Ionizable lipid and application thereof

Abstract

The present invention provides an ionizable lipid, and a drug delivery system comprising the ionizable lipid. Specifically, the invention provides an ionizable lipid with a structure as shown in a formula (I), or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. The 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. # imgabs0 #

Description

Technical Field

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

[0002] In recent years, messenger RNA drugs have become a key therapeutic method for preventing and treating infectious diseases and tumors. Messenger RNA drug technology is recognized by the industry. Thanks to its short R&D cycle, low risk of insertion 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 (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 vectors to protect fragile mRNA molecules and deliver them to the cytoplasm.

[0003] A variety of mRNA delivery vectors have been developed, including lipid nanoparticles (LNP), inorganic nanoparticles, polymer nanoparticles, viral vectors and exosomes. LNP is currently widely used as a drug delivery vector, and its main components include ionizable lipids, phospholipids, cholesterol and lipids containing polyethylene glycol. The most important component in LNP is ionizable lipid. Early permanent positively charged cationic lipids have a lower in vivo circulation time, higher toxicity and severe allergic reactions. This is because their inherent positive charge adsorbs proteins during in vivo circulation and is easily captured and cleared by the reticuloendothelial system; the inherent positive charge will interact with the negatively charged cell membrane, causing cell membrane instability and producing severe toxicity; the permanent positively charged cationic lipid will activate the complement system and cause allergic reactions. Ionizable lipids are not charged under physiological pH conditions, so LNPs prepared from ionizable lipids have relatively high safety. Ionizable lipids endow LNPs with lysosomal escape ability, which causes LNPs to escape and release mRNA into the cytoplasm through the proton sponge effect and membrane fusion mechanism, where it binds to protein-encoding ribosomes and translates the encoded protein.

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

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

[0006] The present invention provides an ionizable lipid with low toxicity and high delivery efficiency.

[0007] In the first aspect of the present invention, an ionizable lipid, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof is provided, characterized in that the ionizable lipid has the following structure of Formula I:

[0008]

[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 -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 2, 3 or 4 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 The structure,

[0022] Among them, R 3a and R 3c Each independently is -(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.

[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 is -(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.

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

[0030] Among them, R 5a and R 5c Each independently is -(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.

[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 is -(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;

[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]

[0042] In another preferred embodiment, the ionizable lipid has a substructure represented by the following formula (I-1):

[0043]

[0044] in,

[0045] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14;

[0046] 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;

[0047] 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;

[0048] R3, R4, R5 and R6 are each independently C2-C 20 Hydrocarbon;

[0049] 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. In another preferred embodiment, X is N, and Y is -CH-.

[0050] In another preferred embodiment, R6 is H.

[0051] In another preferred embodiment, the ionizable lipid has a structure shown in the following formula (I-2):

[0052]

[0053] in,

[0054] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14;

[0055] 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;

[0056] 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;

[0057] 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;

[0058] 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;

[0059] R3b , 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)-, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-;

[0060] R 3e , R 4e , R 5e Each independently is C2-C 20 Hydrocarbon;

[0061] 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.

[0062] In another preferred embodiment, the ionizable lipid has a structure shown in the following formula (I-3):

[0063]

[0064] in,

[0065] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14;

[0066] 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;

[0067] 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;

[0068] 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;

[0069] 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;

[0070] R 3b , R 3d , R 4b , R 4d , R 5b , R 5d , R 6b and R6d 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)-;

[0071] R 3e , R 4e , R 5e , R 6e Each independently is C2-C 20 Hydrocarbon;

[0072] 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.

[0073] In another preferred embodiment, the ionizable lipid has a structure shown in formula (I-1), and in the formula (I-1):

[0074] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 2 to 8;

[0075] L1 is selected from the following functional groups: -(C=O)O-, -O(C=O)-;

[0076] L2 is selected from the following functional groups: -(C=O)O-, -O(C=O)-;

[0077] R3, R4, R5 and R6 are each independently C5-C 20 Hydrocarbon;

[0078] 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.

[0079] In another preferred embodiment, the ionizable lipid has a structure shown in formula (I-2), and in the formula (I-2):

[0080] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 2 to 8;

[0081] L1 is none or -O-(CH2) n -; wherein n is selected from 0, 1, 2, 3 or 4;

[0082] L2 is selected from the following functional groups: -(C=O)O-, -O(C=O)-;

[0083] 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;

[0084] 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 3;

[0085] R 3b and R 4b Each independently selected from the group consisting of: -(C=O)O-, -O(C=O)-;

[0086] R 3d , R 4d , R 5d Each is independently selected from the group consisting of -CH2-, -(SS)-;

[0087] R 5b for nothing;

[0088] R 3e , R 4e , R 5e Each independently is C2-C 15 Hydrocarbon;

[0089] 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.

