Ionizable lipid and use thereof

Abstract

The present invention provides an ionizable lipid and a drug delivery system comprising the ionizable lipid. Specifically, the present invention provides an ionizable lipid having a structure of formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. Lipid nanoparticles constructed by using the ionizable lipid can realize safe and efficient delivery of nucleic acid drugs, small molecule drugs, peptide drugs and protein drugs.

Description

Ionizable lipids and their applications Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an ionizable lipid and its application in drug delivery. Background Art

[0002] In recent years, messenger RNA drugs have become a key therapeutic tool for the prevention and treatment of infectious diseases and tumors. The technology of messenger RNA drugs has been recognized by the industry. Thanks to its short R&D cycle, low risk of insertional mutagenesis and diversity of encoded proteins, mRNA is very suitable for the development of vaccines or therapeutic drugs. However, mRNA itself is very unstable and easily degraded by ubiquitous RNase. In addition, due to the inherent negative charge and large molecular weight of mRNA (usually greater than 10 6 Da), which limits the entry of mRNA molecules into cells. Therefore, it is of great significance to develop suitable delivery vehicles to protect fragile mRNA molecules and deliver them to the cytoplasm.

[0003] A variety of mRNA delivery vehicles have been developed, including lipid nanoparticles (LNPs), inorganic nanoparticles, polymer nanoparticles, viral vectors, and exosomes. LNPs are currently widely used as drug delivery vehicles. Their main components include ionizable lipids, phospholipids, cholesterol, and lipids containing polyethylene glycol. The most important component of LNPs is the ionizable lipid. Early, permanently positively charged cationic lipids suffered from short in vivo circulation times, high toxicity, and severe allergic reactions. This is because their inherent positive charge adsorbs proteins during circulation, making them easily captured and cleared by the reticuloendothelial system. Their inherent positive charge can interact with negatively charged cell membranes, leading to membrane destabilization and severe toxicity. Furthermore, permanently positively charged cationic lipids can activate the complement system and cause allergic reactions. Ionizable lipids are uncharged at physiological pH, and therefore, LNPs prepared from ionizable lipids have a relatively high safety profile. Ionizable lipids give LNPs the ability to escape lysosomes, causing LNPs to escape and release mRNA into the cytoplasm through the proton sponge effect and membrane fusion mechanism, where they bind to protein-encoding ribosomes and translate the encoded protein.

[0004] In short, the development of suitable ionizable lipids is one of the keys to developing LNPs with high safety and lysosomal escape efficiency.

[0005] Therefore, it is of great significance to develop an ionizable lipid with low toxicity and high delivery efficiency.

[0006] Summary of the Invention

[0007] The present invention provides an ionizable lipid with low toxicity and high delivery efficiency.

[0008] In a first aspect of the present invention, an ionizable lipid, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof is provided, wherein the ionizable lipid has the following structure:

[0009] in,

[0010] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14;

[0011] X and Y are each independently -CH- or N;

[0012] L1 has -(L 1a -L 1b )-structure or none, where L 1a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, preferably -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-; L 1b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4;

[0013] L2 has -(L 2a -L 2b )-structure or none; where L 2a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, preferably -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-; L 2b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4;

[0014] R3, R4, R5 and R6 are each independently H, CH3, C2-C 30 Hydrocarbon (such as C2-C 30 Alkyl, C2-C 30 Alkenyl, C2-C 30 Alkynyl), or -(CH2) s -Ra -(CH2) g -R b -(CH2) m -CH3, wherein s and g are each independently selected from a positive integer of 1-20, and m is selected from an integer of 0-20, preferably s+g+m is 2-35;

[0015] R a 、R b Each independently has no or is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, preferably selected from -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-;

[0016] Furthermore, R3 and R4 are not H at the same time; and R5 and R6 are not H at the same time;

[0017] R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1, 2, and 3.

[0018] In another preferred embodiment, R3, R4, R5 and R6 are each independently C4-C 30 Hydrocarbon (such as C4-C 30 Alkyl, C4-C 30 Alkenyl, C4-C 30 Alkynyl), preferably C4-C 20 Hydrocarbon (such as C4-C 20 Alkyl, C4-C 20 Alkenyl, C4-C 20 alkynyl).

[0019] In another preferred embodiment, at least two, three or four of R3, R4, R5 and R6 are C2-C30 hydrocarbon groups (such as C2-C 30 Alkyl, C2-C 30 Alkenyl, C2-C 30 Alkynyl), or -(CH2) s -R a -(CH2) g -R b -(CH2) m -CH3, where s, g, m, R a and R b As defined above.

[0020] In another preferred embodiment, the s+g+m is 3-20, more preferably 4-15.

[0021] In another preferred embodiment, the R3 has -R 3a -R 3b -R 3c -R 3d -R 3e structure,

[0022] Among them, R 3a and R 3c Each independently represents -(CH2) n -, wherein n is a positive integer selected from 1 to 14;

[0023] R 3b and R 3d Each independently has no or is selected from the following functional groups: -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-;

[0024] R 3e C2-C 20 Hydrocarbon group.

[0025] In another preferred embodiment, the R4 has -R 4a -R 4b -R 4c -R 4d -R 4e structure;

[0026] Among them, R 4a and R 4c Each independently represents -(CH2) n -, wherein n is a positive integer selected from 1 to 14;

[0027] R 4b and R 4dEach independently has no or is selected from the following functional groups: -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-;

[0028] R 4e C2-C 20 Hydrocarbon group.

[0029] In another preferred embodiment, the R5 has -R 5a -R 5b -R 5c -R 5d -R 5e structure,

[0030] Among them, R 5a and R 5c Each independently represents -(CH2) n -, wherein n is a positive integer selected from 1 to 14;

[0031] R 5b and R 5d Each independently has no or is selected from the following functional groups: O, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-;

[0032] R 5e C2-C 20 Hydrocarbon group.

[0033] In another preferred embodiment, the R6 has -R 6a -R 6b -R 6c -R 6d -R 6e structure;

[0034] Among them, R6a and R 6c Each independently represents -(CH2) n -, wherein n is a positive integer selected from 1 to 14;

[0035] R 6b and R 6d Each independently has no or is selected from the following functional groups: O, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-;

[0036] R 6e C2-C 20 hydrocarbon group;

[0037] R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1, 2, and 3.

[0038] In another preferred embodiment, X and Y are -CH-.

[0039] In another preferred embodiment, X and Y are N.

[0040] In another preferred embodiment, the ionizable lipid has a structure as shown in the following formula:

[0041] In another preferred embodiment, the ionizable lipid has a substructure represented by the following formula (I-1):

[0042] in,

[0043] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14;

[0044] L1 has -(L 1a -L 1b )-structure or none, where L 1ais selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, preferably -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-; L 1b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4;

[0045] L2 has -(L 2a -L 2b )-structure or none; where L 2a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; preferably -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-; L 2b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4;

[0046] R3, R4, R5 and R6 are each independently C2-C 20 hydrocarbon group;

[0047] R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are independently selected from 1, 2, and 3. In another preferred embodiment, X is N and Y is -CH-.

[0048] In another preferred embodiment, R6 is H.

[0049] In another preferred embodiment, the ionizable lipid has a structure shown in the following formula (I-2):

[0050] in,

[0051] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14;

[0052] L1 has -(L 1a -L 1b )-structure or none, where L 1ais selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, preferably -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-; L 1b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4;

[0053] L2 has -(L 2a -L 2b )-structure or none; where L 2a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, preferably -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-; L 2b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4;

[0054] R3 has -R 3a -R 3b -R 3c -R 3d -R 3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure of R5 has -R 5a -R 5b -R 5c -R 5d -R 5e structure;

[0055] Among them, R 3a 、R 3c 、R 4a 、R 4c 、R 5a 、R 5c Each independently represents -(CH2) n -, wherein n is a positive integer selected from 1 to 14;

[0056] R 3b 、R 3d 、R4b 、R 4d 、R 5b 、R 5d Each independently has no or is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-;

[0057] R 3e 、R 4e 、R 5e Each independently is C2-C 20 hydrocarbon group;

[0058] R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1, 2, and 3.

[0059] In another preferred embodiment, the ionizable lipid has a structure shown in the following formula (I-3):

[0060] in,

[0061] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14;

[0062] L1 has -(L 1a -L 1b )-structure or none, where L 1a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, preferably -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-; L 1b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4;

[0063] L2 has -(L 2a -L2b )-structure or none; where L 2a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, preferably -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-; L 2b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4;

[0064] R3 has -R 3a -R 3b -R 3c -R 3d -R 3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure of R5 has -R 5a -R 5b -R 5c -R 5d -R 5e The structure of R6 has -R 6a -R 6b -R 6c -R 6d -R 6e structure;

[0065] Among them, R 3a 、R 3c 、R 4a 、R 4c 、R 5a 、R 5c 、R 6a and R 6c Each independently represents -(CH2) n -, wherein n is a positive integer selected from 1 to 14;

[0066] R 3b 、R 3d 、R 4b 、R 4d 、R 5b 、R 5d 、R 6b and R 6dEach independently has no or is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-;

[0067] R 3e 、R 4e 、R 5e 、R 6e Each independently is C2-C 20 hydrocarbon group;

[0068] R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1, 2, and 3.

[0069] In another preferred embodiment, the ionizable lipid has a structure shown in formula (I-1), and in the formula (I-1):

[0070] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 2 to 8;

[0071] L1 is selected from the following functional groups: -(C=O)O-, -O(C=O)-;

[0072] L2 is selected from the following functional groups: -(C=O)O-, -O(C=O)-;

[0073] R3, R4, R5 and R6 are each independently C5-C 20 hydrocarbon group;

[0074] R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1 or 2.

