently methyl, ethyl, propyl or butyl, which is optionally substituted with -OH or -SH.

[0055] In one embodiment of the present invention, in the lipid compound represented by the above formula (Ia), R 4 are independently methyl, hydroxymethyl, hydroxyethyl or -OH.

[0056] The present invention provides an ionizable cationic lipid compound represented by formula (Ia) or a pharmaceutically acceptable salt thereof:

[0057] in,

[0058] R 1 and R 2 Each independently is C6-C 20 Alkyl or C6-C 20 alkenyl;

[0059] G1 and G2 are each independently C1-C4 alkylene;

[0060] L1 and L2 are each independently -OC(=O)-, -C(=O)O-, -SC(=O)- or -C(=O)S-;

[0061] R 3 is independently a 5-6 membered saturated heterocyclic group containing 1 or 2 ring heteroatoms independently selected from N, O and S, which is optionally replaced by R 6 replace;

[0062] R 4 are independently C1-C4 alkyl, -OH or -SH, wherein the alkyl group is optionally substituted with -OH or -SH;

[0063] R 6 are independently C1-C6 alkyl, which is optionally substituted with -OH or -SH.

[0064] In one embodiment of the present invention, in the lipid compound represented by the above formula (Ia), R 1 and R 2 Each independently is C 10 -C 20 Alkyl or C10 -C 20 Alkenyl, preferably C 15 -C 20 Alkyl or C 15 -C 20 Alkenyl.

[0065] In one embodiment of the present invention, in the lipid compound represented by the above formula (Ia), R 1 and R 2 Each independently

[0066] In one embodiment of the present invention, in the lipid compound represented by the above formula (Ia), G1 is independently a methylene group, and G2 is independently a methylene group or an ethylene group.

[0067] In one embodiment of the present invention, in the lipid compound represented by the above formula (Ia), R 3 are independently pyrrolidinyl or piperazinyl, which are optionally replaced by R 6 Replace, and R 6 is independently methyl, ethyl, propyl or butyl, which is optionally substituted with -OH or -SH.

[0068] In one embodiment of the present invention, in the lipid compound represented by the above formula (Ia), R 4 are independently methyl, hydroxymethyl, hydroxyethyl or -OH.

[0069] The present invention provides a non-ionizable cationic lipid compound represented by formula (Ib) or a pharmaceutically acceptable salt thereof:

[0070] in,

[0071] R 1 and R 2 Each independently is C6-C 20 Alkyl or C6-C 20 alkenyl;

[0072] G1 and G2 are each independently C1-C4 alkylene;

[0073] L1 and L2 are each independently -OC(=O)-, -C(=O)O-, -SC(=O)- or -C(=O)S-;

[0074] R 3 is independently a 5-6 membered saturated heterocyclic group containing 1 or 2 ring heteroatoms independently selected from N, O and S, which is optionally replaced by R 6 replace;

[0075] R 4are independently C1-C4 alkyl;

[0076] R 5 are independently C1-C4 alkyl;

[0077] R 6 are independently C1-C6 alkyl, which is optionally substituted with -OH or -SH;

[0078] X is a chlorine, bromine or iodine atom.

[0079] In one embodiment of the present invention, in the lipid compound represented by the above formula (Ib), R 1 and R 2 Each independently is C 10 -C 20 Alkyl or C 10 -C 20 Alkenyl, preferably C 15 -C 20 Alkyl or C 15 -C 20 Alkenyl.

[0080] In one embodiment of the present invention, in the lipid compound represented by the above formula (Ib), R 1 and R 2 Each independently

[0081] In one embodiment of the present invention, in the lipid compound represented by the above formula (Ib), G1 is independently a methylene group, and G2 is independently a methylene group or an ethylene group.

[0082] In one embodiment of the present invention, in the lipid compound represented by the above formula (Ib), R 3 are independently pyrrolidinyl or piperazinyl, which are optionally replaced by R 6 Replace, and R 6 is independently methyl, ethyl, propyl or butyl, which is optionally substituted with -OH or -SH.

[0083] In one embodiment of the present invention, in the lipid compound represented by the above formula (Ib), R 4 are independently methyl, and R 5 are independently methyl.

[0084] In one embodiment of the present invention, in the lipid compound represented by the above formula (I), formula (Ia) or formula (Ib), the compound is selected from:

[0085] The present invention also provides a lipid nanoparticle composition comprising the cationic lipid compound represented by the above formula (I), formula (Ia) or formula (Ib) described herein, or a pharmaceutically acceptable salt thereof.

[0086] In one embodiment of the present invention, the above lipid nanoparticle composition further comprises neutral lipids, cholesterol and PEG lipids.

[0087] In one embodiment of the present invention, in the above lipid nanoparticle composition, the neutral lipid is selected from DSPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, SOPE and 1,2-divaleryl-sn-glycero-3-phosphoethanolamine (trans-DOPE), and the PEG lipid is selected from PEG-DMG, PEG-dipalmitoylglycerol, PEG-DSPE, PEG-dilaurylglycerol, Oleamide, PEG-dimyristylglyceramide, PEG-dipalmitoylglyceramide, and PEG-distearoylglyceramide, PEG-cholesterol (1-[8'-(cholest-5-en-3[β]-oxy)carboxamido-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB, mPEG-DMG-2k, PEG2k-DMG, PEG2k-DSPE, PEG2k-DSG, PEG2k-DMA, and PEG2k-DSA.

[0088] In one embodiment of the present invention, in the above lipid nanoparticle composition, the neutral lipid is DSPC or DOPE, and the PEG lipid is mPEG-DMG-2k.

[0089] In one embodiment of the present invention, in the above lipid nanoparticle composition, the cationic lipid compound described herein accounts for 15-60% by mole of the total lipid components in the composition.

[0090] In one embodiment of the present invention, in the above lipid nanoparticle composition, the cationic lipid compound described herein accounts for 25-60% by mole of the total lipid components in the composition.

[0091] In one embodiment of the present invention, in the above lipid nanoparticle composition, the cationic lipid compound described herein accounts for 25-50% by mole of the total lipid components in the composition.

[0092] In one embodiment of the present invention, in the above lipid nanoparticle composition, the cationic lipid compound described herein accounts for 30-50% by mole of the total lipid components in the composition.

[0093] In one embodiment of the present invention, in the above lipid nanoparticle composition, the cationic lipid compound described herein accounts for 40-50% by mole of the total lipid components in the composition.

[0094] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 15-60% of the cationic lipid compound, 5-50% of the neutral lipid, 30-56% of the cholesterol, and 1.5-2.5% of the PEG lipid, and the sum of the molar percentages of the above components is 100%.

[0095] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 25-60% of the cationic lipid compound, 10-45% of the neutral lipid, 30-56% of the cholesterol, and 1.5-2.5% of the PEG lipid, and the sum of the molar percentages of the above components is 100%.

[0096] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 25% of the cationic lipid compound, 43.5% of the neutral lipid, 30% of the cholesterol, and 1.5% of the PEG lipid.

[0097] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 50% of the cationic lipid compound, 10% of the neutral lipid, 38.5% of the cholesterol, and 1.5% of the PEG lipid.

[0098] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 31.5% of the cationic lipid compound, 10% of the neutral lipid, 56% of the cholesterol, and 2.5% of the PEG lipid.

[0099] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 43.3% of the cationic lipid compound, 8.7% of the neutral lipid, 46.5% of the cholesterol, and 1.5% of the PEG lipid.

[0100] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 46.3% of the cationic lipid compound, 9.5% of the neutral lipid, 42.7% of the cholesterol, and 1.5% of the PEG lipid.

[0101] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 50% of the cationic lipid compound, 18.5% of the neutral lipid, 30% of the cholesterol, and 1.5% of the PEG lipid.

[0102] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 57.1% of the cationic lipid compound, 7.1% of the neutral lipid, 34.3% of the cholesterol, and 1.5% of the PEG lipid.

[0103] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 15% of the cationic lipid compound, 48.5% of the neutral lipid, 35% of the cholesterol, and 1.5% of the PEG lipid.

[0104] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 45% of the cationic lipid compound, 10% of the neutral lipid, 43.5% of the cholesterol, and 1.5% of the PEG lipid.

[0105] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 45% of the cationic lipid compound, 15% of the neutral lipid, 38.5% of the cholesterol, and 1.5% of the PEG lipid.

[0106] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 45% of the cationic lipid compound, 18.5% of the neutral lipid, 35% of the cholesterol, and 1.5% of the PEG lipid.

[0107] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 45% of the cationic lipid compound, 22.5% of the neutral lipid, 31% of the cholesterol, and 1.5% of the PEG lipid.

[0108] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 40% of the cationic lipid compound, 12.5% ​​of the neutral lipid, 46% of the cholesterol, and 1.5% of the PEG lipid.