[0090] In another preferred embodiment, the ionizable lipid has a structure shown in formula (I-1), and in the formula (I-1):

[0091] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 2 to 8;

[0092] L1 is selected from the following functional groups: -NH(C=O)-, -(C=O)NH-;

[0093] L2 is selected from the following functional groups: -NH(C=O)-, -(C=O)NH-;

[0094] R3, R4, R5 and R6 are each independently C5-C 20 Hydrocarbon;

[0095] 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.

[0096] In another preferred embodiment, the ionizable lipid has a structure shown in formula (I-1), and in the formula (I-1):

[0097] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 2 to 8;

[0098] L1 is selected from the following functional groups: -(C=O)S-, -S(C=O)-;

[0099] L2 is selected from the following functional groups: -(C=O)S-, -S(C=O)-;

[0100] R3, R4, R5 and R6 are each independently C5-C 20 Hydrocarbon;

[0101] 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.

[0102] In another preferred embodiment, the ionizable lipid has a structure shown in formula (I-3), and in the formula (I-3):

[0103] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 2 to 8;

[0104] L1 is none or -O-(CH2) n -; wherein n is selected from 0, 1, 2, 3 or 4;

[0105] L2 is none or -O-(CH2) n -; wherein n is selected from 0, 1, 2, 3 or 4;

[0106] 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;

[0107] Among them, R 3a , R 3c , R 4a , R 4c , R 5a , R 5c , R 6a , R 6c Each independently is -(CH2) n -, wherein n is a positive integer selected from 1 to 3;

[0108] 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)-;

[0109] R 3d , R 4d , R 5d and R 6d Each is independently selected from the group consisting of -CH2-, -(SS)-;

[0110] R 3e , R 4e , R 5e and R 6e Each independently is C2-C 15 Hydrocarbon;

[0111] 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.

[0112] In another preferred embodiment, R3, R4, R5 and R6 are each independently selected from the following group: C5-C 15 Alkyl, C5-C 15 C5-C 15 The alkynyl group;

[0113] 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.

[0114] In another preferred embodiment, the ionizable lipid has a structure selected from the following Table 1:

[0115] Table 1

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] In another preferred embodiment, R3, R4, R5 and R6 are each independently C5-C 15 In another preferred embodiment, the ionizable lipid has a structure shown in the following formula (I-1):

[0131]

[0132] in,

[0133] R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 4 to 8;

[0134] L1 is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-;

[0135] L2 is selected from the following functional groups: -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-;

[0136] 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;

[0137] 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;

[0138] R 3b , R 3d , R 4b , R 4d , R 5b , R 5d , R 6b and R 6dEach is independently absent or selected from the following functional groups: -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-;

[0139] R 3e , R 4e , R 5e , R 6e Each independently is C2-C 20 Hydrocarbon;

[0140] R7 is a C1-C3 hydrocarbon group, or -(CH2)2-O-(CH2)2-.

[0141] In another preferred embodiment, the ionizable lipid has a structure as shown in the following formula:

[0142]

[0143] 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;

[0144] Wherein, method 1 comprises the following steps:

[0145] (S1) under the protection of inert gas, reacting compound A1 and A2 to obtain compound A3;

[0146] (S2) Under the protection of inert gas, A3 is de-tert-butyloxycarbonyl (Boc) to obtain A4;

[0147] (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));

[0148]

[0149] Wherein, R1 and R2 are each independently selected from -(CH2) n -, where n is a positive integer from 1 to 14;

[0150] G1 and G2 are each independently selected from active functional groups, preferably carbonyl, halogen (fluorine, chlorine, bromine, iodine, etc.), ethylene oxide, etc.;

[0151] 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;

[0152] 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;

[0153] R3, R4, R5 and R6 are each independently C2-C 20 Hydrocarbon.

[0154] 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;

[0155] Method II comprises the following steps:

[0156] (D1) reacting compound B1 with B2 under inert gas protection to obtain compound B3;

[0157] (D2) reacting compound B3 and compound B4 under inert gas protection to obtain compound B5;

[0158] (D3) reacting compounds B5 and B6 under inert gas protection to obtain B7, i.e., the compound shown in (I-2);

[0159]

[0160] Wherein, R1 and R2 are each independently selected from -(CH2) n-, where n is a positive integer from 1 to 14;

[0161] G1 and G2 are each independently selected from active functional groups, preferably carbonyl, halogen (fluorine, chlorine, bromine, iodine, etc.), ethylene oxide, etc.;

[0162] 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;

[0163] 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;

[0164] 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;

[0165] Among them, R 3a , R3c , R 4a , R 4c , R 5a , R 5c Each independently is -(CH2) n -, wherein n is a positive integer selected from 1 to 14;

[0166] 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)-, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-;

[0167] R 3e , R 4e , R 5e Each independently is C2-C 20 Hydrocarbon;

[0168] R6 is H;

[0169] 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;

[0170] Method III comprises the following steps:

[0171] (M1) Under the protection of inert gas, compounds C1 and C2 react to produce C3;

[0172] (M2) Under the protection of inert gas, compound C3 is de-Bocated to generate compound C4;

[0173] (M3) Under the protection of an inert gas, compound C4 reacts with C5 or C6 to generate C7, i.e., a compound represented by formula (I-3);

[0174]

[0175] Wherein, R1 and R2 are each independently selected from -(CH2) n-, where n is a positive integer from 1 to 14;

[0176] G1 and G2 are each independently selected from active functional groups, preferably carbonyl, halogen (fluorine, chlorine, bromine, iodine, etc.), ethylene oxide, etc.;

[0177] 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;

[0178] 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;

[0179] 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 -R6c -R 6d -R 6e structure;

[0180] 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;

[0181] 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)-;

[0182] R 3e , R 4e , R 5e , R 6e Each independently is C2-C 20 Hydrocarbon;

[0183] 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.