[0075] In another preferred embodiment, the ionizable lipid has a structure shown in formula (I-2), and in the formula (I-2):

[0076] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 2 to 8;

[0077] L1 is none or -O-(CH2) n -; wherein n is selected from 0, 1, 2, 3 or 4;

[0078] L2 is selected from the following functional groups: -(C=O)O-, -O(C=O)-;

[0079] R3 has -R 3a -R 3b -R 3c -R 3d -R 3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure of R5 has -R 5a -R 5b -R 5c -R 5d -R 5e structure;

[0080] Among them, R 3a 、R 3c 、R 4a 、R 4c 、R 5a 、R 5c Each independently represents -(CH2) n -, wherein n is a positive integer selected from 1 to 3;

[0081] R 3b and R 4b Each independently selected from the group consisting of: -(C=O)O-, -O(C=O)-;

[0082] R 3d 、R 4d 、R 5d Each is independently selected from the group consisting of: -CH2-, -(SS)-;

[0083] R 5b for nothing;

[0084] R 3e 、R 4e 、R 5e Each independently is C2-C 15 hydrocarbon group;

[0085] R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1 or 2.

[0086] In another preferred embodiment, the ionizable lipid has a structure shown in formula (I-1), and in the formula (I-1):

[0087] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 2 to 8;

[0088] L1 is selected from the following functional groups: -NH(C=O)-, -(C=O)NH-;

[0089] L2 is selected from the following functional groups: -NH(C=O)-, -(C=O)NH-;

[0090] R3, R4, R5 and R6 are each independently C5-C 20 hydrocarbon group;

[0091] R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1 or 2.

[0092] In another preferred embodiment, the ionizable lipid has a structure shown in formula (I-1), and in the formula (I-1):

[0093] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 2 to 8;

[0094] L1 is selected from the following functional groups: -(C=O)S-, -S(C=O)-;

[0095] L2 is selected from the following functional groups: -(C=O)S-, -S(C=O)-;

[0096] R3, R4, R5 and R6 are each independently C5-C 20 hydrocarbon group;

[0097] R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1 or 2.

[0098] In another preferred embodiment, the ionizable lipid has a structure shown in formula (I-3), and in the formula (I-3):

[0099] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 2 to 8;

[0100] L1 is none or -O-(CH2) n -; wherein n is selected from 0, 1, 2, 3 or 4;

[0101] L2 is none or -O-(CH2) n -; wherein n is selected from 0, 1, 2, 3 or 4;

[0102] R3 has -R 3a -R 3b -R 3c -R 3d -R 3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure of R5 has -R 5a -R 5b -R 5c -R 5d -R 5e The structure of R6 has -R 6a -R 6b -R 6c -R 6d -R 6e structure;

[0103] Among them, R 3a 、R 3c 、R 4a 、R 4c 、R 5a 、R 5c 、R 6a 、R 6c Each independently represents -(CH2) n -, wherein n is a positive integer selected from 1 to 3;

[0104] R 3b 、R 4b 、R 5b and R 6b Each independently selected from the group consisting of: -(C=O)O-, -O(C=O)-, -CH(OH)-;

[0105] R 3d 、R 4d 、R 5d and R 6d Each is independently selected from the group consisting of: -CH2-, -(SS)-;

[0106] R 3e 、R 4e 、R 5e and R 6e Each independently is C2-C 15 hydrocarbon group;

[0107] R7 is selected from C1-C5 hydrocarbon group, or -(CH2)m O(CH2) n -, wherein m and n are each independently selected from 1 or 2.

[0108] In another preferred embodiment, R3, R4, R5 and R6 are each independently selected from the following groups: C5-C 15 Alkyl, C5-C 15 C5-C 15 Alkynyl;

[0109] R7 is selected from C1-C5 alkyl, -(CH2)O(CH2)-, or -(CH2)2O(CH2)2-, wherein m and n are each independently selected from 1, 2, and 3.

[0110] In another preferred embodiment, the ionizable lipid has a structure selected from the following Table 1:

[0111] Table 1

[0112] In another preferred embodiment, R3, R4, R5 and R6 are each independently C5-C 15 of alkyl.

[0113] In another preferred embodiment, the ionizable lipid has a structure shown in the following formula (I-1):

[0114] in,

[0115] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 4 to 8;

[0116] L1 is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-;

[0117] L2 is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-;

[0118] R3 has -R 3a -R 3b -R 3c -R 3d -R3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure of R5 has -R 5a -R 5b -R 5c -R 5d -R 5e The structure of R6 has -R 6a -R 6b -R 6c -R 6d -R 6e structure;

[0119] Among them, R 3a 、R 3c 、R 4a 、R 4c 、R 5a 、R 5c 、R 6a and R 6c Each independently represents -(CH2) n -, wherein n is selected from a positive integer of 1-14; and R3, R4, R5 and R6 each independently have 4-20 CH2 structural fragments;

[0120] R 3b 、R 3d 、R 4b 、R 4d 、R 5b 、R 5d 、R 6b and R 6d Each is independently absent or selected from the following functional groups: -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-;

[0121] R 3e 、R 4e 、R 5e 、R 6e Each independently is C2-C 20 hydrocarbon group;

[0122] R7 is a C1-C3 hydrocarbon group, or -(CH2)2-O-(CH2)2-.

[0123] In another preferred embodiment, the ionizable lipid has a structure shown in the following formula:

[0124] The second aspect of the present invention provides a method for preparing the ionizable lipid according to the first aspect of the present invention, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, the preparation method comprising: method I, method II and method III;

[0125] Wherein, method 1 comprises the following steps:

[0126] (S1) Under inert gas protection, reacting compound A1 and A2 to obtain compound A3;

[0127] (S2) Under inert gas protection, A3 is de-tert-butyloxycarbonyl (Boc) to obtain A4;

[0128] (S3) Under inert gas protection, A4 reacts with A5 or A6 to obtain A7 (i.e., the compound shown in (I-1));

[0129] wherein R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14;

[0130] G1 and G2 are each independently selected from active functional groups, preferably carbonyl, halogen (fluorine, chlorine, bromine, iodine, etc.), ethylene oxide, etc.;

[0131] L1 has -(L 1a -L 1b )-structure or none, where L 1a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, preferably -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-; L 1b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4;

[0132] L2 has -(L 2a -L 2b )-structure or none; where L 2ais selected from the following functional groups: O, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, preferably O, -(C=O)O-, -O(C=O)-, -CH(OH)-; L 2b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4;

[0133] R3, R4, R5 and R6 are each independently C2-C 20 Hydrocarbon group.

[0134] R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1, 2, and 3;

[0135] Method II includes the following steps:

[0136] (D1) reacting compound B1 with B2 under inert gas protection to obtain compound B3;

[0137] (D2) reacting compounds B3 and B4 under inert gas protection to obtain compound B5;

[0138] (D3) Under inert gas protection, reacting compound B5 and B6 to obtain B7, the compound shown in (I-2);

[0139] wherein R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14;

[0140] G1 and G2 are each independently selected from active functional groups, preferably carbonyl, halogen (fluorine, chlorine, bromine, iodine, etc.), ethylene oxide, etc.;

[0141] L1 has -(L 1a -L 1b )-structure or none, where L 1a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, preferably -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-; L1b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4;

[0142] L2 has -(L 2a -L 2b )-structure or none; where L 2a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, preferably -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-; L 2b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4;

[0143] R3 has -R 3a -R 3b -R 3c -R 3d -R 3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure of R5 has -R 5a -R 5b -R 5c -R 5d -R 5e structure;

[0144] Among them, R 3a 、R 3c 、R 4a 、R 4c 、R 5a 、R 5c Each independently represents -(CH2) n -, wherein n is a positive integer selected from 1 to 14;

[0145] R 3b 、R 3d 、R 4b 、R 4d 、R 5b 、R 5dEach independently has no or is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-;

[0146] R 3e 、R 4e 、R 5e Each independently is C2-C 20 hydrocarbon group;

[0147] R6 is H;

[0148] R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1, 2, and 3;

[0149] Method III comprises the following steps:

[0150] (M1) Under inert gas protection, compounds C1 and C2 react to produce C3;

[0151] (M2) Under inert gas protection, compound C3 is de-Bocized to generate compound C4;

[0152] (M3) Under inert gas protection, compound C4 reacts with C5 or C6 to produce C7, i.e., the compound represented by formula (I-3);

[0153] wherein R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14;

[0154] G1 and G2 are each independently selected from active functional groups, preferably carbonyl, halogen (fluorine, chlorine, bromine, iodine, etc.), ethylene oxide, etc.;

[0155] L1 has -(L 1a -L 1b )-structure or none, where L 1ais selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, preferably -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-; L 1b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4;

[0156] L2 has -(L 2a -L 2b )-structure or none; where L 2a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, preferably -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-; L 2b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4;

[0157] R3 has -R 3a -R 3b -R 3c -R 3d -R 3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure of R5 has -R 5a -R 5b -R 5c -R 5d -R 5e The structure of R6 has -R 6a -R 6b -R 6c -R 6d -R 6e structure;

[0158] Among them, R 3a 、R 3c 、R 4a 、R 4c 、R 5a 、R 5c 、R 6a and R6c Each independently represents -(CH2) n -, wherein n is a positive integer selected from 1 to 14;

[0159] R 3b 、R 3d 、R 4b 、R 4d 、R 5b 、R 5d 、R 6b and R 6d Each independently has no or is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-;

[0160] R 3e 、R 4e 、R 5e 、R 6e Each independently is C2-C 20 hydrocarbon group;

[0161] R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1, 2, and 3.