[0109] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 40% of the cationic lipid compound, 15% of the neutral lipid, 43.5% of the cholesterol, and 1.5% of the PEG lipid.

[0110] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 40% of the cationic lipid compound, 20% of the neutral lipid, 38.5% of the cholesterol, and 1.5% of the PEG lipid.

[0111] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 36.5% of the cationic lipid compound, 16% of the neutral lipid, 46% of the cholesterol, and 1.5% of the PEG lipid.

[0112] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 50% of the cationic lipid compound, 10% of the neutral lipid, 38.5% of the cholesterol, and 1.5% of the PEG lipid.

[0113] In one embodiment of the present invention, the above lipid nanoparticle composition further comprises a nucleic acid molecule selected from mRNA, siRNA, antisense oligonucleotide (ASO), saRNA, miRNA and DNA. Specifically, the mRNA is selected from FFluc mRNA (SEQ ID NO. 1) and HPV mRNA (SEQ ID NO. 2).

[0114] In one embodiment of the present invention, in the above lipid nanoparticle composition, the mass ratio of the sum of all lipid components of the composition to the nucleic acid molecule is 15:1-40:1, preferably 20:1 or 40:1.

[0115] The present invention also provides a method for preparing the above lipid nanoparticle composition, which comprises: adding the prepared cationic lipid compound, DSPC, cholesterol, and mPEG-DMG-2k described in the present application to an ethanol solution in a certain proportion, wherein the proportion is the molar percentage of each lipid component as described above in the present application to the total lipid component of the composition, and the total lipid concentration is 23 mg / ml, and mixing thoroughly; using malic acid or sodium acetate buffer (pH = 3 or 4) to dilute mRNA to 0.2-0.4 mg / ml; mixing the above lipid ethanol solution and the mRNA buffer aqueous solution in a volume ratio of 1:3-1:5 on a microfluidic device, at a flow rate of 10-15 ml / min and a mixing temperature of 50-60° C., and purifying the obtained lipid nanoparticles by ultrafiltration or dialysis to obtain the final lipid nanoparticles.

[0116] In one embodiment of the present invention, in the above method for preparing the lipid nanoparticle composition, the volume ratio of the lipid ethanol solution to the mRNA aqueous solution is 1:3, the flow rate is 10 mL / min, and the mixing temperature is 50°C.

[0117] The present invention also provides a method for delivering a nucleic acid into a cell, comprising delivering the lipid nanoparticles described above into the cell.

[0118] In one embodiment of the present invention, the cell is a mammalian cell, preferably the cell is a human cell.

[0119] The present invention also provides a use of the lipid nanoparticles described above in the present application in the preparation of a drug or vaccine for treating a disease. Preferably, the disease is cancer, tuberculosis, Helicobacter pylori, human papillomavirus (HPV), herpes zoster, dyslipidemia, etc.

[0120] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be construed as indefinite or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0121] The term "pharmaceutically acceptable" as used in this application refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0122] The term "pharmaceutically acceptable salt" as used herein refers to a salt of a compound of the invention, prepared from a compound having a specific substituent discovered by the present invention and a relatively nontoxic acid or base. When a compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such compound with a sufficient amount of a base in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, organic acid salts, salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid (see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds of the present invention contain basic and acidic functional groups and can be converted into either a base or acid addition salt.

[0123] The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. Generally, the salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two.

[0124] The term "alkyl" or "alkylene" as used herein is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, "C1-C 10 Alkyl (or alkylene) is intended to include C1, C2, C3, C4, C5, C6, C7, C8, C9 and C 10 Alkyl (or alkylene). Alkyl groups may be unsubstituted or substituted, wherein at least one hydrogen is replaced by another chemical group. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl). Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, and butylene.

[0125] Specifically, the term "C6-C 20 "Alkyl" includes C6, C7, C8, C9, C 10 、C 11 、C 12 、C13 、C 14 、C 15 、C 16 、C 17 、C 18 、C 19 and C 20 Alkyl; preferably, the "C 10 -C 20 "Alkyl" includes C 10 、C 11 、C 12 、C 13 、C 14 、C 15 、C 16 、C 17 、C 18 、C 19 and C 20 Alkyl; and more preferably, the "C 15 -C 20 "Alkyl" includes C 15 、C 16 、C 17 、C 18 、C 19 and C 20 In addition, the term "C2-C5 alkyl" used in this application includes C2, C3, C4 and C5 alkyl. In addition, the term "C 16 -C 20 "Alkyl" includes C 16 、C 17 、C 18 、C 19 and C 20 alkyl.

[0126] As used herein, the term "alkenyl" is intended to include hydrocarbon chains having a straight or branched configuration and having one or more carbon-carbon double bonds that may be present at any stable point along the chain. For example, "C2-C6 alkenyl" is intended to include C2, C3, C4, C5, and C6 alkenyl groups. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, and hexenyl.

[0127] Specifically, the term "C6-C 20 "Alkenyl" includes C6, C7, C8, C9, C 10 、C 11 、C 12 、C 13 、C 14 、C 15 、C 16 、C 17 、C 18 、C 19and C 20 Preferably, the "C 10 -C 20 "Alkenyl" includes C 10 、C 11 、C 12 、C 13 、C 14 、C 15 、C 16 、C 17 、C 18 、C 19 and C 20 and more preferably, the "C 15 -C 20 "Alkenyl" includes C 15 、C 16 、C 17 、C 18 、C 19 and C 20 Alkenyl.

[0128] As used herein, the term "heterocycle" or "heterocyclyl" refers to a stable monocyclic or bicyclic ring containing a heteroatom or heteroatom group, which may be saturated, partially unsaturated, or unsaturated (aromatic), and which comprises carbon atoms and 1, 2, 3, or 4 ring heteroatoms independently selected from N, O, and S. The nitrogen atom may be substituted or unsubstituted. Exemplary monocyclic heterocyclic groups include azetidinyl, pyrrolidinyl, oxetanyl, imidazolinyl, oxazolidinyl, isoxazolinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, azepinyl, 1-pyridonyl, 4-piperidonyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxolane and tetrahydro-1,1-dioxothiphenyl. Exemplary bicyclic heterocyclic groups include quinuclidine. The "heterocycle" employed in the present invention is preferably a 5-6 membered saturated heterocycle containing 1 or 2 ring heteroatoms independently selected from N, O and S, more preferably a piperazine ring.

[0129] The term "neutral lipid" as used herein refers to any of a variety of lipids present in the form of uncharged or neutral zwitterions at physiological pH. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin and cerebroside. In addition, the neutral lipid of the present invention can also be selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and oleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane ‐1 carboxylate (DOPE‐mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16‐O‐monomethyl PE, 16‐O‐dimethyl PE, 18‐1‐trans PE, 1‐stearoyl‐2‐oleoylphosphatidylethanolamine (SOPE) and 1,2‐divaleryl‐sn‐glycero‐3‐phosphoethanolamine (trans DOPE), preferably, the neutral lipid is selected from DSPC or DOPE.

[0130] As used herein, the term "PEG lipid" may be selected from the group consisting of PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE), PEG-dilaurylglyceramide, PEG-dimyristylglyceramide, PEG-dipalmitoylglyceramide and PEG-distearoylglyceramide, PEG-cholesterol (1-[8'-(cholester-5-en-3[β]-oxy)carboxamido-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol) ethanol), PEG-DMB (3,4-tetradecanoylbenzyl-[ω]-methyl-poly(ethylene glycol) ether), mPEG-DMG-2k (dimyristoylglycerol-polyethylene glycol 2000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (Cat. No. 880120C, obtained from Avanti The PEG lipids are selected from the group consisting of PEG-DMG-2k (Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG; GS-020, NOF Tokyo, Japan), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), and 1,2-distearoyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). Preferably, the PEG lipid is selected from mPEG-DMG-2k.

[0131] The terms "nucleic acid" or "nucleic acid molecule" as used herein will be recognized and understood by those of ordinary skill in the art, for example, to mean a molecule comprising, preferably consisting of, a nucleic acid component. The term nucleic acid molecule preferably refers to a DNA or RNA molecule. It is preferably used synonymously with the term polynucleotide. Preferably, a nucleic acid or nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers that are covalently linked to each other via phosphodiester bonds of a sugar / phosphate backbone. Preferably, the nucleic acid molecule is selected from mRNA, siRNA, antisense oligonucleotide (ASO), saRNA or miRNA.