[0184] The third aspect of the present invention provides a lipid nanoparticle (LNP), wherein the lipid nanoparticle comprises the ionizable lipid as described in the first aspect of the present invention, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0185] In another preferred embodiment, the lipid nanoparticles further comprise auxiliary lipids.

[0186] In another preferred embodiment, in the lipid nanoparticles, the content of the ionizable lipid is 30-65% by mole of the total lipid content.

[0187] In another preferred embodiment, the auxiliary lipid comprises an auxiliary phospholipid, a sterol, a polymer-conjugated lipid, or a combination thereof.

[0188] In another preferred embodiment, the auxiliary lipid is a combination of auxiliary phospholipids, sterols and polymer-conjugated lipids.

[0189] 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), dioleoyl phosphatidylcholine (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.

[0190] In another preferred embodiment, the sterol includes cholesterol or a cholesterol derivative.

[0191] In another preferred embodiment, the polymer-conjugated lipid is a polyethylene glycol (PEG) lipid.

[0192] 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-(polypeptide, protein, amino acid, vitamin and other active substances) or a combination thereof.

[0193] 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).

[0194] In another preferred embodiment, the lipid nanoparticles further comprise bioactive substances encapsulated in the lipid nanoparticles.

[0195] In another preferred embodiment, the bioactive substance is selected from the following group: nucleic acid, protein, polypeptide, small molecule, or a combination thereof.

[0196] In another preferred embodiment, the nucleic acid includes DNA, plasmid, messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotide, small RNA, ribosomal RNA, micro RNA and transfer RNA, preferably mRNA.

[0197] A fourth aspect of the present invention provides a lipid nanoparticle pharmaceutical preparation, wherein the lipid nanoparticle pharmaceutical preparation comprises:

[0198] i) The lipid nanoparticles according to the third aspect of the present invention;

[0199] ii) a biologically active substance encapsulated in the lipid nanoparticle; and

[0200] iii) a pharmaceutically acceptable carrier.

[0201] In another preferred embodiment, the bioactive substance is selected from the following group: nucleic acid, protein, polypeptide, small molecule, or a combination thereof.

[0202] In another preferred embodiment, the nucleic acid includes DNA, plasmid, messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotide, small RNA, ribosomal RNA, micro RNA and transfer RNA, preferably mRNA.

[0203] In another preferred embodiment, the bioactive substance is a nucleic acid, and in the lipid nanoparticle drug, 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, and more preferably (4-8):1.

[0204] 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.

[0205] 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.

[0206] In another preferred embodiment, the dosage form of the lipid nanoparticle pharmaceutical preparation is selected from the following group: injection, lyophilized agent, nebulizer inhalation, and smearable agent.

[0207] In another preferred embodiment, the lipid nanoparticle pharmaceutical preparation is administered by injection, i.e., intravenous, intramuscular, intradermal, subcutaneous, intrathecal, intraduodenal or intraperitoneal injection.

[0208] In another preferred embodiment, the lipid nanoparticle pharmaceutical preparation is administered by inhalation, such as intranasal administration.

[0209] In another preferred embodiment, the lipid nanoparticle drug preparation is administered transdermally, such as transdermal application or electrode introduction.

[0210] The 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:

[0211] (a) mixing the ionizable lipid or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof according to the first aspect of the present invention and an optional auxiliary lipid with an organic solvent to obtain a lipid organic phase;

[0212] (b) mixing the biologically active substance with an aqueous solvent to obtain an aqueous phase containing the biologically active substance;

[0213] (c) mixing the lipid organic phase in step (a) with the aqueous phase in step (b) to obtain the lipid nanoparticle drug.

[0214] In another preferred embodiment, the organic solvent includes ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, tetrahydrofuran or a combination thereof.

[0215] In another preferred embodiment, the aqueous solvent is a buffer solution.

[0216] In another preferred embodiment, the aqueous solvent is a buffer solution with a pH range of 3-7.

[0217] In another preferred embodiment, the acidic buffer is a citrate buffer with a pH of 4.0.

[0218] 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).

[0219] In another preferred embodiment, in step (c), the lipid organic phase and the aqueous phase are mixed through a microfluidic chip.

[0220] In another preferred embodiment, the method further comprises step (d): purifying, concentrating and filtering and sterilizing the lipid nanoparticle drug obtained in step (c).