[0162] The third aspect of the present invention provides a lipid nanoparticle (LNP), which comprises the ionizable lipid as described in the first aspect of the present invention, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0163] In another preferred embodiment, the lipid nanoparticles further comprise helper lipids.

[0164] In another preferred embodiment, in the lipid nanoparticles, the content of the ionizable lipid is 30-65% by mole of the total lipid content.

[0165] In another preferred embodiment, the helper lipid comprises a helper phospholipid, a sterol, a polymer-conjugated lipid, or a combination thereof.

[0166] In another preferred embodiment, the helper lipid is a combination of helper phospholipids, sterols and polymer-conjugated lipids.

[0167] In another preferred embodiment, the auxiliary phospholipid is preferably selected from: 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine, 1,2-myristoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, 1,2-palmitoylphosphatidylglycerol, 1-palmitoyl-2-oleoyl phosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, 1-stearoyl-2-oleoyl-phosphatidylethanolamine, or a combination thereof.

[0168] In another preferred embodiment, the sterol includes cholesterol or a cholesterol derivative.

[0169] In another preferred embodiment, the polymer-conjugated lipid is a PEGylated lipid.

[0170] In another preferred embodiment, the PEGylated lipid is preferably selected from the following group: DMG-PEG2000, DSPE-PEG2000, DSG-PEG2000, DSPE-PEG-Mannose, DMG-PEG2000-(polypeptides, proteins, amino acids, vitamins and other active substances) or a combination thereof.

[0171] In another preferred embodiment, the lipid nanoparticles contain ionizable lipids, DSPC, cholesterol and DMG-PEG2000, wherein the molar ratio of ionizable lipids:DSPC:cholesterol:DMG-PEG2000 is (30-65):(5-30):(30-55):(1-5), preferably (40-50):(10-15):(38-45):(1.5-2).

[0172] In another preferred embodiment, the lipid nanoparticles further comprise a bioactive substance encapsulated in the lipid nanoparticles.

[0173] In another preferred embodiment, the bioactive substance is selected from the group consisting of nucleic acids, proteins, polypeptides, small molecules, or a combination thereof.

[0174] In another preferred embodiment, the nucleic acid includes DNA, plasmid, messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotide, small RNA, ribosomal RNA, microRNA and transfer RNA, preferably mRNA.

[0175] A fourth aspect of the present invention provides a lipid nanoparticle pharmaceutical preparation, comprising:

[0176] i) the lipid nanoparticles according to the third aspect of the present invention;

[0177] ii) a bioactive substance encapsulated in the lipid nanoparticles; and

[0178] iii) a pharmaceutically acceptable carrier.

[0179] In another preferred embodiment, the bioactive substance is selected from the group consisting of nucleic acids, proteins, polypeptides, small molecules, or a combination thereof.

[0180] In another preferred embodiment, the nucleic acid includes DNA, plasmid, messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotide, small RNA, ribosomal RNA, microRNA and transfer RNA, preferably mRNA.

[0181] In another preferred embodiment, the bioactive substance is a nucleic acid, and in the lipid nanoparticle drug, the molar ratio of the ionizable nitrogen atom in the ionizable lipid molecule to the phosphate group in the nucleic acid molecule is (2-10):1, more preferably (4-8):1.

[0182] In another preferred embodiment, the hydrated particle size of the lipid nanoparticle drug is 50-200 nm, preferably 70-150 nm, and most preferably 75-110 nm.

[0183] In another preferred embodiment, the lipid nanoparticle pharmaceutical preparation can be used for the treatment and / or prevention of tumors, infectious diseases and rare diseases.

[0184] In another preferred embodiment, the dosage form of the lipid nanoparticle pharmaceutical preparation is selected from the following group: injection, lyophilized preparation, nebulized inhalation preparation, and smear-type preparation.

[0185] In another preferred embodiment, the lipid nanoparticle pharmaceutical preparation is administered by injection, i.e., intravenous, intramuscular, intradermal, subcutaneous, intrathecal, intraduodenal or intraperitoneal injection.

[0186] In another preferred embodiment, the lipid nanoparticle pharmaceutical preparation is administered by inhalation, such as intranasal administration.

[0187] In another preferred embodiment, the lipid nanoparticle pharmaceutical preparation is administered transdermally, such as by transdermal application or electrode introduction.

[0188] A fifth aspect of the present invention provides a method for preparing the lipid nanoparticle pharmaceutical preparation according to the fourth aspect of the present invention, the method comprising:

[0189] (a) mixing the ionizable lipid according to the first aspect of the present invention or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof and an optional helper lipid with an organic solvent to obtain a lipid organic phase;

[0190] (b) mixing the bioactive substance with an aqueous solvent to obtain an aqueous phase containing the bioactive substance;

[0191] (c) mixing the lipid organic phase in step (a) with the aqueous phase in step (b) to obtain the lipid nanoparticle drug.

[0192] In another preferred embodiment, the organic solvent includes ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, tetrahydrofuran or a combination thereof.

[0193] In another preferred embodiment, the aqueous solvent is a buffer solution.

[0194] In another preferred embodiment, the aqueous solvent is a buffer solution with a pH range of 3-7.

[0195] In another preferred embodiment, the acidic buffer is a citrate buffer with a pH of 4.0.

[0196] In another preferred embodiment, the volume ratio of the lipid organic phase to the aqueous phase containing the bioactive substance is 1:(2-5), preferably 1:(3-4).

[0197] In another preferred embodiment, in step (c), the lipid organic phase and the aqueous phase are mixed through a microfluidic chip.

[0198] In another preferred embodiment, the method further comprises step (d): purifying, concentrating and filtering and sterilizing the lipid nanoparticle drug obtained in step (c).

[0199] The sixth aspect of the present invention provides a use of the ionizable lipid as described in the first aspect of the present invention, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof for preparing a drug delivery system.

[0200] In another preferred embodiment, the delivery system includes lipid nanoparticles (LNP), liposomes, polymer nanoparticles, etc., which are preferably used to prepare lipid nanoparticles.

[0201] In another preferred embodiment, the drug delivery system is used to deliver drugs for treating and / or preventing tumors, infectious diseases and rare diseases.

[0202] The seventh aspect of the present invention provides a use of the lipid nanoparticles as described in the third aspect of the present invention in the preparation of drugs for treating and / or preventing tumors, infectious diseases and rare diseases.

[0203] In an eighth aspect, the present invention provides the use of the ionizable lipid or a pharmaceutically acceptable salt thereof according to the first aspect of the present invention for preparing a lipid nanoparticle pharmaceutical preparation, wherein the lipid nanoparticle pharmaceutical preparation is used to deliver a biologically active substance to cells in a subject in need thereof.

[0204] In another preferred embodiment, the bioactive substance is selected from the group consisting of DNA, RNA (mRNA, tRNA, rRNA, miRNA), natural and synthetic oligonucleotides (including antisense oligonucleotides (ASO), interfering RNA (RNAi) and small interfering RNA (siRNA)).

[0205] In another preferred embodiment, the bioactive substance is mRNA.

[0206] In another preferred embodiment, the mRNA is transfected into cells and expressed in the cells.

[0207] In another preferred embodiment, the cells are immune cells.

[0208] In another preferred embodiment, the cells include T cells, B cells, NK cells, or a combination thereof.

[0209] In another preferred embodiment, the T cells are selected from the following group: human primary T cells, JM cells, Jurkat cells, or a combination thereof.

[0210] In another preferred embodiment, the human primary T cells include helper T cells, regulatory T cells, and memory T cells.

[0211] In another preferred embodiment, the ionizable lipid has a structure shown in the following formula:

[0212] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0213] Figure 1 shows the particle size characterization results of LNP-mRNA with different formulations composed of AXT-8, with a particle size range of 80-130 nm, demonstrating that the LNPs prepared above have similar physicochemical properties (Note: in the above legend, LNP(AXT-8)-Luc-1 indicates that LNP composed of the molecule AXT-8 encapsulates Luc mRNA, 1 indicates formulation 1 in the LNP series encapsulating Luc mRNA, and so on; LNP(AXT-8)-hEPO-1 indicates that LNP composed of the molecule AXT-8 encapsulates EPO mRNA, 1 indicates formulation 1 in the LNP series encapsulating EPO mRNA, and so on 11; LNP(AXT-8)-eGFP-1 indicates that LNP composed of the molecule AXT-8 encapsulates eGFP mRNA, 1 indicates formulation 1 in the LNP series encapsulating eGFP mRNA; LNP(AXT-8)-mCherry-1 indicates that LNP composed of the molecule AXT-8 encapsulates mCherry mRNA, 1 indicates formulation 1 in the LNP series encapsulating mCherry mRNA).

[0214] Figure 2 shows the particle size distribution characterization results of different formulations of LNP-mRNA composed of AXT-8. PDI < 0.2 proves that the particle size distribution of nanoparticles composed of AXT-8 is small.

[0215] Figure 3 shows the encapsulation efficiency characterization results of LNP-mRNA with different formulations composed of AXT-8. The encapsulation efficiency is >90%, which proves that the encapsulation results are good.

[0216] FIG4 shows the transfection results of LNP-mRNA with different ratios composed of AXT-8 in 293T cells. As shown in the figure, with the increase of mRNA concentration, the cell expression results of multiple LNP (AXT-8)-mRNA formulas are better than lipofectamine.

[0217] Figure 5 shows the cytotoxicity results of LNP (AXT-8)-mRNA composed of AXT-8. As shown in the figure, the cell inhibition rate of LNP (AL-8) series products is almost 0 when the mRNA concentration is lower than 1ug / mL.