[0132] The term "cancer" as used herein refers to a class of diseases in which a group of cells show uncontrolled growth (division beyond the normal range), invade (invade and destroy adjacent tissues) and sometimes metastasize (spread to other locations in the body via lymph or blood). Most cancers form tumors, which are swellings or lesions formed by the abnormal growth of cells (called neoplastic cells or tumor cells), but some (such as leukemias) do not. Term cancers according to the present invention include triple-negative breast cancer, leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat cancer (ENT), breast cancer, prostate cancer, uterine cancer, ovarian cancer and lung cancer and their metastases. Examples thereof are triple negative breast cancer, lung cancer, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, cervical cancer or metastases of the aforementioned cancer types or tumors.The term cancer according to the invention also includes cancer metastases.

[0133] The cationic lipid provided by the present invention can specifically be a non-ionized cationic lipid structure, wherein the head group is selected from a protonable heterocycle, the middle connecting chain is selected from a non-ionized quaternary ammonium salt, the connecting bond is selected from an ester bond that is easily biodegradable, and the tail is selected from a fatty chain of different lengths, different saturations, and different numbers of tail chains. Such a structural design enables the non-ionized cationic lipid to achieve higher in vivo mRNA transfection efficiency and the advantages of only local distribution and expression. Therefore, the cationic lipid of the present invention, more specifically the non-ionized cationic lipid compound, has the following advantages: (1) the LNP preparation prepared therefrom has better expression in vivo and in vitro than the DOTAP LNP preparation; (2) the LNP preparation prepared therefrom has a uniform and controllable particle size, and the potential is lower than that of the DOTAP preparation at the same ratio, which can reduce the safety of the system brought by the non-ionized cationic material to a certain extent.

[0134] The cationic lipid provided by the present invention can also be specifically an ionizable cationic lipid structure, wherein the head group is selected from a protonable heterocycle, the middle connecting chain is selected from a protonable tertiary amine, the connecting bond is selected from an ester bond that is easily biodegradable, and the tail is selected from a fatty chain of different lengths, different saturations and different numbers of tail chains. Such a structural design enables the ionizable cationic lipid to achieve a higher in vivo mRNA transfection efficiency. Therefore, the cationic lipid of the present invention, more specifically the ionizable cationic lipid compound, has the following advantages: (1) based on the characteristics and structure-activity relationship of the phospholipid itself, it is independently designed and synthesized and breaks through the foreign material patent blockade; (2) through the optimization of a certain prescription ratio and process, the existing invention compound LNP preparation can produce specific benefits in distribution, and this characteristic can be given targeted treatment according to different clinical indications and needs; (3) the transfection effect of the LNP preparation prepared by the compound in vivo is slightly better than or equivalent to the LNP effect prepared by the commercially available head product. BRIEF DESCRIPTION OF THE DRAWINGS

[0135] Figure 1: Hela cell transfection results of lipid nanoparticles LNP-1 to LNP-9 prepared with different ratios of compounds 1 and 2 of the present invention, LNP-19 prepared with compound 9, LNP-20 prepared with compound 10, and LNP-21 prepared with compound 11, relative to LNP-10 prepared with MC3.

[0136] Figure 2: Animal transfection results of lipid nanoparticles LNP-1 to LNP-3 and LNP-7 to LNP-8 prepared using compounds 1 and 2 of the present invention, LNP-19 prepared using compound 9, LNP-20 prepared using compound 10, and LNP-21 prepared using compound 11, relative to LNP-10 prepared using MC3.

[0137] Figure 3: In vivo animal imaging of lipid nanoparticles LNP-1 to LNP-3 and LNP-7 to LNP-8 prepared using compounds 1 and 2 of the present invention, LNP-19 prepared using compound 9, LNP-20 prepared using compound 10, LNP-21 prepared using compound 11, and LNP-10 prepared using MC3.

[0138] FIG4 : Animal transfection results of lipid nanoparticles LNP-11 prepared using compound 3 of the present invention compared with LNP-12 prepared using DOTAP.

[0139] FIG5 : In vivo animal imaging of lipid nanoparticles LNP-11 prepared using compound 3 of the present invention and LNP-12 prepared using DOTAP.

[0140] Figure 6: Animal transfection results of lipid nanoparticles LNP-2 and LNP-22 to LNP-30 prepared using compound 1 of the present invention, and LNP-8 and LNP-31 to LNP-34 prepared using compound 2, relative to LNP-10 prepared using MC3.

[0141] Figure 7: In vivo animal imaging of lipid nanoparticles LNP-2 and LNP-22 to LNP-30 prepared using compound 1 of the present invention, LNP-8 and LNP-31 to LNP-34 prepared using compound 2, and LNP-10 prepared using MC3.

[0142] Figure 8: Animal transfection results of lipid nanoparticles LNP-8 prepared using compound 2 of the present invention, LNP-35 prepared using compound 17, LNP-2 prepared using compound 1, and LNP-36 prepared using compound 18, relative to LNP-10 prepared using MC3.

[0143] Figure 9: In vivo animal imaging of lipid nanoparticles LNP-8 prepared using compound 2 of the present invention, LNP-35 prepared using compound 17, LNP-2 prepared using compound 1, LNP-36 prepared using compound 18, and LNP-10 prepared using MC3.

[0144] FIG10 : Immunogenicity results of LNP-37 prepared using compound 1 of the present invention and LNP-38 prepared using commercially available SM-102 in mice. DETAILED DESCRIPTION

[0145] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are merely illustrative and illustrative of the present invention and should not be construed as limiting the scope of protection of the present invention. Any technical solutions implemented based on the content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0146] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0147] Synthesis of different cationic lipids

[0148] Example 1: Preparation of Compound 1

[0149] Synthesis of compound 1c: Compound 1a (18.5 g) was dissolved in 180 ml of isopropanol. Compound 1b (4 g of glycidol) was added dropwise with stirring at 50-60°C. The reaction was allowed to proceed at controlled temperature. After completion of the reaction as detected by TLC, the reaction system was concentrated under reduced pressure at 50-60°C to obtain 10.21 g of a brown oil.

[0150] Synthesis of Compound 1d: Compound 1c (10.21 g) was added to a reaction flask and dissolved in 100 g of water. 1.8 g of formic acid and 3.2 g of 37% formaldehyde solution were then added dropwise. After the additions were complete, the temperature was raised to 40-50°C. After TLC, the reaction was completed, the pH was adjusted to 8-9 with sodium hydroxide, and the mixture was extracted with dichloromethane. The organic phase was concentrated under reduced pressure to yield 9.0 g of a yellow oil.

[0151] Synthesis of Compound 1: Dissolve 14.58 g of Compound 1e in 140 ml of chloroform with stirring. Then, add N,N-carbonyldiimidazole and 5 g of Compound 1d. Stir and react at 50-60°C. After completion of the reaction, monitor the reaction by TLC. Remove the solvent by rotary evaporation under reduced pressure. Purify the mixture by column chromatography (silica gel, eluent: DCM:MeOH = 100:1 (volume ratio)) to obtain 3.4 g of Compound 1 as a pale yellow oil. 1H NMR (400MHz, CDCl3) δ5.18-5.16(m,1H),4.42-4.40(m,1H),4.07-4.04(m,1H),2.65-2.54(m,7H),2.53(t ,J=2.7Hz,2H),2.34-2.29(m,5H),1.80(s,4H),1.56-1.44(m,8H),1.26(s,41H),0.88(t,J=7.0Hz,12H). MS m / z(ESI): 680.8[M+H] + .

[0152] Example 2: Preparation of Compound 2

[0153] Synthesis of compound 2c: Compound 2a (22 g) was dissolved in 220 ml of isopropanol. Compound 2b (4 g of glycidol) was added dropwise at 50-60°C. The reaction was allowed to proceed at this temperature. After completion of the reaction as detected by TLC, the reaction system was concentrated under reduced pressure at 60-70°C to obtain 11.8 g of a light yellow oil.

[0154] Synthesis of Compound 2d: Compound 2c (11.8 g) was added to a reaction flask and dissolved in 110 g of water. 1.8 g of formic acid and 3.2 g of 37% formaldehyde solution were then added dropwise. After the additions were complete, the temperature was raised to 40-50°C. After TLC, the pH was adjusted to 8-9 with sodium hydroxide. The mixture was extracted with dichloromethane, and the organic phase was concentrated under reduced pressure to yield 4.0 g of a yellow oil.

[0155] Synthesis of Compound 2: Dissolve 10.2 g of Compound 2e in 100 ml of chloroform with stirring. Then, add N,N-carbonyldiimidazole and 4 g of Compound 2d. Stir and react at 50-60°C. After completion of the reaction, monitor the reaction by TLC. Remove the solvent by rotary evaporation under reduced pressure. Purify the mixture by column chromatography (silica gel, eluent: DCM:MeOH = 100:1 (volume ratio)) to obtain 2.29 g of Compound 2 as a pale yellow oil. 1H NMR (400MHz, CDCl3) δ5.18-5.14(quint,J=7.1Hz,1H),4.42-4.39(d,1H),4.07-4.04(d,J=7.1Hz,1H),2. 58-2.43(m,8H),2.28(s,6H),1.60-1.56(m,4H),1.43-1.42(m,4H),1.26(s,42H),0.88(t,J=7.0Hz,12H). MS m / z(ESI):709.5[M+H] + .