[0221] 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.

[0222] In another preferred embodiment, the delivery system includes lipid nanoparticles (LNP), liposomes, polymer nanoparticles, etc., and is preferably used to prepare lipid nanoparticles.

[0223] In another preferred embodiment, the drug delivery system is used to deliver drugs for treating and / or preventing tumors, infectious diseases and rare diseases.

[0224] 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.

[0225] 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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0226] Figure 1 The particle size characterization results of LNP-mRNA with different formulations composed of AXT-8 are shown, with a particle size range of 80-130 nm, proving that the physicochemical properties of the LNPs prepared above are similar (Note: In the above legend, LNP (AXT-8) -Luc-1 means that LNP composed of the molecule AXT-8 encapsulates Luc mRNA, 1 means formula 1 in the LNP series encapsulating Luc mRNA, and the same applies to the others; LNP (AXT-8) -hEPO-1 means that LNP composed of the molecule AXT-8 encapsulates EPO mRNA, 1 means formula 1 in the LNP series encapsulating EPO mRNA, and the same applies to the others 11).

[0227] Figure 2 The particle size distribution characterization results of different formulations of LNP-mRNA composed of AXT-8 are shown, with PDI < 0.2, proving that the particle size distribution of nanoparticles composed of AXT-8 is smaller.

[0228] Figure 3 The encapsulation efficiency characterization results of different formulations of LNP-mRNA composed of AXT-8 are shown, and the encapsulation efficiency is >90%, proving that the encapsulation results are good.

[0229] Figure 4The results of transfection of LNP-mRNA with different ratios composed of AXT-8 in 293T cells are shown. As shown in the figure, with the increase of mRNA concentration, the cell expression results of multiple formulas of LNP (AXT-8)-mRNA are better than lipofectamine.

[0230] Figure 5 The cytotoxicity results of LNP (AXT-8)-mRNA composed of AXT-8 are shown. As shown in the figure, the cell inhibition rate of LNP (AL-6) series products is almost 0 when the mRNA concentration is lower than 1ug / mL.

[0231] Figure 6 The in vivo hEPO expression results of LNP-mRNA with different formulations composed of AXT-8 are shown. 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 at around 6 hours. DETAILED DESCRIPTION

[0232] The inventors have found an ionizable lipid by accident for the first time after extensive and in-depth research, and the ionizable lipid has the advantages of stable physicochemical properties and low toxicity, and the drug delivery system obtained by encapsulating drug load (such as mRNA) with ionizable lipids of the present invention has high delivery efficiency and low toxicity, and the drug load is efficiently delivered, while the expression amount of the drug load is increased, the safety of the drug delivery system is improved, and the prevention and treatment effects of the drug delivery system are more prominent. On this basis, the present invention has been completed.

[0233] the term

[0234] 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.

[0235] 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 for the carbon chain. Examples of alkyl include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl and tert-butyl, pentyl, hexyl, heptyl, octyl, and the like.

[0236] 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".

[0237] In the claims of the present invention, when describing "C1-C30 hydrocarbon group", it means that the group can be an alkane group, an alkene group or an alkyne group having 1-30 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). 30 Hydrocarbon", "C2-C 30 "Hydrocarbon" has a similar meaning.

[0238] When describing "alkyl", it means that the group can be a saturated hydrocarbon 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 a straight chain or have a branched structure, usually a chain group without a ring structure. Alkyl groups that meet the aforementioned number of carbon atoms are all within the scope of the term description.

[0239] 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 a 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 the term description. 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).

[0240] 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).

[0241] 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 ring structure.

[0242] 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.

[0243] Ionizable lipids

[0244] 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.

[0245] Ionizable lipids are protonated and converted into cationic lipids at low pH values, and then converted into auxiliary phospholipids at normal physiological pH values. The auxiliary phospholipids interact less with the anionic cell membranes of blood cells, improving the biocompatibility of lipid nanoparticles. When lipid nanoparticles are internalized by cells, the pH value in the endosome is low, the lipids are protonated and positively charged, and the stability of the membrane structure deteriorates or even is destroyed, which is conducive to the escape of lipid nanoparticles from the endosome. In general, the pH-sensitive properties of lipids are conducive to the in vivo delivery of lipid nanoparticles that encapsulate bioactive ingredients (such as mRNA molecules).

[0246] Helper lipids

[0247] As used herein, the term "helper lipid" refers to other types of lipids in lipid nanoparticles, except ionizable lipids, including helper phospholipids, sterols, polymer-conjugated lipids, or combinations thereof. Helper lipids are mainly used to improve the performance of lipid nanoparticles, such as stability, delivery efficiency, tolerance, and biological separation.

[0248] 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), 1,2-dioleoylphosphatidylcholine (DOPC), 1,2-diole ... amine, 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-oleoyl phosphatidylethanolamine, distearoylphosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, 1-stearoyl-2-oleoyl-phosphatidylethanolamine, or a combination thereof.