[0218] FIG6 shows the in vivo hEPO expression results of LNP-mRNA of different AXT-8 formulations. As can be seen from the figure, for different formulations, the in vivo mRNA expression trend is to increase first and then decrease, and all reach the maximum around 6 hours.

[0219] Figure 7 shows the expression results of LNP-mRNA composed of AXT-8 in JM cells and Jurkat cells in vitro transfection. As can be seen from the figure, LNP (AXT-8)-mRNA can be successfully expressed in JM cells and Jurkat cells, and the expression levels are better than lipofectamine Max.

[0220] Figure 8 shows the expression results of LNP-mRNA composed of AXT-8 in vitro transfection of human primary T cells. As can be seen from the figure, LNP (AXT-8)-mRNA can be successfully expressed in human primary T cells, and the expression level is better than lipofectamine Max. DETAILED DESCRIPTION

[0221] The inventor has unexpectedly discovered a kind of ionizable lipid through extensive and in-depth research for the first time, and the ionizable lipid has the advantages of stable physicochemical properties, low toxicity, and the drug delivery system obtained by using the ionizable lipid encapsulated drug load (such as mRNA) of the present invention has high delivery efficiency and low toxicity, and in the efficient delivery of drug load, while improving the expression amount of drug load, improves the safety of drug delivery system, makes the prevention and treatment effect of drug delivery system more outstanding.On this basis, complete the present invention.

[0222] the term

[0223] In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning given below.

[0224] The term "alkyl" refers to a saturated carbon chain having 1 to 20 carbon atoms, which may be straight or branched, or a combination thereof, unless otherwise defined. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl and tert-butyl, pentyl, hexyl, heptyl, octyl, and the like.

[0225] Unless otherwise specified in the specification, an alkyl group is optionally substituted. The term "unsaturated hydrocarbon group" means a group containing at least one C=C double bond (alkenyl) or at least one C≡C triple bond (alkynyl). "Alkyl," "alkenyl," and "alkynyl" may be collectively referred to as "hydrocarbon group."

[0226] In the claims of the present invention, when describing a "C1-C30 hydrocarbon group", it means that the group may be an alkane group, an alkene group or an alkyne group having 1 to 30 carbon atoms (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms). "C1-C5 hydrocarbon group", "C4-C30 Hydrocarbon", "C2-C 30 "Hydrocarbon" has a similar meaning.

[0227] When describing an "alkyl group," it is intended that the group may be a saturated hydrocarbon group having a given number of carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms), which may be straight-chain or branched, and is typically a chain group without a cyclic structure. Alkyl groups meeting the aforementioned number of carbon atoms are included within the scope of this term.

[0228] When describing "alkenyl", it means that the group can be an alkene group with a given number of carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms), which can be straight chain or have a branched structure, usually a chain group without a cyclic structure. Alkenyl groups that meet the aforementioned number of carbon atoms are all within the scope of this term. In different embodiments of the present invention, the alkenyl group can be a group formed by a monoolefin or a polyolefin (such as a diene).

[0229] When describing “C2-C 30 When the term "straight-chain or branched olefin group" is used, it means that the group can be an olefin group with a given number of carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms), which can be straight-chain or have a branched structure, usually a chain group without a cyclic structure. Olefin groups that meet the aforementioned number of carbon atoms are within the scope of the description of this term. In different embodiments of the present invention, the olefin group can be a monoolefin or a polyolefin (such as a diene).

[0230] When describing “C1-C 10 When the term "alkylene" is used, it means that the group may be an alkylene group having 1-10 carbon atoms (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), and the alkylene group may be a straight chain or branched chain structure, usually a chain group without a cyclic structure.

[0231] In the present application, when the definition of a divalent group includes “none”, it means that adjacent structural fragments of the divalent group are directly connected by chemical bonds.

[0232] Ionizable lipids

[0233] As used herein, the terms "ionizable lipid of the present invention" and "ionizable cationic lipid of the present invention" are used interchangeably and refer to a lipid compound having the structure of Formula I, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0234] Ionizable lipids are protonated and converted into cationic lipids at low pH values, and then converted into auxiliary phospholipids at normal physiological pH values. These auxiliary phospholipids interact less with the anionic cell membranes of blood cells, improving the biocompatibility of lipid nanoparticles. Once lipid nanoparticles are internalized by cells, the pH in the endosomes is low, and the lipids are protonated, becoming positively charged. This destabilizes or even destroys the membrane structure, facilitating the escape of lipid nanoparticles from endosomal structures. In short, the pH-sensitive nature of lipids facilitates the in vivo delivery of lipid nanoparticles loaded with bioactive ingredients (such as mRNA molecules).

[0235] Helper lipids

[0236] As used herein, the term "helper lipid" refers to other types of lipids in lipid nanoparticles, in addition to ionizable lipids, including helper phospholipids, sterols, polymer-conjugated lipids, or combinations thereof. Helper lipids are primarily used to improve the performance of lipid nanoparticles, such as stability, delivery efficiency, tolerability, and bioavailability.

[0237] In some embodiments, the auxiliary phospholipids include (but are not limited to) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DSPC), 1,2-diole ... 1-Hydroxy- ...

[0238] In a preferred embodiment of the present invention, the auxiliary phospholipid is DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine, also known as distearoylphosphatidylcholine). DSPC is a commonly used phosphatidylcholine. The tail group of DSPC is a saturated alkane chain, with a melting point of -54°C. It has a cylindrical morphology and forms a lamellar structure in the lipid nanoparticles, making the lipid nanoparticles more stable.

[0239] In a preferred embodiment of the present invention, the auxiliary phospholipid is DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, also known as dioleoylphosphatidylethanolamine). DOPE is a commonly used phosphatidylethanolamine. The tail groups of DOPE are two unsaturated alkane chains. It has a melting point of -30°C and a conical morphology. It easily forms an inverted hexagon in lipid nanoparticles, causing endosomal membrane instability and facilitating the endosomal escape of lipid nanoparticles.

[0240] In some embodiments, the sterol includes (but is not limited to) cholesterol or cholesterol derivatives. Cholesterol can adjust the integrity and hardness of the lipid membrane and enhance the stability of lipid nanoparticles, while the morphology of cholesterol derivatives can affect the delivery efficiency and biodistribution of lipid nanoparticles, such as the chain length of the hydrophobic tail group of the cholesterol analog, the flexibility of the sterol ring, the polarity of the hydroxyl group, etc. Cholesterol also affects the morphology of lipid nanoparticles. Cholesterol derivatives allow the formed lipid nanoparticles to have a multi-layered polyhedral structure divided by lipids rather than a spherical morphology. At the same time, cholesterol affects the selectivity of lipid nanoparticles for targets: lipid nanoparticles containing cholesterol oleate have higher selectivity for liver endothelial cells than for hepatocytes; when containing cholesterol with an oxidized tail group, the content of lipid nanoparticles in liver endothelial cells and Kupffer cells is higher than that in hepatocytes.

[0241] In some embodiments, the polymer-conjugated lipid is a polyethylene glycol (PEG)-conjugated lipid, also known as a PEGylated lipid or PEGylated lipid. PEGylated lipids have multiple effects on the properties of lipid nanoparticles: the amount of PEGylated lipid affects the particle size and potential of lipid nanoparticles; particle aggregation is reduced, thereby improving the stability of lipid nanoparticles; the particle clearance rate mediated by the kidneys and the mononuclear phagocyte system (MPS) is reduced, thereby prolonging the particle circulation time; and surface functional groups can be modified with ligands to enhance targeted delivery. The molar mass and lipid length influence the properties of PEGylated lipids. DMG-PEG2000 and DSG-PEG2000 are both neutral phospholipids with saturated alkyl chain lengths of C14, C16, or C18, respectively. However, DMG-PEG2000 dissociates from lipid nanoparticles more quickly, facilitating cellular uptake and endosomal escape of the nanoparticles. Therefore, the delivery efficiency of DMG-PEG2000 is superior to that of DSG-PEG2000.

[0242] In a preferred embodiment of the present invention, the helper lipid is a combination of DSPC, cholesterol and DMG-PEG2000.

[0243] Lipid nanoparticles (LNPs)

[0244] As used herein, the terms "lipid nanoparticle," "lipid nanoparticle," or "LNP" refer to particles having a diameter of about 5 to 500 nm. In some embodiments, the lipid nanoparticle contains one or more active agents (biologically active substances). In some embodiments, the lipid nanoparticle includes nucleic acids. In some embodiments, the nucleic acids are condensed in the interior of the nanoparticle with cationic lipids, polymers, or multivalent small molecules and an outer lipid coating that interacts with the biological environment. Due to the repulsive forces between the phosphate groups, nucleic acids are naturally rigid polymers that prefer an elongated configuration. In cells, in order to cope with volume constraints, DNA can package itself with the help of ions and other molecules under appropriate solution conditions. Generally, DNA condensation is defined as the collapse of an extended DNA chain into a compact, ordered particle containing only one or a few molecules. By binding to phosphate groups, cationic lipids can condense DNA by neutralizing the phosphate charge and causing it to pack tightly.

[0245] In some embodiments, the bioactive substance is encapsulated into the LNP. In some embodiments, the bioactive substance can be an anionic compound, including but not limited to DNA, RNA (messenger RNA, transfer RNA, ribosomal RNA, microRNA, etc.), natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNA and small interfering RNA), nucleoproteins, peptides, nucleic acids, ribozymes, DNA-containing nucleoproteins, such as complete or partially deproteinized virus particles (virions), oligomeric and polymeric anionic compounds other than DNA (such as acidic polysaccharides and glycoproteins). In some embodiments, the bioactive substance can be mixed with an adjuvant.