[0156] Example 3: Preparation of Compound 3

[0157] Synthesis of compound 3c: 3 g of compound 3b was dissolved in 30 ml of ethanol, and compound 3a was added. The temperature was raised to 45-50°C and the reaction was monitored by TLC. After the reaction was complete, the mixture was concentrated under reduced pressure to obtain 4.81 g of a light yellow oil.

[0158] Synthesis of Compound 3: Dissolve 11.6 g of Compound 3d in 110 ml of dichloromethane with stirring. Add N,N-carbonyldiimidazole and 4.81 g of Compound 3c sequentially, and stir at 50-60°C. After TLC monitoring of the reaction, remove the solvent by rotary evaporation under reduced pressure. Purification by column chromatography (silica gel column, eluent: DCM:MeOH = 50:1; DCM:MeOH = 30:1 (volume ratio)) yields 1.13 g of Compound 3 as a light yellow oil. 1 H NMR (400MHz, CDCl3) δ5.62(s,1H),4.67-4.50(m,2H),4.10(dd,J=12.3,5.8Hz,1H),3.95(s,2H),3.77(dd,J=14.3,7.8Hz,1H),3.52(d,J=5.0 Hz, 6H), 3.20-2.83 (m, 2H), 2.54 (t, J = 48.6Hz, 4H), 2.41-2.24 (m, 3H), 1.79 (s, 4H), 1.68-1.34 (m, 8H), 1.26 (s, 41H), 0.88 (t, J = 6.6Hz, 12H). MS m / z(ESI): 693.6[M-Cl]+ .

[0159] Example 4: Preparation of Compound 4

[0160] The preparation method of compound 4 is the same as that of compound 3. 1 H NMR (400MHz, CDCl3) δ5.15 (1H, tt, J = 6.0, 5.7Hz), 4.47-4.36 (2H, 4.42 (d, J = 6.0Hz), 3.37-3.24 (4H, 3.30 (d, J = 5.7Hz)), 3.14-2.83 (12H, 2.89 (t, J=6.8Hz),2.33-2.20(4H,2.26(t,J=7.4Hz),1.99-1.79(4H),1.63-1.49(4H),1.34-1.17(24H,1.23(quint,J=7.0Hz),0.90(t,J=6.8Hz,6H).MS m / z(ESI): 525.83[M-Cl]+.

[0161] Example 5: Preparation of Compound 5

[0162] The preparation method of compound 5 is the same as that of compound 4. 1 H NMR (400MHz, CDCl3) δ5.42-5.31(m,5H),5.25-5.15(m,1H),4.36(dd,J=11.9,3.1Hz,1H),4.0 3(ddd,J=19.0,14.3,8.6Hz,4H),3.96-3.79(m,4H),3.32(d,J=5.9Hz,4H),3.03-2.86(m,2H) ,2.61(qd,J=13.4,6.4Hz,3H),2.44(d,J=10.7Hz,3H),2.40-2.35(m,2H),2.34-2.16(m,7H), 2.03(d,J=5.6Hz,10H),1.64-1.59(m,4H),1.30(d,J=13.5Hz,41H),0.89(d,J=7.0Hz,6H).MS m / z(ESI): 745.67[M-Cl]+.

[0163] Example 6: Preparation of Compound 6

[0164] The preparation method of compound 6 is the same as that of compound 3. 1H NMR(400MHz, CDCl3)δ5.64(s,1H),5.45-5.28(m,8H),4.61-4.44(m,2H),4.13(dd ,J=12.1,5.8Hz,1H),3.92(t,J=11.4Hz,3H),3.51(t,J=8.2Hz,6H),3.14-2.96(m ,2H),2.84-2.71(m,4H),2.63(s,4H),2.42-2.28(m,4H),2.06(q,J=6.7Hz,8H),1 .90-1.74(m,4H),1.70-1.54(m,4H),1.43-1.23(m,28H),0.90(t,J=6.8Hz,6H).MS m / z(ESI):741.1[M-Cl] + .

[0165] Example 7: Preparation of Compound 7

[0166] The preparation method of compound 7 is the same as that of compounds 3 and 4, using 1-Boc-4-(2-chloroethyl)piperazine as the starting material and finally removing Boc to obtain it. 1 H NMR (400MHz, CDCl3) δ5.65 (s, 1H), 5.42-5.31 (m, 4H), 4.58-4.46 (m, 2H), 4.13 (dd, J= 12.1,5.9Hz,1H),3.97(s,1H),3.88(dd,J=14.4,8.6Hz,2H),3.48(d,J=2.9Hz,6H),2 .97(s,3H),2.91(d,J=5.2Hz,2H),2.64(s,4H),2.36(dt,J=15.4,7.8Hz,4H),2.05(t ,J=15.9Hz,15H),1.63(d,J=7.0Hz,4H),1.37-1.23(m,48H),0.90(t,J=6.7Hz,6H).MS m / z(ESI):761.37[M-Cl] + .

[0167] Example 8: Preparation of Compound 8

[0168] The preparation method of compound 8 is the same as that of compound 7. 1H NMR (400MHz, CDCl3) δ5.65 (s, 1H), 5.45-5.29 (m, 8H), 4.53-4.37 (m, 2H), 4.11 (dt, J = 1 6.3,8.2Hz,1H),3.88(ddd,J=31.9,14.2,8.7Hz,3H),3.45(s,6H),2.88(dd,J=12.4,8 .0Hz,6H),2.79(t,J=6.3Hz,4H),2.57(d,J=31.5Hz,4H),2.43-2.26(m,4H),2.07(dd, J=13.6,6.7Hz,10H),1.62(d,J=7.0Hz,4H),1.41-1.10(m,33H),0.91(t,J=6.8Hz,6H). MS m / z(ESI): 756.97[M-Cl] + .

[0169] Example 9: Preparation of Compound 9

[0170] The preparation method of compound 9 is the same as that of compound 1. 1 H NMR (400MHz, CDCl3) δ7.30-7.09(m,1H),5.44-5.26(m,11H),5.30-5.12(m,1H),4.35 (dd,J=11.9,3.1Hz,1H),4.09(dd,J=11.9,6.4Hz,1H),2.85-2.69(m,12H),2.69-2.62 (m,2H),2.55(d,J=6.5Hz,2H),2.35-2.27(m,8H),2.23(t,J=7.6Hz,1H),2.06(q,J=6. 7Hz, 11H), 1.86 (s, 4H), 1.67-1.56 (m, 5H), 1.39-1.27 (m, 38H), 0.90 (t, J = 6.8Hz, 6H). MS m / z(ESI):728.10[M+H] + .

[0171] Example 10: Preparation of Compound 10

[0172] The preparation method of compound 10 is the same as that of compound 2. 1H NMR (400MHz, CDCl3) δ5.46-5.28(m,9H),5.19(qd,J=6.4,3.1Hz,1H),4.37(dd,J=11.9,3.0Hz,1H),4.11(dd,J=11.9,6.5Hz,1H),2.93 (d,J=6.0Hz,1H),2.85-2.63(m,5H),2.68-2.35(m,15H),2.40-2.24(m,12H),2.06(q,J=6.7Hz,10H),1.71-1.52(m,5H),1.42-1.22(m, 37H), 0.90 (t, J=6.8Hz, 6H). MS m / z(ESI):757.24[M+H] + .

[0173] Example 11: Preparation of Compound 11

[0174] Synthesis of Compound 11c: Compound 11a (2.7 g) was dissolved in 100 ml of acetonitrile, and 11.5 g of potassium carbonate and 0.345 g of potassium iodide were added. The mixture was stirred and heated to 50°C. Compound 11b (5 g) was added to the mixture, and the reaction was incubated. TLC was used for analysis. After completion of the reaction, the inorganic salts were removed by filtration, and the mother liquor was concentrated under reduced pressure to yield 4.23 g of a pale yellow oil.

[0175] Synthesis of compound 11f: 11e (30 g) was dissolved in 300 ml of dichloromethane. Oxalyl chloride (29.7 g) was added dropwise to the dichloromethane solution at room temperature. A small amount of bubbles and smoke were generated during the addition. After the addition was complete, the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain 31.68 g of a light yellow oil. 11d (6 g) was dissolved in 60 ml of dichloromethane, and 16.4 g of triethylamine was added. The mixture was stirred at room temperature. The 31.68 g of the light yellow oil obtained above was added dropwise to the above solution. After the addition was complete, the reaction was stirred for 2 hours. After the reaction was completed, the reaction solution was washed three times with an equal volume of water. The organic phase was collected, dried, and concentrated to obtain 11f (34.9 g) as a crude oil. The crude product was purified by column chromatography (dichloromethane) to obtain 22.44 g of 11f as an oil.