[0249] 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, the melting point is -54°C, and it is cylindrical in shape. It forms a layered structure in the lipid nanoparticles, making the structure of the lipid nanoparticles more stable.

[0250] 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 group of DOPE is two unsaturated alkane chains, the melting point is -30°C, and it is conical in shape. It is easy to form an inverted hexagon in lipid nanoparticles, causing instability of the endosomal membrane, which helps the lipid nanoparticles to escape from the endosomal structure.

[0251] In some embodiments, the sterol includes (but is not limited to) cholesterol or cholesterol derivatives. Cholesterol can adjust the integrity and hardness of lipid membranes 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 cholesterol analogs, 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 be multilayered polyhedral structures divided by lipids rather than spherical shapes. 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 oxidized tail groups, the content of lipid nanoparticles in liver endothelial cells and Kupffer cells is higher than that in hepatocytes.

[0252] In some embodiments, the polymer-conjugated lipid is a polyethylene glycol (PEG)-conjugated lipid, also known as a polyethylene glycol lipid or a PEGylated lipid. PEGylated lipids have multiple effects on the properties of lipid nanoparticles: the amount of PEGylated lipids affects the particle size and potential of lipid nanoparticles; reduces particle aggregation and improves the stability of lipid nanoparticles; reduces the particle clearance rate mediated by the kidney and the mononuclear phagocyte system (MPS), prolongs the circulation time of particles; and the surface functional groups can be modified with ligands to improve the targeted delivery ability. The molar mass and the length of the lipid affect the properties of PEGylated lipids. Both DMG-PEG2000 and DSG-PEG2000 are neutral phospholipids, and the length of the saturated alkyl chain is C14, C16 or C18, respectively. However, DMG-PEG2000 can separate from lipid nanoparticles faster, which is beneficial to the cell's uptake of nanoparticles and endosomal escape, so the delivery efficiency of DMG-PEG2000 is better than that of DSG-PEG2000.

[0253] In a preferred embodiment of the present invention, the helper lipid is a combination of DSPC, cholesterol and DMG-PEG2000.

[0254] Lipid Nanoparticles (LNP)

[0255] As used herein, the term "lipid nanoparticle", "lipid nanoparticle" or "LNP" refers to a particle with 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 with cationic lipids, polymers or multivalent small molecules inside the nanoparticle and an external lipid coating that interacts with the biological environment. Due to the repulsive forces between phosphate groups, nucleic acids are naturally rigid polymers that prefer elongated configurations. 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 extended DNA chains into compact, ordered particles 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 be tightly packed.

[0256] In some embodiments, the bioactive substance is encapsulated into LNP. In some embodiments, the bioactive substance can be anionic compounds, 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, nucleoproteins containing DNA, 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.

[0257] In LNP vaccine products, bioactive substances are usually contained inside the LNP. In some embodiments, the bioactive substances include nucleic acids. Typically, water-soluble nucleic acids are condensed with cationic lipids or polycationic polymers inside the particles, and the particle surface is rich in auxiliary phospholipids or PEG lipid derivatives. Additional ionizable cationic lipids may also be located on the surface. After entering the cell lysosome, the ionizable cationic lipids are ionized by the acidic environment of the lysosome and are positively charged, interacting with the lysosomal membrane to promote endosomal escape.

[0258] About LNP, ionizable lipid can have different properties or functions.Due to the pKa of amino group, when the 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 form 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 removal circulation, hemolytic toxicity, including immune activation relevant (Filion et al. Biochim Biophys Acta. October 23, 1997; 1329 (2): 345-56).

[0259] In some embodiments, the pKa can be high enough so that the ionizable cationic lipid can adopt a positively charged form at an acidic endosomal pH. In this way, the cationic lipid can be combined with an endogenous endosomal anionic lipid to promote membrane lysis non-bilayer structure, such as hexagonal HII phase, thereby producing more effective intracellular delivery. In some embodiments, the pKa ranges between 6.2-6.5. For example, the pKa can be about 6.2, about 6.3, about 6.4, about 6.5. The unsaturated tail also contributes to the ability of the lipid to adopt a non-bilayer structure. (Jayaraman et al., Angew Chem Int Ed Engl. 2012 August 20; 51(34):8529-33).

[0260] The release of nucleic acids from LNP formulations, as well as other characteristics such as liposomal clearance and circulation half-life, can be altered 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.

[0261] In one aspect of the present invention, a lipid nanoparticle (LNP) is provided, wherein the lipid nanoparticle comprises 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 also comprises one or more auxiliary lipids, wherein the auxiliary lipids include auxiliary phospholipids, steroids, and polymer-conjugated lipids.

[0262] Lipid nanoparticle drug preparations

[0263] In another aspect of the present invention, a lipid nanoparticle pharmaceutical preparation (or lipid nanoparticle pharmaceutical combination or LNP composition) is provided, wherein the lipid nanoparticle pharmaceutical preparation comprises the lipid nanoparticle as described in the third aspect of the present invention, and a bioactive substance encapsulated in the lipid nanoparticle, 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.