[0246] In LNP vaccine products, the bioactive substance is typically contained within the LNP. In some embodiments, the bioactive substance includes a nucleic acid. Typically, the water-soluble nucleic acid is condensed with a cationic lipid or polycationic polymer within the particle, and the particle surface is enriched with auxiliary phospholipids or PEG lipid derivatives. Additional ionizable cationic lipids may also be located on the surface. Upon entering the cell lysosome, the ionizable cationic lipids are ionized by the acidic environment of the lysosome and become positively charged, interacting with the lysosomal membrane and promoting endosomal escape.

[0247] About LNP, ionizable lipid can have different properties or functions.Due to the pKa of amino group, when external pH value is lower than the pKa of lipid molecule, it can be protonated and positively charged.Under these conditions, lipid molecule can be electrostatically bound to the phosphate group of nucleic acid, which makes LNP formation and nucleic acid be encapsulated, and the surface charge of LNP in the biological fluid (such as blood) of physiological pH value is basically neutral.High LNP surface charge and toxicity, fixation and free macrophage rapid clearance circulation, hemolytic toxicity, including immune activation relevant (people such as Filion Biochim Biophys Acta. October 23, 1997; 1329 (2): 345-56).

[0248] In some embodiments, the pKa can be high enough so that the ionizable cationic lipid can adopt a positively charged form at acidic endosomal pH. In this way, the cationic lipid can be combined with endogenous endosomal anionic lipids to promote membrane lysis and non-bilayer structures, such as hexagonal HII phases, thereby resulting in more effective intracellular delivery. In some embodiments, the pKa range is between 6.2 and 6.5. For example, the pKa can be about 6.2, about 6.3, about 6.4, or about 6.5. Unsaturated tails also contribute to the ability of lipids to adopt non-bilayer structures. (Jayaraman et al., Angew Chem Int Ed Engl. 2012 Aug 20;51(34):8529-33).

[0249] The release of nucleic acids from LNP formulations, as well as other characteristics such as liposomal clearance and circulation half-life, can be modified by the presence of polyethylene glycol and / or sterols (e.g., cholesterol) or other potential additives in the LNP, as well as the overall chemical structure, including the pKa of any ionizable cationic lipids that are part of the formulation.

[0250] In one aspect of the present invention, a lipid nanoparticle (LNP) is provided, comprising the ionizable lipid described in the first aspect of the present invention, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. Further, the lipid nanoparticle further comprises one or more helper lipids, including helper phospholipids, steroids, and polymer-conjugated lipids.

[0251] Lipid nanoparticle drug preparations

[0252] In another aspect of the present invention, a lipid nanoparticle pharmaceutical preparation (or lipid nanoparticle pharmaceutical combination or LNP composition) is provided, comprising the lipid nanoparticles as described in the third aspect of the present invention, a bioactive substance encapsulated in the lipid nanoparticles, and a pharmaceutically acceptable carrier. The lipid nanoparticle pharmaceutical preparation is used to deliver the bioactive substance to cells in a subject in need thereof.

[0253] In some embodiments, the bioactive substance is encapsulated into the LNP. In some embodiments, the bioactive substance can be an anionic compound, including but not limited to DNA, RNA (messenger RNA, transfer RNA, ribosomal RNA, microRNA, etc.), natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNA and small interfering RNA), nucleoproteins, peptides, nucleic acids, ribozymes, DNA-containing nucleoproteins, such as complete or partially deproteinized virus particles (virions), oligomeric and polymeric anionic compounds other than DNA (such as acidic polysaccharides and glycoproteins). In some embodiments, the bioactive substance can be mixed with an adjuvant.

[0254] In some embodiments, the LNP composition comprises: a nucleic acid, an ionizable cationic lipid having a structure as shown in formula (I), an auxiliary phospholipid (e.g., DSPC, DOPE, DOPC, or a combination thereof), a sterol (e.g., cholesterol or a cholesterol derivative, or a plant sterol such as β-sitosterol) and a polymer-conjugated lipid (e.g., DMG-PEG2000). In some embodiments, the LNP composition comprises: a nucleic acid; an ionizable cationic lipid having a structure as shown in formula I, the content of which is 30-65% (molar ratio, the same below) of the total lipids of the composition; an auxiliary phospholipid (e.g., DSPC, DOPE, DOPC, or a combination thereof) the content of which is 5-30% of the total lipids of the composition; a sterol (e.g., cholesterol or a cholesterol derivative, or a plant sterol such as β-sitosterol) the content of which is 30-55% of the total lipids of the composition; and a polymer-conjugated lipid (e.g., DMG-PEG2000) the content of which is 1-5% of the total lipids of the composition. Furthermore, in the LNP composition, the molar ratio of the ionizable N atom in the ionizable lipid molecule to the phosphate group in the nucleic acid molecule is (2-10):1, more preferably (4-8):1.

[0255] In a preferred embodiment of the present invention, the LNP composition comprises: a nucleic acid, an ionizable cationic lipid having a structure represented by formula (I), an auxiliary phospholipid (e.g., DSPC, DOPE, DOPC, etc., or a combination thereof), a sterol (e.g., cholesterol or a cholesterol derivative, or a phytosterol such as β-sitosterol), and a polymer-conjugated lipid (e.g., DMG-PEG2000, etc.). In a more preferred embodiment of the present invention, the LNP composition comprises: a nucleic acid, an ionizable cationic lipid having a structure represented by formula (I), an auxiliary phospholipid (e.g., DSPC, DOPE, DOPC, etc., or a combination thereof), a sterol (e.g., cholesterol or a cholesterol derivative, or a phytosterol such as β-sitosterol), and a polymer-conjugated lipid (e.g., DMG-PEG2000, etc.).

[0256] As used herein, the terms "encapsulate" and "encapsulated" refer to mRNA, DNA, siRNA, or other nucleic acid drugs within or associated with lipid nanoparticles. As used herein, the term "encapsulate" refers to complete encapsulation or partial encapsulation. For example, mRNA can be selected to treat and / or prevent a disease associated with the administration of a lipid nanoparticle composition comprising the mRNA to a subject in need thereof.

[0257] As used herein, the term "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, vehicle, excipient, scintillant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the Food and Drug Administration for use in humans or veterinary animals.

[0258] Preparation method of lipid nanoparticle pharmaceutical preparation

[0259] In another aspect of the present invention, a method for preparing a lipid nanoparticle pharmaceutical preparation is provided, comprising: (a) mixing the ionizable lipid and optional auxiliary lipid described in the first aspect of the present invention with an organic solvent to obtain a lipid organic phase; (b) mixing a bioactive substance with an aqueous solvent to obtain an aqueous phase containing the bioactive substance; and (c) mixing the lipid organic phase from step (a) with the aqueous phase from step (b) to obtain the lipid nanoparticle pharmaceutical. Furthermore, the method further comprises the step (d) of purifying, concentrating, and filtering and sterilizing the lipid nanoparticle pharmaceutical obtained in step (c).

[0260] In some embodiments, the organic solvent includes, but is not limited to, ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane or tetrahydrofuran, or a combination thereof. In some embodiments, the lipid organic phase includes a small percentage of water or pH buffer. The lipid organic phase may include up to 60% by volume of water, for example, up to about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% by volume of water. In one embodiment, the lipid organic phase includes about 0.05% to 60% water by volume, for example, about 0.05% to 50%, about 0.05% to 40%, or about 5% to 20% water by volume.

[0261] In some embodiments, the lipid organic phase includes a single type of lipid, for example, an ionizable cationic lipid, an auxiliary phospholipid, a sterol or a polymer-conjugated lipid. In some embodiments, the lipid organic phase includes a plurality of lipids. In one embodiment of the invention, the lipid organic phase comprises an ionizable cationic lipid having a structure shown in Formula I, an auxiliary phospholipid (for example, DSPC, DOPE, DOPC or a combination thereof), a sterol (for example, cholesterol or a cholesterol derivative, or a plant sterol such as β-sitosterol) and a polymer-conjugated lipid (for example, DMG-PEG2000). In a preferred embodiment of the invention, the lipid organic phase includes an ionizable cationic lipid having a structure shown in Formula (I), an auxiliary phospholipid (for example, DSPC, DOPE, DOPC or a combination thereof), a sterol (for example, cholesterol or a cholesterol derivative, or a plant sterol such as β-sitosterol) and a polymer-conjugated lipid (for example, DMG-PEG2000). In a more preferred embodiment of the present invention, the lipid organic phase includes an ionizable cationic lipid having a structure shown in formula (I), an auxiliary phospholipid (e.g., DSPC, DOPE, DOPC or a combination thereof), a sterol (e.g., cholesterol or a cholesterol derivative, or a phytosterol such as β-sitosterol) and a polymer-conjugated lipid (e.g., DMG-PEG2000). In a specific embodiment of the present invention, the lipid organic phase includes an ionizable cationic lipid having a structure shown in formula (I), DSPC, cholesterol and DMG-PEG2000.