[0176] Synthesis of Compound 11: Dissolve 3.5 g of Compound 11c in 150 ml of N,N-dimethylformamide by stirring. Add 12.6 g of potassium carbonate and 0.42 g of potassium iodide. Raise the temperature to 40°C. Add 11f (10.5 g) dropwise to the system containing 11c. After addition, control the temperature at 40-50°C and monitor the reaction by TLC. After completion of the reaction, filter to remove insoluble matter, and concentrate the mother liquor under reduced pressure to obtain 13.54 g of a crude pale yellow oil. Purify by column chromatography to obtain 400 mg of Compound 11 as a pale yellow oil. 1 H NMR (400MHz, CDCl3) δ5.08-4.67(m,1H),3.71-3.60(m,1H),3.50(dt,J=33.6,7.2Hz,1H),3.03(d,J=15.8Hz,1H), 2.69-2.47(m,6H),1.61(dd,J=13.3,7.7Hz,1H),1.43(dd,J=13.2,6.1z,2H),1.25(s,14H),0.87(t,J=6.2Hz,4H). MS m / z(ESI):739.04[M+H] + .

[0177] Example 12: Preparation of Compound 17

[0178] Synthesis of compound 17c: Compound 17a (5.0 g) was added to 17b (50 ml) and refluxed for 8 hours. After the reaction was complete as detected by TLC, the mixture was concentrated under reduced pressure to give a light yellow oil (6.49 g), which was used directly in the next step.

[0179] Synthesis of compound 17d: Compound 17c (6.49 g) was dissolved in 70 ml of tetrahydrofuran. Lithium aluminum hydride (LAH) (1.5 eq) was added in batches at room temperature, releasing gas and heat. The temperature was controlled at 30-40°C using an ice-water bath. After the addition, the mixture was brought to reflux for reaction. After TLC monitoring, the reaction solution was cooled to 5-10°C and water, sodium hydroxide, and water were added in sequence to quench the reaction. After quenching, the mixture was directly filtered, the filter cake was washed with tetrahydrofuran, and the filtrate was concentrated under reduced pressure to obtain 5.5 g of a yellow oil, which was used directly in the next reaction.

[0180] Synthesis of compound 17f: Compound 17d (5.50 g) was dissolved in 60 ml of isopropanol. At room temperature, the isopropanol solution of 17e (2 eq) was added dropwise to the reaction mixture of 17d. After the addition was complete, the reaction was allowed to react at room temperature overnight. After completion of the reaction monitored by TLC, the product was purified by column separation (silica gel column, eluent: dichloromethane:methanol = 40:1 (volume ratio)) to give 3.5 g of an orange-red oil.

[0181] Synthesis of compound 17i: Compound 17g (20 g) was dissolved in dichloromethane. At room temperature, 17h (0.5 eq), N,N-diisopropylethylamine (DIPEA) (1 eq), 4-dimethylaminopyridine (DMAP) (0.5 eq) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (1 eq) were added in sequence. After the addition, the mixture was stirred at room temperature and reacted overnight. After TLC detection, the reaction solution was directly washed with dilute hydrochloric acid to separate the layers. The organic phase was washed once with equal volumes of purified water and sodium chloride aqueous solution. The organic phase was concentrated under reduced pressure to obtain an oily substance, which was then purified by column separation (silica gel column, eluent: DCM:MeOH = 100:1 (volume ratio)) to obtain 21.2 g of a light yellow oily substance.

[0182] Synthesis of Compound 17: Compound 17i (4 g) was dissolved in 20 ml of dichloromethane, and a few drops of DMF were added dropwise. The mixture was cooled to 0-5°C in an ice-water bath, and oxalyl chloride (1.5 eq) was added dropwise. After the addition was complete, the mixture was reacted at room temperature. After TLC monitoring, the reaction was concentrated to dryness under reduced pressure. Compound 17f (0.5 eq) was dissolved in dichloromethane, and triethylamine (2 eq) was added. The mixture was cooled to 0-5°C in an ice-water bath. The above-reduced dry product was dissolved in an appropriate amount of dichloromethane and added dropwise to the reaction system of compound 17f. The reaction was allowed to react overnight at room temperature. After TLC monitoring, the reaction solution was washed once with aqueous citric acid and once with saturated aqueous sodium chloride. The organic phase was concentrated to obtain a light yellow oil. The product was purified by column chromatography (silica gel column, eluent: DCM:MeOH = 80:1 (volume ratio)) to obtain 1.21 g of a light yellow oil. 1 H NMR (400MHz, CDCl3) δ5.51(d,J=4.9Hz,1H),4.86(dd,J=6.1,1.4Hz,2H),4.40(s,1H),4.07(dd,J=12.1,5.4Hz,1H),3.96-3.28(m,6H),3.16( s,4H),2.82(dd,J=13.0,7.4Hz,10H),2.50-2.25(m,8H),1.66(s,8H),1.51(d,J=5.7Hz,8H),1.28(d,J=15.4Hz,48H),0.89(t,J=6.8Hz,12H). MS m / z(ESI):964.82[M+H] + .

[0183] Example 13: Preparation of Compound 18

[0184] Synthesis of compound 18c: Compound 18a (6.4 g) was dissolved in 100 ml of ethanol, and compound 18b (3.7 g of glycidol) was added dropwise under reflux. The reaction was controlled at the temperature. After completion of the reaction by TLC, the mixture was concentrated under reduced pressure to obtain a light yellow oil. The oil was then purified by column separation (silica gel column, eluent: DCM:MeOH = 100:1 (volume ratio)) to obtain 4.5 g of an oil.

[0185] Synthesis of compound 18f: Compound 18d (20 g) was dissolved in dichloromethane. At room temperature, 18e (0.5 eq), DIPEA (1 eq), DMAP (0.5 eq) and EDCI (1 eq) were added sequentially. The mixture was stirred at room temperature and allowed to react overnight. After the reaction was complete, TLC was performed, and the reaction solution was directly washed with dilute hydrochloric acid to separate the layers. The organic phase was washed once with equal volumes of purified water and then with an aqueous sodium chloride solution. The organic phase was concentrated under reduced pressure to obtain an oil, which was then purified by column separation (silica gel column, eluent: DCM:MeOH = 100:1 (volume ratio)) to obtain 24.2 g of a light yellow oil.

[0186] Synthesis of compound 18: Compound 18f (0.8 g) was dissolved in 10 ml of dichloromethane. At room temperature, 18c (0.4 g), DIEA (0.39 g), DMAP (0.1 g), and EDCI (0.5 g) were added sequentially. The mixture was stirred at room temperature overnight. TLC analysis showed that only a small amount of 18f had not reacted completely. The reaction solution was concentrated under reduced pressure to obtain an oil, which was then purified by column separation (silica gel column, eluent: petroleum ether:ethyl acetate = 1:10 (volume ratio)) to obtain 0.6 g of a light yellow oil. 1 H NMR (400MHz, CDCl3) δ5.26-5.14(m,1H),4.85(d,J=1.4Hz,2H),4.35(dd,J=12.0,3.1Hz,1H),4.13-4.03(m,1H),2.59(s,10H) ,2.33(dd,J=11.9,4.9Hz,11H),2.13(s,4H),1.66(s,8H),1.50(d,J=5.5Hz,8H),1.32-1.22(m,48H),0.88(t,J=6.8Hz,12H). MS m / z(ESI):935.96[M+H] + .

[0187] Preparation of different lipid nanoparticles (LNPs) from the cationic lipids prepared above

[0188] Formulation Example 1: Preparation of lipid nanoparticles (LNP-1) containing 25 mol% of compound 1

[0189] A certain amount of compound 1, DSPC (Lipoid), cholesterol (Nanjing Green Leaf), and mPEG-DMG-2k (Sinopeg) were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 25:43.5:30:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-1 preparation.

[0190] Formulation Example 2: Preparation of lipid nanoparticles (LNP-2) containing 50 mol% of compound 1

[0191] A certain amount of compound 1, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 50:10:38.5:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50 ° C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-2 preparation.

[0192] Formulation Example 3: Preparation of lipid nanoparticles (LNP-3) containing 25 mol% of compound 2

[0193] A certain amount of compound 2, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 25:43.5:30:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50 ° C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-3 preparation.

[0194] Formulation Example 4: Preparation of lipid nanoparticles (LNP-4) containing 31.5 mol% of compound 2

[0195] A certain amount of compound 2, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 31.5:10:56:2.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-4 preparation.

[0196] Formulation Example 5: Preparation of lipid nanoparticles (LNP-5) containing 43.3 mol% of compound 2

[0197] A certain amount of compound 2, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 43.3:8.7:46.5:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-5 preparation.