[0264] In some embodiments, the bioactive substance is encapsulated into LNP. In some embodiments, the bioactive substance can be anionic compounds, 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, nucleoproteins containing DNA, 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.

[0265] In some embodiments, the LNP composition includes: nucleic acid, ionizable cationic lipid having a structure shown in formula (I), auxiliary phospholipid (e.g., DSPC, DOPE, DOPC or a combination thereof), sterol (e.g., cholesterol or cholesterol derivatives, or plant sterols such as β-sitosterol) and polymer-conjugated lipid (e.g., DMG-PEG2000). In some embodiments, the LNP composition includes: nucleic acid; ionizable cationic lipid having a structure shown in formula I, whose content is 30-65% (molar ratio, the same below) of the total lipid of the composition; auxiliary phospholipid (e.g., DSPC, DOPE, DOPC or a combination thereof) whose content is 5-30% of the total lipid of the composition; sterol (e.g., cholesterol or cholesterol derivatives, or plant sterols such as β-sitosterol) whose content is 30-55% of the total lipid of the composition; and polymer-conjugated lipid (e.g., DMG-PEG2000) whose content is 1-5% of the total lipid 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, and more preferably (4-8):1.

[0266] In a preferred embodiment of the present invention, the LNP composition comprises: nucleic acid, ionizable cationic lipid having a structure shown in formula (I), auxiliary phospholipid (e.g., DSPC, DOPE, DOPC, etc. or a combination thereof), sterol (e.g., cholesterol or cholesterol derivatives, or plant sterols such as β-sitosterol) and polymer-conjugated lipid (e.g., DMG-PEG2000, etc.). In a more preferred embodiment of the present invention, the LNP composition comprises: nucleic acid, ionizable cationic lipid having a structure shown in formula (I), auxiliary phospholipid (e.g., DSPC, DOPE, DOPC, etc. or a combination thereof), sterol (e.g., cholesterol or cholesterol derivatives, or plant sterols such as β-sitosterol) and polymer-conjugated lipid (e.g., DMG-PEG2000, etc.).

[0267] As used herein, the terms "encapsulation" and "encapsulated" refer to mRNA, DNA, siRNA or other nucleic acid drugs within or in combination with lipid nanoparticles. As used herein, the term "encapsulation" refers to complete encapsulation or partial encapsulation. For example, mRNA can be selected to treat and / or prevent related diseases when a lipid nanoparticle composition comprising mRNA is administered to a subject in need.

[0268] As used herein, the term "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, scintillator, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the Food and Drug Administration for use in humans or livestock.

[0269] Preparation method of lipid nanoparticle pharmaceutical preparation

[0270] In another aspect of the present invention, a method for preparing a lipid nanoparticle drug preparation is provided, the method 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; (c) mixing the lipid organic phase in step (a) with the aqueous phase in step (b) to obtain the lipid nanoparticle drug. Further, the method further comprises step (d): purifying, concentrating and filtering and sterilizing the lipid nanoparticle drug obtained in step (c).

[0271] In some embodiments, organic solvents include, but are 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 water between about 0.05% and 60% by volume, for example, between about 0.05% and 50%, between about 0.05% and 40%, or between about 5% and 20% by volume.

[0272] 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 variety of lipids. In one embodiment of the present invention, the lipid organic phase comprises an ionizable cationic lipid of the 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 plant sterol such as β-sitosterol) and a polymer-conjugated lipid (e.g., DMG-PEG2000). In a 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 plant sterol such as β-sitosterol) and a polymer-conjugated lipid (e.g., 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.

[0273] 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.) aqueous buffer.Bioactive substances such as nucleic acid (such as mRNA) are dissolved in the aqueous solvent, thereby obtaining the aqueous phase containing bioactive substances.The aqueous phase can include a small percentage of water-miscible organic solvents.The aqueous phase can include at least one organic solvent miscible with water of up to 60% by volume, for example, at 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 any volume % between the two A kind of organic solvent (for example, water-miscible organic solvent). 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%, between about 0.05% and 40%, or between about 5% and 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, etc. In some embodiments, the aqueous buffer is a citrate buffer having a pH between 4-6 (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.

[0274] In some embodiments, the solution of the mixture comprising the lipid organic phase and the aqueous phase containing the bioactive substance can be diluted, and the mixture comprises the LNP suspension. In some embodiments, the pH of the solution of the mixture comprising the lipid organic phase of the LNP suspension and the aqueous phase containing the bioactive substance can be adjusted. The pH of the LNP suspension can be diluted or adjusted by adding water, acid, alkali or aqueous buffer. In some embodiments, the dilution or adjustment of the pH of the LNP suspension is not carried out. In some embodiments, the pH of the LNP suspension is diluted and adjusted.

[0275] In some embodiments, excessive reagents, solvents, unencapsulated nucleic acids 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 by 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.

[0276] The main advantages of the present invention include:

[0277] (a) The ionizable lipids of the present invention and other components, 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 by other groups / or PEG derivatives, etc.), can form stable nanoparticles.