[0262] In some embodiments, aqueous solvent is water.In some embodiments, aqueous solvent is pH between 3-8 (for example, pH is about 3, about 4, about 5 or about 6 etc.).Bioactive substance such as nucleic acid (for example mRNA) is dissolved in described aqueous solvent, thus obtain the water containing bioactive substance.Described aqueous phase can comprise the water miscible organic solvent of small percentage.Described aqueous phase can comprise at least one organic solvent miscible with water of 60 volume % at the most, for example, at the most about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or a kind of organic solvent (for example, water miscible organic solvent) of any volume % between the two. In one embodiment, the aqueous phase comprises an organic solvent between about 0.05% and 60% by volume, for example, an organic solution (e.g., a water-miscible organic solvent) between about 0.05% and 50%, about 0.05% to 40%, or about 5% to 20% by volume. The aqueous phase buffer can be a citrate buffer, a Tris-HCl buffer solution, a sodium acetate buffer solution, a PBS buffer solution, or a combination thereof. In some embodiments, the aqueous buffer solution is a citrate buffer having a pH between 4 and 6 (e.g., a pH of about 4, about 5, or about 6). In one embodiment, the aqueous buffer solution is a citrate buffer having a pH of about 4.

[0263] In some embodiments, a solution comprising a mixture of a lipid organic phase and an aqueous phase containing a bioactive substance can be diluted, the mixture comprising an LNP suspension. In some embodiments, the pH of a solution comprising a mixture of a lipid organic phase and an aqueous phase containing a bioactive substance can be adjusted. The pH of the LNP suspension can be diluted or adjusted by adding water, an acid, an alkali, or an aqueous buffer. In some embodiments, the pH of the LNP suspension is not diluted or adjusted. In some embodiments, the pH of the LNP suspension is diluted and adjusted.

[0264] In some embodiments, excessive reagent, solvent, unencapsulated nucleic acid can be removed from the LNP suspension by tangential flow filtration (TFF) (e.g., diafiltration). Organic solvents (e.g., ethanol) and buffers can also be removed from the LNP suspension with TFF. In some embodiments, the LNP suspension is dialyzed. In some embodiments, the LNP suspension is TFFed. In some embodiments, the LNP suspension is dialyzed and TFFed.

[0265] The main advantages of the present invention include:

[0266] (a) The ionizable lipids of the present invention can form stable nanoparticles with other ingredients, such as auxiliary phospholipids (DSPC, DOPE, DOPC, DOPS, etc.), sterols (such as cholesterol or cholesterol derivatives), PEG derivatives (such as DMG-PEG lipids, or DMG-PEG substances modified with other groups / or PEG derivatives).

[0267] (b) The ionizable lipids of the present invention form nanoparticles with other ingredients to encapsulate mRNA, resulting in uniform particle size, high encapsulation efficiency, and good stability. This can improve the transfection efficiency of mRNA in target tissues or cells and has low toxicity, thereby making the preventive and therapeutic effects of mRNA vaccines / drugs more prominent.

[0268] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which no specific conditions are specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0269] Example 1

[0270] Preparation of ionizable lipids

[0271] 1.1 Synthesis steps and characterization of compound AXT-8:

[0272] Synthesis flow chart of AXT-8

[0273] 1.1.1 Synthesis and characterization of product 8-31

[0274] Tert-butyl N-[(3R)-pyridin-3-yl]carbamate (1.2 g, 6.443 mmol, 1 equiv), 2-bromoethanol (1.21 g, 9.665 mmol, 1.5 equiv), Na2CO3 (1.37 g, 12.886 mmol, 2 equiv) and CH3CN (24 mL) were placed in a 40 mL round-bottom flask, and the resulting solution was stirred at 65 ° C overnight under inert nitrogen protection.

[0275] Afterwards, the reaction mixture was allowed to cool to ambient temperature and filtered, and the filtrate was concentrated under reduced pressure. The residue was solvated with 50 mL of water, and the resulting solution was extracted with 3×50 mL of ethyl acetate and the organic layer was combined, and the organic phase was dried over anhydrous sodium sulfate and concentrated.

[0276] The result was tert-butyl N-[(3R)-1-(2-hydroxyethyl)pyrrolidin-3-yl]carbamate (0.72 g, 48.52% yield) as a colorless oil. The mass spectrum and NMR data are shown below:

[0277] LC-MS-8-31:(ES,m / z):231[M+H]+;

[0278] H-NMR-8-31:1H NMR(300MHz,Chloroform-d,ppm)δ4.998(brs,1H),4.249(brs,1H),3.702(t,J=5.4Hz,2H),3.025(brs,1 H), 2.776-2.725 (m, 4H), 2.495-2.381 (m, 2H), 2.353-2.294 (m, 1H), 1.688 (t, J = 8.4Hz, 1H), 1.464 (s, 9H).

[0279] 1.1.2 Synthesis and characterization of product 8-32

[0280] Tert-butyl N-[(3R)-1-(2-hydroxyethyl)pyridin-3-yl]carbamate (0.72 g, 3.126 mmol, 1 equiv) and DCM (7.2 ml) were placed in a 100 ml 3-necked round-bottom flask and protected with inert nitrogen.

[0281] Then, trifluoroacetaldehyde (1.45 mL) was added dropwise at 0° C., and the resulting solution was stirred at 0° C. for 30 minutes and monitored by LCMS. The resulting mixture was concentrated.

[0282] The results were 2-[(3R)-3-aminopyrrolidin-1-yl]ethanol and trifluoroacetaldehyde (0.5 g, yield: 70.08%) as a light yellow oil. The mass spectrometry and NMR characterization results are shown below:

[0283] LC-MS-8-32:(ES,m / z):131[M+H]+;

[0284] H-NMR-8-32:1H NMR (300MHz, Chloroform-d, ppm) δ4.128 (brs, 1H), 3.830 (t, J = 4.8Hz, 2H), 3.65 3-3.392(m,4H),3.353-3.279(m,2H),2.612-2.488(m,1H),2.235-2.142(m,1H).

[0285] 1.1.3 Synthesis and characterization of the final product

[0286] Synthesis steps:

[0287] 1) 2-[(3R)-3-Aminopyridin-1-yl]ethanol was placed in a 500 ml three-necked round-bottom flask and protected with inert nitrogen. Trifluoroacetaldehyde (300 mg, 1.315 mmol, 1 equiv), 6-oxohexyl-2-hexyldecanoate (1025.43 mg, 2.893 mmol, 2.2 equiv), DCM (15 mL) and THF (15 mL) were added successively. The resulting solution was stirred at room temperature for 0.5 hour.

[0288] 2) Diacetyl peroxide and sodium acetate borate (835.82 mg, 3.945 mmol, 3 equiv) were added dropwise at 0°C over 5 minutes.

[0289] 3) Continue stirring at 0°C for 0.5 hours, then stir at room temperature overnight;

[0290] 4) Add 50 mL of saturated NH4Cl to quench the reaction;

[0291] 5) The resulting solution was extracted with 3×50 mL of DCM and the organic layers were combined;

[0292] 6) the organic phase was dried over anhydrous sodium sulfate and concentrated;

[0293] 7) The residue was further purified by flash-prepare-hplc under the following conditions (IntelFlash-1): column, C18 silica gel; mobile phase: A: H2O (0.05% TFA) B: CH3CN = 55, increased to H2O (0.05% TFA) B: CH3CN = 95 within 15 min, held for 10 min; detector, UV 220 nm; and

[0294] 8) The qualified fractions were combined and concentrated; the residue was dissolved in DCM (50 mL), and the organic phase was washed with 2×50 mL NaHCO 3 (aq, 2M), 1×50 mL water, and brine (50 mL), and dried over anhydrous Na 2 SO 4 .

[0295] The filtered filtrate was concentrated under reduced pressure. This yielded 6-({6-[(2-hexyldecyl)oxy]hexyl}[(3R)-1-(2-hydroxyethyl)pyrrolidin-3-yl]amino)hexylhexyldecanoate (0.2321 g, 21.87% yield) as a yellow oil. Mass spectrometry, NMR, and HPLC characterization results are shown below:

[0296] LC-MS:(ES,m / z):807.8[M+H] + ;

[0297] H-NMR:1H NMR (300MHz, Chloroform-d, ppm) δ4.062 (t, J = 6.6 Hz, 4H), 3.639 (t, J = 5.1 Hz, 2H), 3.419 (t, J = 6.9 Hz, 1H), 2.745-2.569 (m, 6H), 2.473 (t, J = 7. 8Hz,4H),2.337-2.279(m,2H),2.062-1.926(m,1H),1.822-1.708(m,1H ),1.692-1.504(m,8H),1.492-1.188(m,56H),0.876(t,J=5.7Hz,12H);

[0298] The purity test results of AXT-8 by HPLC-CAD showed that the product peak time was 18.039 min and the purity reached more than 94%.

[0299] Example 2

[0300] Encapsulation of mRNA into LNP-mRNA using ionizable lipids

[0301] The present inventors used ionizable lipids to encapsulate mRNA and assembled it into LNP-mRNA, the steps including:

[0302] 1) Ionizable lipid (AXT-8), DSPC (distearoylphosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, and DMG-PEG2000 were mixed in ethanol at different ratios and thoroughly mixed to form the organic phase;

[0303] 2) mRNA was dissolved in sodium citrate (pH = 4.0) and mixed thoroughly as the aqueous phase;

[0304] 3) Transfer the aqueous phase and organic phase into appropriate BD syringes respectively, removing as many bubbles as possible;

[0305] 4) using a PNI nanoparticle preparation apparatus, mixing the aqueous phase and the organic phase at a volume ratio of 3:1 and a flow rate of 12 mL / min, and sequentially purifying, concentrating, and filtering to sterilize to obtain a lipid nanoparticle (LNP-mRNA) product encapsulating mRNA; and

[0306] 5) Perform physical and chemical quality control on the final product LNP-mRNA.

[0307] Physical and chemical quality control methods and results of LNP-mRNA:

[0308] 1) Particle size and PDI (distribution): The particle size and distribution of LNPs were measured using a nanoparticle size analyzer. The results are shown in Figures 1 and 2.