[0198] Formulation Example 6: Preparation of lipid nanoparticles (LNP-6) containing 46.3 mol% of compound 2

[0199] A certain amount of compound 2, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 46.3:9.5:42.7:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-6 preparation.

[0200] Formulation Example 7: Preparation of lipid nanoparticles (LNP-7) containing 50 mol% of compound 2

[0201] A certain amount of compound 2, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 50:18.5:30:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50 ° C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-7 preparation.

[0202] Formulation Example 8: Preparation of lipid nanoparticles (LNP-8) containing 50 mol% of compound 2

[0203] A certain amount of compound 2, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 50:10:38.5:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50 ° C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-8 preparation.

[0204] Formulation Example 9: Preparation of lipid nanoparticles (LNP-9) containing 57.1 mol% of compound 2

[0205] A certain amount of compound 2, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 57.1:7.1:34.3:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-9 preparation.

[0206] Preparation Control Example 10: Preparation of Lipid Nanoparticles (LNP-10) Containing 50 mol% MC3

[0207] A certain amount of 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA / MC3, Shanghai Langxu Biotechnology), DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 50:10:38.5:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed with PBS at pH = 7.4 for 24 hours to obtain the final LNP-10 preparation.

[0208] Formulation Example 11: Preparation of lipid nanoparticles (LNP-11) containing 25 mol% of compound 3

[0209] A certain amount of compound 3, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 25:43.5:30:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50 ° C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-11 preparation.

[0210] Preparation Control Example 12: Preparation of Lipid Nanoparticles (LNP-12) Containing 25 mol% DOTAP

[0211] A certain amount of (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP, Lipoid), DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 25:43.5:30:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 hours using PBS at pH = 7.4 to obtain the final LNP-12 preparation.

[0212] Formulation Example 13: Preparation of lipid nanoparticles (LNP-13) containing 25 mol% of compound 4

[0213] A certain amount of compound 4, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 25:43.5:30:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-13 preparation.

[0214] Formulation Example 14: Preparation of lipid nanoparticles (LNP-14) containing 25 mol% of compound 5

[0215] A certain amount of compound 5, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 25:43.5:30:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-14 preparation.

[0216] Formulation Example 15: Preparation of lipid nanoparticles (LNP-15) containing 25 mol% of compound 6

[0217] A certain amount of compound 6, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 25:43.5:30:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-15 preparation.

[0218] Formulation Example 16: Preparation of lipid nanoparticles (LNP-16) containing 25 mol% of compound 6

[0219] A certain amount of compound 6, DOPE, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 25:43.5:30:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed with PBS at pH = 7.4 for 24 hours to obtain the final LNP-16 preparation.

[0220] Formulation Example 17: Preparation of lipid nanoparticles (LNP-17) containing 25 mol% of compound 7

[0221] A certain amount of compound 7, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 25:43.5:30:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-17 preparation.

[0222] Formulation Example 18: Preparation of lipid nanoparticles (LNP-18) containing 25 mol% of compound 8

[0223] A certain amount of compound 8, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 25:43.5:30:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-18 preparation.

[0224] Formulation Example 19: Preparation of lipid nanoparticles (LNP-19) containing 25 mol% of compound 9

[0225] A certain amount of compound 9, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 25:43.5:30:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-19 preparation.

[0226] Formulation Example 20: Preparation of lipid nanoparticles (LNP-20) containing 25 mol% of compound 10

[0227] A certain amount of compound 10, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 25:43.5:30:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-20 formulation.

[0228] Formulation Example 21: Preparation of lipid nanoparticles (LNP-21) containing 25 mol% of compound 11

[0229] A certain amount of compound 11, DOPE, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 25:43.5:30:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed with PBS at pH = 7.4 for 24 hours to obtain the final LNP-21 preparation.

[0230] Formulation Example 22: Preparation of lipid nanoparticles (LNP-22) containing 45 mol% of compound 1

[0231] A certain amount of compound 1, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 45:10:43.5:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-22 preparation.

[0232] Formulation Example 23: Preparation of lipid nanoparticles (LNP-23) containing 45 mol% of compound 1

[0233] A certain amount of compound 1, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 45:15:38.5:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 40:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-23 preparation.

[0234] Formulation Example 24: Preparation of lipid nanoparticles (LNP-24) containing 45 mol% of compound 1

[0235] A certain amount of compound 1, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 45:18.5:35:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 40:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-24 preparation.

[0236] Formulation Example 25: Preparation of lipid nanoparticles (LNP-25) containing 45 mol% of compound 1

[0237] A certain amount of compound 1, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 45:22.5:31:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 40:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-25 preparation.

[0238] Formulation Example 26: Preparation of lipid nanoparticles (LNP-26) containing 43.3 mol% of compound 1

[0239] A certain amount of compound 1, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 43.3:8.7:46.5:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-26 preparation.

[0240] Formulation Example 27: Preparation of lipid nanoparticles (LNP-27) containing 40 mol% of compound 1

[0241] A certain amount of compound 1, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 40:12.5:46:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-27 preparation.

[0242] Formulation Example 28: Preparation of lipid nanoparticles (LNP-28) containing 40 mol% of compound 1

[0243] A certain amount of compound 1, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 40:15:43.5:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 40:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-28 preparation.

[0244] Formulation Example 29: Preparation of lipid nanoparticles (LNP-29) containing 40 mol% of compound 1

[0245] A certain amount of compound 1, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 40:20:38.5:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 40:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-29 preparation.

[0246] Formulation Example 30: Preparation of lipid nanoparticles (LNP-30) containing 36.5 mol% of compound 1

[0247] A certain amount of compound 1, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 36.5:16:46:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-30 preparation.

[0248] Formulation Example 31: Preparation of lipid nanoparticles (LNP-31) containing 45 mol% of compound 2

[0249] A certain amount of compound 2, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 45:10:43.5:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 40:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50 ° C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-31 preparation.

[0250] Formulation Example 32: Preparation of lipid nanoparticles (LNP-32) containing 45 mol% of compound 2

[0251] A certain amount of compound 2, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 45:15:38.5:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 40:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-32 preparation.

[0252] Formulation Example 33: Preparation of lipid nanoparticles (LNP-33) containing 45 mol% of compound 2

[0253] A certain amount of compound 2, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 45:18.5:35:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 40:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-33 preparation.

[0254] Formulation Example 34: Preparation of lipid nanoparticles (LNP-34) containing 40 mol% of compound 2

[0255] A certain amount of compound 2, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 40:20:38.5:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 40:1. The above-mentioned lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50 ° C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-34 preparation.

[0256] Formulation Example 35: Preparation of lipid nanoparticles (LNP-35) containing 50 mol% of compound 17

[0257] A certain amount of compound 17, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 50:10:38.5:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-35 preparation.

[0258] Formulation Example 36: Preparation of lipid nanoparticles (LNP-36) containing 50 mol% of compound 18

[0259] A certain amount of compound 18, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 50:10:38.5:1.5. FFluc mRNA (SEQ ID NO.1) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to FFluc mRNA was 20:1. The above lipid ethanol solution and the FFluc mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 10 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-36 preparation.

[0260] Formulation Example 37: Preparation of lipid nanoparticles (LNP-37) containing 36.5 mol% of compound 1

[0261] A certain amount of compound 1, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 36.5:16:46:1.5. HPV mRNA (SEQ ID NO.2) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to HPV mRNA was 20:1. The above-mentioned lipid ethanol solution and HPV mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50 ° C and a flow rate of 12 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-37 preparation.

[0262] Preparation Control Example 38: Preparation of Lipid Nanoparticles (LNP-38) Containing 50 mol% SM-102

[0263] A certain amount of heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanoxy)hexyl)amino)octanoate) (SM-102, Sinopeg), DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 50:10:38.5:1.5. HPV mRNA (SEQ ID NO.2) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to HPV mRNA was 20:1. The above-mentioned lipid ethanol solution and HPV mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50°C and a flow rate of 12 mL / min. The obtained lipid nanoparticles were dialyzed for 24 hours using PBS at pH = 7.4 to obtain the final LNP-38 preparation.

[0264] Formulation Example 39: Preparation of lipid nanoparticles (LNP-39) containing 43.3 mol% of compound 1

[0265] A certain amount of compound 1, DSPC, cholesterol, and mPEG-DMG-2k were weighed and a lipid ethanol solution with a total lipid concentration of 23 mg / mL was prepared at a molar ratio of 43.3:8.7:46.5:1.5. HPV mRNA (SEQ ID NO.2) was diluted with malic acid buffer at pH = 4.0, and the mass ratio of lipid to HPV mRNA was 20:1. The above-mentioned lipid ethanol solution and HPV mRNA aqueous solution were mixed at a volume ratio of 1:3 on a microfluidic device (Miana, INano L) at a temperature of 50 ° C and a flow rate of 12 mL / min. The obtained lipid nanoparticles were dialyzed for 24 h using PBS at pH = 7.4 to obtain the final LNP-39 preparation.