[0278] (b) After the ionizable lipids of the present invention form nanoparticles with other ingredients, mRNA is encapsulated, and the particle size is uniform, the encapsulation efficiency is high, and the stability is good. The transfection efficiency of mRNA in the target tissue or cell can be improved, and the toxicity is low, thereby making the prevention and treatment effects of mRNA vaccines / drugs more prominent.

[0279] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0280] Example 1

[0281] Preparation of ionizable lipids

[0282] 1.1 Synthesis steps and characterization of compound AXT-8:

[0283]

[0284] Synthesis flow chart of AXT-8

[0285] 1.1.1 Synthesis and characterization of product 8-31

[0286]

[0287] 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.

[0288] 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 x 50 mL of ethyl acetate and the organic layer combined, and the organic phase was dried over anhydrous sodium sulfate and concentrated.

[0289] As a result, tert-butyl N-[(3R)-1-(2-hydroxyethyl)pyrrolidin-3-yl]carbamate (0.72 g, yield 48.52%) was obtained as a colorless oil. The mass spectrum and NMR data are shown below:

[0290] LC-MS-8-31:(ES,m / z):231[M+H]+;

[0291] 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).

[0292] 1.1.2 Synthesis and characterization of product 8-32

[0293]

[0294] Place tert-butyl N-[(3R)-1-(2-hydroxyethyl)pyridin-3-yl]carbamate (0.72 g, 3.126 mmol, 1 equiv) and DCM (7.2 ml) in a 100 ml 3-necked round bottom flask and protect with inert nitrogen.

[0295] Next, 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.

[0296] As a result, 2-[(3R)-3-aminopyrrolidin-1-yl]ethanol and trifluoroacetaldehyde (0.5 g, yield: 70.08%) were obtained as light yellow oil. The results of mass spectrometry and NMR characterization are shown below:

[0297] LC-MS-8-32:(ES,m / z):131[M+H]+;

[0298] 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).

[0299] 1.1.3 Synthesis and characterization of the final product

[0300]

[0301] Synthesis steps:

[0302] 1) 2-[(3R)-3-aminopyridin-1-yl]ethanol was placed in a 500 ml three-necked round-bottom flask, protected with inert nitrogen, and 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, and the resulting solution was stirred at room temperature for 0.5 hours;

[0303] 2) At 0°C, diacetyl peroxide and sodium acetate borate (835.82 mg, 3.945 mmol, 3 equiv) were added dropwise over 5 minutes.

[0304] 3) Continue stirring at 0°C for 0.5 hours, then stir at room temperature overnight;

[0305] 4) Add 50 mL of saturated NH4Cl to quench the reaction;

[0306] 5) The resulting solution was extracted with 3×50 mL DCM and the organic layers were combined;

[0307] 6) the organic phase was dried over anhydrous sodium sulfate and concentrated;

[0308] 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, maintained for 10 min; detector, UV 220 nm; and

[0309] 8) The qualified fractions were combined and concentrated; the residue was dissolved with DCM (50 mL), and the organic phase was washed with 2×50 mL NaHCO3 (aq, 2M), 1×50 mL water, and brine (50 mL), and dried over anhydrous Na2SO4.

[0310] The filtrate after filtration was concentrated under reduced pressure. Thus, 6-({6-[(2-hexyldecyl)oxy]hexyl}[(3R)-1-(2-hydroxyethyl)pyrrolidin-3-yl]amino)hexylhexyldecanoate (0.2321 g, yield 21.87%) was obtained as a yellow oil. The results of mass spectrometry, nuclear magnetic resonance and HPLC characterization are shown below:

[0311] LC-MS: (ES, m / z): 807.8 [M+H] + ;

[0312] 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);

[0313] The purity test result of AXT-8 by HPLC-CAD showed that the peak time of the product was 18.039min and the purity was over 94%.

[0314] Example 2

[0315] Encapsulation of mRNA using ionizable lipids to form LNP-mRNA

[0316] The inventors use ionizable lipids to encapsulate mRNA and assemble it into LNP-mRNA, the steps include:

[0317] 1) Ionizable lipid (AXT-8), DSPC (distearoylphosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, and DMG-PEG2000 were mixed in different proportions in an ethanol phase and mixed thoroughly to form an organic phase;

[0318] 2) mRNA was dissolved in sodium citrate solution (pH = 4.0) and mixed thoroughly as the aqueous phase;

[0319] 3) Transfer the aqueous phase and organic phase into appropriate BD syringes respectively, and try to remove all bubbles;

[0320] 4) using a PNI nano preparation instrument, 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

[0321] 5) Perform physical and chemical quality control on the final product LNP-mRNA.

[0322] Physical and chemical quality control methods and results of LNP-mRNA:

[0323] 1) Particle size and PDI (distribution): The particle size and distribution of LNP were measured using a nanoparticle size analyzer. The results are detailed in Figure 1 and Figure 2 .