[0309] 2) Encapsulation efficiency (EE%): Total mRNA and free mRNA were stained with RiboGreen, respectively, and then detected using a microplate reader to calculate the encapsulation efficiency. The results are shown in Figure 3.

[0310] 3) pH: Use a pH meter to detect the final product pH = 7.2-7.4

[0311] 4) Osmotic pressure: The osmotic pressure of LNP-mRNA was measured using an ice point osmometer. The osmotic pressure at a concentration of 100 μg / mL was 280-310 mOsmol / kg.

[0312] 5) mRNA integrity: Agilent Fragment Analyzers were used to detect the integrity of the encapsulated mRNA (ie, mRNA purity). The purity was greater than 90% as determined using an RNA kit (15nt).

[0313] Example 3

[0314] In vitro expression detection of LNP-mRNA

[0315] The present invention also conducted an LNP-mRNA cell expression screening experiment, and the specific steps are as follows:

[0316] 1) Cell plating: After 293T cells were digested, the cell density was adjusted to 2×10 4 / well was plated in a 96-well plate, 100 μL / well, and placed in a cell culture incubator overnight.

[0317] 2) Experimental grouping: LNP (AXT-8)-Luc series products were used as the sample group, Lipofectamine Messenger MAX was used as the positive control group, and only 100 μL of the mixture of culture medium and cells was added as the negative control group.

[0318] 3) Cell Transfection: LNP-Luc does not require transfection. A three-fold serial dilution series is performed, starting with 100 ng of mRNA per well, for a total of six steps. Each sample is plated in triplicate. 10 μL of each dilution of LNP-Luciferase is evenly added to a 96-well cell plate, mixed thoroughly, and incubated in an incubator.

[0319] 4) Kit detection: 48 hours after transfection, according to ONE-Glo TM EX Luciferase Assay System kit instructions were followed. Luciferase luminescence intensity was detected using a BioTek SYNERGY microplate reader, and the average OD values ​​were taken for comparison.

[0320] The results are shown in Figure 4. All LNP (AXT-8)-Luc samples with different formulations were expressed normally in cells, with expression levels increasing with increasing mRNA concentration. This indicates that the LNP-mRNA prepared from AXT-8 is highly expressed in vitro.

[0321] Example 4

[0322] In vitro toxicity testing of LNP-mRNA

[0323] Cytotoxicity assay, the specific steps are as follows:

[0324] 1) Cell plating: After digestion, 293T cells were plated at a density of 2 × 104 cells / well in a 96-well plate at 100 μL / well and placed in a cell culture incubator overnight.

[0325] 2) Experimental grouping: LNP(AXT-8)-Luc was used as the sample group, 100 μL of culture medium alone was added as the blank control group, 100 μL of a mixture of culture medium and cells alone was added as the negative control group, and 100 μL of a mixture of culture medium and cells plus Staurosporine (STS), an apoptosis inducer, was used as the positive control group.

[0326] 3) Cell Transfection: LNP-Luc does not require transfection. A three-fold serial dilution (buffer dilution) is performed, with 100 ng of mRNA per well as the highest concentration, for a total of four serial dilutions. 10 μL of each LNP-Luc dilution factor is evenly added to a 96-well cell plate, with three replicates set for each sample. Mix thoroughly and incubate in an incubator. 10 μL of 100 μM STS (diluted in PBS) is added to a 96-well cell plate, with six replicates set, mixed thoroughly, and incubated in an incubator.

[0327] 4) Kit detection: culture for 48 hours, press Luminescent Cell V(I)bility Assay Kit instructions were followed. Luminescence intensity was measured using a BioTek SYNERGY microplate reader. OD values ​​were averaged for comparison. The cell inhibition rate was calculated using the formula "(negative control group - sample group / positive control group) ÷ (negative control group - blank control group) × 100%" and a dose-response-inhibition curve was plotted.

[0328] The results are shown in Figure 5. The series of LNP-mRNA products formed by AXT-8 had no significant effect on cell proliferation when the concentration was less than 1 μg / mL, indicating that there was no obvious toxicity within the detected concentration range.

[0329] Example 5

[0330] In vivo expression detection of LNP-mRNA

[0331] To elucidate the in vivo expression capacity and toxicity of LNP-mRNA assembled using AXT-8, we further designed hEPO ELISA expression detection experiments and toxicity experiments to measure its expression and toxicity in mice with intact innate immunity. The steps are as follows:

[0332] 1) LNP-mRNA Encapsulation: hEPO mRNA was dissolved in an aqueous buffer and mixed thoroughly to form the aqueous phase. Ionizable lipids (AXT-8), DSPC, cholesterol, and DMG-PEG2000 were dissolved in anhydrous ethanol and mixed thoroughly in a specific proportion to form the organic phase. The aqueous and organic phases were transferred to syringes and prepared using a PNI microfluidic nanopreparation instrument with a 3:1 volume ratio of aqueous to organic phase and a flow rate of 12 mL / min. The LNP-hEPO was concentrated, purified, sterilized, and filtered, and injected into mice after passing quality control.

[0333] 2) Tail Vein Injection: Immobilize mice and inject into the tail vein. Inject 5 μg of LNP-hEPO. Use LNP(AXT-8)-hEPO as the sample group and solvent-only injection as the negative control group. Inject two mice per LNP-hEPO sample.

[0334] 3) Submandibular blood collection: 6 and 24 hours after injection, collect blood in EDTA anticoagulant tubes, gently mix, and number the tubes for later use.

[0335] 4) Serum extraction: After obtaining whole blood, centrifuge at 2000g for 10 minutes. The supernatant is plasma. Aliquot and store at -80°C.

[0336] 5) Detect hEPO expression using a human erythropoietin ELISA kit: Follow the instructions, setting up two replicate wells for each serum sample. Measure the OD value using a microplate reader, and calculate the expression level based on the standard curve and the dilution ratio. The kit's IU to μg conversion unit is as follows: 1650 IU = 11 μg.

[0337] The physicochemical properties of the in vivo formulation prepared by AXT-8 and successfully expressed in mice and the hEPO expression concentration in vivo are shown in Table 2.

[0338] Table 2

[0339] C: ≥0.1 and <1;

[0340] D:<0.1

[0341] The results are shown in Figure 6. The LNP (AXT-8)-hEPO series products prepared by AXT-8 can be successfully expressed in mice. This experiment shows that the LNP formed by AXT-8 can efficiently mediate the expression of mRNA in vivo.

[0342] Example 6

[0343] Detection of LNP-mRNA expression in T cell lines

[0344] To demonstrate the ability of LNP-mRNA assembled with AXT-8 to transfect human T cell lines in vitro, an experiment was designed to transfect JM cells and Jurkat cells with LNP-eGFP. TM MessengerMAX TM Lipofectamine MAX (purchased from Thermo Fisher Scientific, catalog number: LMRNA001) was used as a positive control group to detect the expression level of eGFP in cells. The steps are as follows:

[0345] 1) Cell plating: AO / PI cell counting, no double antibody added to the culture medium, only FBS. Adjust JM cells or Jurkat cells to 1*10 6 / ml, 24-well plate, 200ul cells per well, i.e. 2*10 5 2) Cell transfection: Add 6 μg / well of AXT-8-encapsulated mRNA and add culture medium to 1 ml. The culture medium contains only FBS and no double-antibody.

[0346] 3) Flow cytometry: Culture at 37°C for 24 hours before flow cytometry. Collect the cells into 1.5 ml EP tubes, label the sample names, centrifuge at 400 g for 5 minutes, and discard the supernatant. Wash the cells once with 1 ml 2% FBS (prepared in PBS), centrifuge at 400 g for 5 minutes, and discard the supernatant. Add the corresponding antibody to the cell suspension and stain for 20 minutes. Wash the cells once with 1 ml 2% FBS (prepared in PBS), centrifuge at 400 g for 5 minutes, and discard the supernatant. Resuspend the cells in 50 ul of 2% FBS (prepared in PBS) and detect on a flow cytometer. As shown in Figure 7, LNP-eGFP prepared by AXT-8 can be successfully expressed in JM cells and Jurkat cells.

[0347] Example 7

[0348] Detection of LNP-mRNA expression in primary human T cells

[0349] To demonstrate the ability of AXT-8-assembled LNP-mRNA to transfect human T cell lines in vitro, an experiment was designed to transfect human primary T cells with LNP-mCherry. TM MessengerMAX TM (Lipofectamine MAX) was used as a positive control group to detect the expression level of mCherry in cells. The steps are as follows:

[0350] 1) Isolation of primary T cells from human PBMC: magnetic bead labeling (hereinafter referred to as 10 7 For example, add the corresponding reagents in proportion to the number of cells. 7 Resuspend the total cells in 80 μL of buffer, add 20 μL of CD3 MicroBeads, incubate in the refrigerator for 15 minutes (2-8°C), wash the cells with buffer, and resuspend up to 10 μL of cells in 500 μL of buffer. 8 Place the MS column on a magnetic stand and use it to separate T cells.

[0351] 2) AXT-8 transfected primary T cells: T cells were adjusted to 1*10 6 / ml, 24-well plate, 200ul cells per well, i.e. 2*10 5 AL-08 (6 μg / ml) was added to each well, and X-vivo medium was added to 1 ml. IL-2 was added to each well to a final concentration of 100 IU / ml. Culture at 37°C for 24 hours, followed by flow cytometry analysis.