[0266] Test example

[0267] Experimental Example 1: Formulation Characterization of Different Lipid Nanoparticles mRNA-LNPs Prepared Using Different Cationic Lipid Compounds

[0268] The particle size and PDI of the mRNA-LNPs were characterized by dynamic light scattering using a particle size analyzer (Malvern Zetasizer Nano-ZS). The encapsulation efficiency of mRNA (SEQ ID NO. 1 and SEQ ID NO. 2) was determined using a Ribogreen RNA quantification kit (Thermo Fisher) on a microplate reader (Molecular Devices, SpectraMax i3x) with an excitation wavelength of 480 nm and an emission wavelength of 525 nm. The pKa of the lipid nanoparticles was determined using the 2-(p-toluidinyl)-6-naphthalenesulfonic acid (TNS) dye-binding method on a microplate reader (Molecular Devices, SpectraMax i3x) with an excitation wavelength of 325 nm and an emission wavelength of 435 nm. The formulation characteristics are shown in Tables 1 and 2 below.

[0269] Table 1. Particle size, PDI, Zeta potential, encapsulation efficiency, and pKa of mRNA-LNPs prepared using different cationic lipids

[0270] Table 2. Particle size, PDI, Zeta potential, and encapsulation efficiency of mRNA-LNPs prepared using different cationic lipids

[0271] The results in Tables 1 and 2 indicate that by adjusting the formulation ratio and process parameters, such as adjusting the ratio of cationic lipids to cholesterol, all compounds can be prepared into LNPs with small PDI, uniform particle size, and high encapsulation efficiency.

[0272] Experimental Example 2: Cell transfection assay of different lipid nanoparticles prepared using different cationic lipid compounds

[0273] Hela cells (Beina Biotech) in good growth condition and in the logarithmic growth phase were digested and resuspended, and then evenly inoculated into 96-well cell culture plates at a cell density of 2×10 4 Cells / well were placed in a cell culture incubator (Thermo Fisher, 1379) at 37°C and 5% CO2 and incubated overnight. 10 μL of sample diluent at different concentrations was added to a 96-well cell culture plate so that each well contained 100 ng of FFluc mRNA (SEQ ID NO.1), and 4 replicates were set for each sample. After incubation in a cell culture incubator for 24 h, 60 μL of lysis buffer containing D-luciferin potassium salt (Vazyme, DD1201-02) was added to each well to lyse the cells and release the luciferase. The cells were mixed by pipetting and incubated at room temperature for 6 min, then transferred to a white opaque bottom plate and the fluorescence intensity was detected using a microplate reader. The results are shown in Tables 3 and 4 below and in Figure 1.

[0274] Table 3. Transfection efficiency of lipid nanoparticles prepared with different cationic lipids

[0275] The results in Table 3 and Figure 1 show that compared with the existing commercially available cationic lipid MC3, the lipid nanoparticles prepared from ionizable cationic lipid materials in different proportions of the present invention all showed significantly higher in vitro FFluc mRNA (SEQ ID NO. 1) transfection efficiency in Hela cells in vitro. For example, the in vitro FFluc mRNA transfection efficiency of LNP-7, in which the molar ratio of cationic lipid compound 2 was 50%, was 23.23 times that of MC3 LNP-10; the in vitro FFluc mRNA transfection efficiency of LNP-9, in which the molar ratio of cationic lipid compound 2 was 57.1%, was 17.95 times that of MC3 LNP-10; and the in vitro FFluc mRNA transfection efficiency of LNP-5, in which the molar ratio of cationic lipid compound 2 was 43.3%, was 16.84 times that of MC3 LNP-10.

[0276] Table 4. Transfection efficiency of lipid nanoparticles prepared with different cationic lipids

[0277] The results in Table 4 show that, compared to the commercially available cationic lipid DOTAP, LNP-11 prepared from cationic lipid compound 3 of the present invention exhibited significantly higher in vitro FFluc mRNA transfection efficiency in Hela cells, which was 47.67 times that of DOTAP LNP-12. LNP-16 prepared from cationic lipid compound 6 exhibited 123.71 times that of DOTAP LNP-12.

[0278] Experimental Example 3: Animal transfection experiment of different lipid nanoparticles prepared with different cationic lipid compounds (24 h)

[0279] Balb / c mice (male, 6-8 weeks old, 20-24 g, source: Jiangwei Tonglihua Laboratory Animal Technology Co., Ltd.) were randomly divided into groups of 5. The prepared lipid nanoparticles were injected intramuscularly in the legs at a dose of 0.1 mg / kg (calculated based on FFluc mRNA (SEQ ID NO. 1)). In vivo imaging was performed 24 hours later. The abdominal and leg injection sites of the Balb / c mice were prepared. After anesthesia, D-luciferin potassium salt was injected intraperitoneally. The mice were observed and images were captured using an IVIS in vivo imager (PerkinElmer, Series III). In vivo imaging was performed within 15 minutes after substrate injection. The results are shown in Tables 5 and 6 below and in Figures 2-5.

[0280] Table 5. Animal transfection efficiency results of lipid nanoparticles prepared with different cationic lipids

[0281] The results in Table 5 and Figures 2 and 3 demonstrate that, compared to the commercially available cationic lipid MC3, the lipid nanoparticles (LNPs) prepared from the ionizable cationic lipid materials Compounds 1 and 2 of the present invention exhibited significantly higher transfection efficiency in thigh muscle at the injection site than the commercially available cationic lipid MC3. For example, the photon intensity at the injection site for LNP-1 prepared from Compound 1 was 2.820 times that of MC3 LNP-10, while the photon intensity at the injection site for LNP-3 prepared from Compound 2 was 1.607 times that of MC3 LNP. Furthermore, the in vivo transfection efficiency of FFluc mRNA at a 50% concentration of Compound 2 was comparable to that of MC3, and the in vivo transfection efficiency of Compound 1 at a 50% concentration was 1.668 times that of MC3.

[0282] Table 6. Animal transfection efficiency results of lipid nanoparticles prepared with different cationic lipids

[0283] The results in Table 6 and Figures 4 and 5 show that the LNP-11 prepared from the cationic lipid compound 3 of the present invention has significantly better in vivo transfection efficiency than the existing commercially available cationic lipid DOTAP LNP. The systemic luciferase expression is 1.41 times that of DOTAP LNP-12, and the photon intensity at the injection site is 3.12 times that of DOTAP LNP-12. This further demonstrates that the LNP-11 prepared from compound 3 of the present invention is mainly expressed at the injection site and has very weak expression in organs. Compared with DOTAP, it is expected to have an enhanced efficacy and reduced toxicity effect.

[0284] Experimental Example 4: Animal transfection experiment of different lipid nanoparticles prepared with different cationic lipid compounds (12 h)

[0285] Balb / c mice (male, 6-8 weeks old, 20-24 g, source: Jiangwei Tonglihua Laboratory Animal Technology Co., Ltd.) were randomly divided into groups of 5. The prepared lipid nanoparticles were injected intramuscularly in the legs at a dose of 0.1 mg / kg (calculated based on FFluc mRNA (SEQ ID NO. 1)). In vivo imaging was performed 12 hours later. The abdominal and leg injection sites of the Balb / c mice were prepared. After anesthesia, D-luciferin potassium salt was injected intraperitoneally. The mice were observed and images were captured using an IVIS in vivo imager (PerkinElmer, Series III). In vivo imaging was performed within 15 minutes after substrate injection. The results are shown in Tables 7 and 8 and Figures 6-9 below.

[0286] Table 7. Animal transfection efficiency results of lipid nanoparticles prepared with different cationic lipids

[0287] Compounds 1 and 2 of the present invention have certain advantages over the existing commercially available cationic lipid MC3. Specifically, the present invention explored the transfection characteristics of lipid nanoparticles LNP prepared by different formulation ratios compared to the commercially available MC3 formulation 12 hours after administration. As shown in Table 7 and Figures 6-7, the accumulation of all formulations in the thigh muscle at the injection site is not inferior to MC3 LNP, and specific formulations are significantly higher than MC3 LNP. For example, the photon intensity of LNP-27 prepared from compound 1 at the injection site is 11.23 times that of MC3 LNP; the photon intensity of LNP-33 prepared from compound 2 at the injection site is 3.48 times that of MC3 LNP. In addition, the expression of all formulations in the liver is close to that of MC3 LNP, indicating that while maintaining high transfection ability, it has similar liver toxicity and side effects as MC3 LNP.

[0288] Table 8. Animal transfection efficiency results of lipid nanoparticles prepared with different cationic lipids

[0289] As can be seen from Table 8 and Figures 8-9, 12 hours after administration, the accumulation of LNPs prepared from Compound 1, Compound 2, Compound 17, and Compound 18 of the present invention in the thigh muscle at the injection site was not inferior to that of MC3 LNP, and the LNPs prepared from Compound 17 and Compound 18 had almost no expression in the liver, indicating that the LNPs prepared from Compound 17 and Compound 18 achieved local transfection equivalent to that of MC3 LNP while completely avoiding liver toxicity.

[0290] Experimental Example 5: Immunogenicity Experiment of Different Lipid Nanoparticles Prepared with Different Cationic Lipid Compounds

[0291] C57BL / 6 mice (animal source: Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd., female, 6-8 weeks old, 20-22 g) were randomly divided into 3 groups, 5 mice in each group, and physiological saline, LNP-38, and LNP-39 were administered to the inner thigh muscles of the mice, respectively. The dosage was 10 μg / mouse according to the HPV mRNA (SEQ ID NO. 2). One week after immunization, the spleens of the mice were aseptically removed and the mouse spleen cell suspension (1×10 6 cells / mL) for later use.

[0292] IFN-γ secretion was detected using an ELISPOT kit. Mouse spleen cell suspensions were added to ELISPOT plates at a volume of 100 μL per well. Stimulants were mixed with the corresponding cell suspensions in the wells of the plates. No stimulant was added as a negative control (NS), PMA was used as a positive control, and the HPV 16 E7 peptide pool was used as the experimental group (E7-PepLib). The ELISPOT plates were incubated overnight in a cell culture incubator (Thermo Fisher, 1379) at 37°C and 5% CO2.

[0293] Remove the ELISPOT plate, discard all liquid, and wash. Add 100 μL of the prepared antibody solution to each well and incubate at room temperature for 2 hours. Then, discard the liquid and wash the plate. Add 100 μL of the prepared Streptavidin-ALP solution to each well and incubate at room temperature for 1 hour. Discard the liquid and wash the plate again. Add 100 μL of the colorimetric reagent to each well. After developing for 7 minutes, rinse with tap water to terminate the color development. Repeat three times and air-dry the ELISPOT plate. Count the spots using a spot analyzer.

[0294] As shown in Figure 10 , the immunogenicity results for LNP-37 prepared from Compound 1 of the present invention and LNP-38 prepared from the commercially available SM-102 showed no significant difference in mouse immunogenicity. This suggests that Compound 1 can serve as an effective carrier for mRNA vaccines, replacing the commercially available cationic lipid SM-102, effectively stimulating the immune response and achieving effective prevention and treatment of target diseases.

[0295] Experimental Example 6: Antitumor Experiments of Different Lipid Nanoparticles Prepared with Different Cationic Lipid Compounds

[0296] C3.43 cervical cancer cell suspension (1×10 7 cells / mL, 200 μL, cell source: Keck School of Medicine, University of Southern California; cells are HPV16 transgenic cell line, derived from C57BL / 6) were inoculated into the No. 3 site and under the right axillary fat pad of C57BL / 6 mice (animal source: Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd., female, 6-8 weeks old, 20-22 g) 10-12 days after inoculation. 3 At the same time, tumor-bearing mice were randomly divided into three groups, with five mice in each group. Normal saline, LNP-38, and LNP-39 were administered into the inner thigh muscle of the mice, respectively. The second administration was performed 7 days later. The dosage was calculated based on HPV mRNA (SEQ ID NO. 2), and the tumor inhibition rate was 10 μg / mouse. The results are shown in Table 9.

[0297] Table 9. Antitumor effects of lipid nanoparticles prepared with different cationic lipids

[0298] As shown in Table 9, after two administrations, LNP-39 prepared from Compound 1 of the present invention exhibited similar tumor inhibition effects as LNP-38 prepared from the commercially available cationic lipid SM-102. This suggests that Compound 1 can serve as an effective carrier for HPV mRNA vaccines, replacing the commercially available cationic lipid SM-102, effectively stimulating the body's immune response and inhibiting HPV-related tumor growth, offering new possibilities for future cancer treatments.

Claims

1. A cationic lipid compound represented by formula (I) or a pharmaceutically acceptable salt thereof: in, R 1 and R 2 Each independently is C6-C 20 Alkyl or C6-C 20 Alkenyl, or substituted C2-C5 alkyl, the substituent is C 16 -C 20 Alkyl-substituted -OC(=O)-, -C(=O)O-, -SC(=O)-, or -C(=O)S-; G1 and G2 are each independently C1-C4 alkylene; L1 and L2 are each independently -OC(=O)-, -C(=O)O-, -SC(=O)- or -C(=O)S-; R 3 is independently a 5-6 membered saturated heterocyclic group containing 1 or 2 ring heteroatoms independently selected from N, O and S, which is optionally replaced by R 6 replace; R 4 are independently C1-C4 alkyl, -OH or -SH, wherein the alkyl is optionally substituted by -OH or -SH; R 5 are independently absent or C1-C4 alkyl; R 6 are independently C1-C6 alkyl, which is optionally substituted with -OH or -SH.

2. The lipid compound according to claim 1, wherein R 1 and R 2 Each independently is C6-C 20 Alkyl or C6-C 20 Alkenyl, preferably C 10 -C 20 Alkyl or C 10 -C 20 Alkenyl, more preferably C 15 -C 20 Alkyl or C 15 -C 20 Alkenyl.

3. The lipid compound according to claim 1 or 2, wherein R 1 and R 2 Each independently 4. The lipid compound according to claim 1, wherein R 1 and R 2 are each independently a substituted C2-C5 alkyl group, preferably a substituted C4 alkyl group, wherein the substituent is a C 16 -C 20 Alkyl-substituted -OC(=O)-, -C(=O)O-, -SC(=O)-, or -C(=O)S-.

5. The lipid compound according to claim 1 or 4, wherein R 1 and R 2 Each independently 6. The lipid compound according to any one of claims 1 to 5, wherein G1 is independently a methylene group, and G2 is independently a methylene group or an ethylene group.

7. The lipid compound according to any one of claims 1 to 6, wherein R 3 are independently pyrrolidinyl or piperazinyl, which are optionally replaced by R 6 Replace, and R 6 is independently methyl, ethyl, propyl or butyl, which is optionally substituted with -OH or -SH.

8. The lipid compound according to any one of claims 1 to 7, wherein R 4 are independently methyl, hydroxymethyl, hydroxyethyl or -OH, and R 5 Does not exist.

9. The lipid compound according to any one of claims 1 to 7, wherein R 4 are independently methyl, and R 5 are independently methyl.

10. The lipid compound according to any one of claims 1 to 9, which is an ionizable cationic lipid compound represented by the following formula (Ia) or a pharmaceutically acceptable salt thereof:

11. The lipid compound according to any one of claims 1 to 9, which is a non-ionizable cationic lipid compound represented by the following formula (Ib) or a pharmaceutically acceptable salt thereof: in, X is a chlorine, bromine or iodine atom.

12. The lipid compound according to any one of claims 1 to 11, which is a cationic lipid compound or a pharmaceutically acceptable salt thereof selected from any one of the following:

13. A lipid nanoparticle composition comprising the cationic lipid compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof.

14. The lipid nanoparticle composition according to claim 13, further comprising a neutral lipid, cholesterol and a PEG lipid, wherein the neutral lipid is selected from the group consisting of DSPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, SOPE and 1,2-divaleryl-sn-glycero-3-phosphoethanolamine (trans-DOPE), and the PEG lipid is selected from the group consisting of PEG-DMG, PEG-dipalmitoylglycerol, PEG-DSPE, P EG-dilaurylglyceramide, PEG-dimyristylglyceramide, PEG-dipalmitoylglyceramide, and PEG-distearoylglyceramide, PEG-cholesterol (1-[8'-(cholest-5-en-3[β]-oxy)carboxamido-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB, mPEG-DMG-2k, PEG2k-DMG, PEG2k-DSPE, PEG2k-DSG, PEG2k-DMA, and PEG2k-DSA.

15. The lipid nanoparticle composition according to any one of claims 13 to 14, wherein the cationic lipid compound accounts for 15-60% by mole of the total lipid components in the composition.

16. The lipid nanoparticle composition according to any one of claims 13 to 15, further comprising a nucleic acid molecule selected from the group consisting of mRNA, siRNA, antisense oligonucleotides (ASO), saRNA and miRNA.

17. A method for delivering a nucleic acid into a cell, comprising delivering the lipid nanoparticle composition of any one of claims 13 to 16 into the cell, wherein the cell is a mammalian cell, preferably a human cell.

18. Use of the lipid nanoparticle composition according to any one of claims 13 to 16 in the preparation of a medicament for treating a disease.

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