[0352] 3) Flow cytometry: Collect cells into individual 1.5 ml EP tubes, label them, centrifuge at 400 g for 5 min, and discard the supernatant. Wash cells once with 1 ml of 2% FBS (in PBS), centrifuge at 400 g for 5 min, and discard the supernatant. Add the corresponding antibody to the cell suspension and stain for 20 min. Wash cells once with 1 ml of 2% FBS (in PBS), centrifuge at 400 g for 5 min, and discard the supernatant. Resuspend cells in 50 μl of 2% FBS (in PBS) and analyze on a flow cytometer.

[0353] As shown in Figure 8 , LNP-mCherry was prepared from AXT-8 and successfully expressed in primary human T cells.

[0354] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An ionizable lipid, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, characterized in that: The ionizable lipid has the following structure: in, R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14; X and Y are each independently -CH- or N; L1 has -(L 1a -L 1b )-structure or none, where L 1a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; L 1b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4; L2 has -(L 2a -L 2b )-structure or none; where L 2a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; L 2b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4; R3, R4, R5 and R6 are each independently H, CH3, C2-C 30 Hydrocarbyl, or -(CH2) s -R a -(CH2) g -R b -(CH2) m -CH3, wherein s and g are each independently selected from a positive integer of 1-20, and m is selected from an integer of 0-20; R a , R b Each independently has no or is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; Furthermore, R3 and R4 are not H at the same time; and R5 and R6 are not H at the same time; R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1, 2, and 3.

2. The ionizable lipid according to claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: The ionizable lipid has a substructure shown in the following formula (I-1): in, R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14; L1 has -(L 1a -L 1b )-structure or none, where L 1a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; L 1b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4; L2 has -(L 2a -L 2b )-structure or none; where L 2a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; L 2b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4; R3, R4, R5 and R6 are each independently C2-C 20 Hydrocarbon; R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1, 2, and 3.

3. The ionizable lipid according to claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: The ionizable lipid has a structure shown in the following formula (I-2): in, R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14; L1 has -(L 1a -L 1b )-structure or none, where L 1a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; L 1b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4; L2 has -(L 2a -L 2b )-structure or none; where L 2a Selected from the following functional groups: -O-, L 2b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4; R3 has -R 3a -R 3b -R 3c -R 3d -R 3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure of R5 has -R 5a -R 5b -R 5c -R 5d -R 5e structure; Among them, R 3a , R 3c , R 4a , R 4c , R 5a , R 5c Each independently is -(CH2) n -, wherein n is a positive integer selected from 1 to 14; R 3b , R 3d , R 4b , R 4d , R 5b , R 5d Each independently has no or is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-; R 3e , R 4e , R 5e Each independently is C2-C 20 Hydrocarbon; R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1, 2, and 3.

4. The ionizable lipid according to claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: The ionizable lipid has a structure shown in the following formula (I-3): in, R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14; L1 has -(L 1a -L 1b )-structure or none, where L 1a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; L 1b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4; L2 has -(L 2a -L 2b )-structure or none; where L 2a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; L 2b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4; R3 has -R 3a -R 3b -R 3c -R 3d -R 3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure of R5 has -R 5a -R 5b -R 5c -R 5d -R 5e The structure of R6 has -R 6a -R 6b -R 6c -R 6d -R 6e structure; Among them, R 3a , R 3c , R 4a , R 4c , R 5a , R 5c , R 6a and R 6c Each independently is -(CH2) n -, wherein n is a positive integer selected from 1 to 14; R 3b , R 3d , R 4b , R 4d , R 5b , R 5d , R 6b and R 6d Each independently has no or is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-; R 3e , R 4e , R 5e , R 6e Each independently is C2-C 20 Hydrocarbon; R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1, 2, and 3.

5. The ionizable lipid according to claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, characterized in that: R3, R4, R5 and R6 are each independently C5-C 15 of alkyl.

6. The ionizable lipid according to claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: The ionizable lipid has a structure shown in the following formula (I-1): in, R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 4 to 8; L1 is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-; L2 is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-; R3 has -R 3a -R 3b -R 3c -R 3d -R 3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure of R5 has -R 5a -R 5b -R 5c -R 5d -R 5e The structure of R6 has -R 6a -R 6b -R 6c -R 6d -R 6e structure; Among them, R 3a , R 3c , R 4a , R 4c , R 5a , R 5c , R 6a and R 6c Each independently is -(CH2) n -, wherein n is selected from a positive integer of 1-14; and R3, R4, R5 and R6 each independently have 4-20 CH2 structural fragments; R 3b , R 3d , R 4b , R 4d , R 5b , R 5d , R 6b and R 6d Each is independently absent or selected from the following functional groups: -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-; R 3e , R 4e , R 5e , R 6e Each independently is C2-C 20 Hydrocarbon; R7 is a C1-C3 hydrocarbon group, or -(CH2)2-O-(CH2)2-.

7. The ionizable lipid according to claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: The ionizable lipid has a structure as shown in the following formula:

8. A method for preparing the ionizable lipid according to claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, characterized in that: The preparation method includes: method I, method II and method III; Wherein, method 1 comprises the following steps: (S1) under the protection of inert gas, reacting compound A1 and A2 to obtain compound A3; (S2) Under the protection of inert gas, A3 is de-tert-butyloxycarbonyl (Boc) to obtain A4; (S3) Under the protection of an inert gas, A4 reacts with A5 or A6 to obtain A7 (i.e., the compound shown in (I-1)); Wherein, R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14; G1 and G2 are each independently selected from reactive functional groups; L1 has -(L 1a -L 1b )-structure or none, where L 1a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; L 1b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4; L2 has -(L 2a -L 2b )-structure or none; where L 2a is selected from the following functional groups: O, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; L 2b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4; R3, R4, R5 and R6 are each independently C2-C 20 Hydrocarbon; R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1, 2, and 3; Method II comprises the following steps: (D1) reacting compound B1 with B2 under inert gas protection to obtain compound B3; (D2) reacting compound B3 and compound B4 under inert gas protection to obtain compound B5; (D3) reacting compounds B5 and B6 under inert gas protection to obtain B7, i.e., the compound shown in (I-2); Wherein, R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14; G1 and G2 are each independently selected from reactive functional groups; L1 has -(L 1a -L 1b )-structure or none, where L 1a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; L 1b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4; L2 has -(L 2a -L 2b )-structure or none; where L 2a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; L 2b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4; R3 has -R 3a -R 3b -R 3c -R 3d -R 3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure of R5 has -R 5a -R 5b -R 5c -R 5d -R 5e structure; Among them, R 3a , R 3c , R 4a , R 4c , R 5a , R 5c Each independently is -(CH2) n -, wherein n is a positive integer selected from 1 to 14; R 3b , R 3d , R 4b , R 4d , R 5b , R 5d Each independently has no or is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-; R 3e , R 4e , R 5e Each independently is C2-C 20 Hydrocarbon; R6 is H; R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1, 2, and 3; Method III comprises the following steps: (M1) Under the protection of inert gas, compounds C1 and C2 react to produce C3; (M2) Under the protection of inert gas, compound C3 is de-Bocated to generate compound C4; (M3) Under the protection of inert gas, compound C4 reacts with C5 or C6 to generate C7, i.e., compound C7 represented by formula (I-3): Compounds; Wherein, R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14; G1 and G2 are each independently selected from reactive functional groups; L1 has -(L 1a -L 1b )-structure or none, where L 1a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; L 1b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4; L2 has -(L 2a -L 2b )-structure or none; where L 2a is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; L 2b -(CH2) n -, wherein n is selected from 0, 1, 2, 3 or 4; R3 has -R 3a -R 3b -R 3c -R 3d -R 3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure of R5 has -R 5a -R 5b -R 5c -R 5d -R 5e The structure of R6 has -R 6a -R 6b -R 6c -R 6d -R 6e structure; Among them, R 3a , R 3c , R 4a , R 4c , R 5a , R 5c , R 6a and R 6c Each independently is -(CH2) n -, wherein n is a positive integer selected from 1 to 14; R 3b , R 3d , R 4b , R 4d , R 5b , R 5d , R 6b and R 6d Each independently has no or is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-; R 3e , R 4e , R 5e , R 6e Each independently is C2-C 20 Hydrocarbon; R7 is selected from C1-C5 hydrocarbon group, or -(CH2) m O(CH2) n -, wherein m and n are each independently selected from 1, 2, and 3.

9. A lipid nanoparticle (LNP), characterized in that The lipid nanoparticle comprises the ionizable lipid of claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

10. A lipid nanoparticle pharmaceutical preparation, characterized in that: The lipid nanoparticle pharmaceutical preparation comprises: i) The lipid nanoparticle according to claim 9; ii) a biologically active substance encapsulated in the lipid nanoparticle; and iii) a pharmaceutically acceptable carrier.

11. A method for preparing the lipid nanoparticle pharmaceutical preparation according to claim 10, characterized in that: The method comprises: (a) mixing the ionizable lipid according to claim 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof and optionally a helper lipid with an organic solvent to obtain a lipid organic phase; (b) mixing the biologically active substance with an aqueous solvent to obtain an aqueous phase containing the biologically active substance; (c) mixing the lipid organic phase in step (a) with the aqueous phase in step (b) to obtain the lipid nanoparticle drug.

12. Use of the ionizable lipid according to claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof for preparing a drug delivery system.

13. Use of the lipid nanoparticles according to claim 9 in the preparation of drugs for treating and / or preventing tumors, infectious diseases and rare diseases.

14. The use of the ionizable lipid or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Used to prepare lipid nanoparticle pharmaceutical preparations, which are used to deliver biologically active substances to cells in a subject in need.

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