[0324] 2) Encapsulation efficiency (EE%): Total mRNA and free mRNA were stained with RiboGreen, respectively, and then detected with an ELISA reader to calculate the encapsulation efficiency. The results are detailed in Figure 3 .

[0325] 3) pH: Use a pH meter to test the final product pH = 7.2-7.4

[0326] 4) Osmotic pressure: The osmotic pressure of LNP-mRNA was detected using an ice point osmometer. The osmotic pressure was 280-310 mOsmol / kg when the detection concentration was 100 μg / mL.

[0327] 5) mRNA integrity: Agilent Fragment Analyzers were used to detect the integrity of the encapsulated mRNA (ie, mRNA purity), and the purity was greater than 90% using RNA kit (15nt).

[0328] Example 3

[0329] In vitro expression detection of LNP-mRNA

[0330] The present invention also conducted a LNP-mRNA cell expression screening experiment, and the specific steps are as follows:

[0331] 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.

[0332] 2) Experimental grouping: LNP (AXT-8) -Luc series products were used as the sample group, Lipofectamine MessengerMAX was set as the positive control group, and only 100 μL of the mixture of culture medium and cells was added as the negative control group.

[0333] 3) Cell transfection: LNP-Luc does not need transfection. The highest concentration of mRNA in each well is 100ng. Three-fold gradient dilution is performed, with a total of six gradients, and three replicate wells are set for each sample. 10μL of LNP-Luciferase at different dilution multiples is evenly added to the 96-well cell plate, mixed and placed in the incubator for culture.

[0334] 4) Kit detection: 48 hours after transfection, follow the instructions of the ONE-GloEX Luciferase Assay System kit and use a BioTek SYNERGY microplate reader to detect the luminescence intensity of Luciferase, and take the average OD value for comparison.

[0335] The results are as follows Figure 4 As shown, LNP (AXT-8)-Luc samples with different formulations can be expressed normally in cells, and the expression amount increases with the increase of mRNA concentration. It can be seen that the LNP-mRNA prepared by AXT-8 is highly expressed in vitro.

[0336] Example 4

[0337] In vitro toxicity assay of LNP-mRNA

[0338] Cytotoxicity experiment, the specific steps are as follows:

[0339] 1) Cell plating: After 293T cells were digested, the cell density was adjusted to 2×104 / well and plated on a 96-well plate at 100 μL / well and placed in a cell culture incubator overnight.

[0340] 2) Experimental grouping: LNP (AXT-8) -Luc was used as the sample group, only 100 μL of culture medium was added as the blank control group, only 100 μL of the mixture of culture medium and cells was added as the negative control group, and Staurosporine (STS) apoptosis inducer was added to 100 μL of the mixture of culture medium and cells as the positive control group.

[0341] 3) Cell transfection: LNP-Luc does not need to be transfected. The highest concentration of mRNA in each well is 100ng, and three-fold gradient dilution (buffer dilution) is performed, with a total of four gradients. 10μL of LNP-Luc with different dilution multiples is evenly added to a 96-well cell plate, three replicate wells are set for each sample, and the mixture is placed in an incubator for culture. 10μL STS (PBS dilution) of 100μM is added to a 96-well cell plate, six replicate wells are set, and the mixture is placed in an incubator for culture.

[0342] 4) Kit detection: culture for 48 hours, according to The Luminescent Cell V(I)bilityAssay Kit was operated according to the instructions. The luminescence intensity was detected using a BioTek SYNERGY microplate reader, and the OD values ​​were averaged for comparison. The cell inhibition rate was calculated according to the formula "(negative control group - sample group / positive control group) ÷ (negative control group - blank control group) × 100%" and the dose-response-inhibition curve was drawn.

[0343] The results are as follows Figure 5 As shown, 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.

[0344] Example 5

[0345] In vivo expression detection of LNP-mRNA

[0346] In order to clarify the in vivo expression ability and toxicity of LNP-mRNA assembled using AXT-8, hEPO ELISA expression detection experiment and toxicity experiment were further designed to measure its expression level and toxicity in mice with sound innate immunity. The steps are as follows:

[0347] 1) Encapsulation of LNP-mRNA: Dissolve hEPO mRNA in aqueous buffer and mix well as the aqueous phase; ionizable lipids (AXT-8), DSPC, cholesterol and DMG-PEG2000 are dissolved in anhydrous ethanol, mixed well in a certain proportion and used as the organic phase. The aqueous phase and the organic phase are transferred to syringes respectively, and the parameters are set using the PNI microfluidic nanopreparation instrument (the volume ratio of the aqueous phase to the organic phase is 3:1, and the flow rate is 12mL / min) to prepare hEPO-LNP. LNP-hEPO is concentrated, purified, sterilized and filtered, and injected into mice after passing the quality control.

[0348] 2) Mouse tail vein injection: Fix the mouse and select the tail vein for injection. The LNP-hEPO injection amount is 5 μg. LNP (AXT-8)-hEPO is set as the sample group, and the negative control group is injected with only the solvent. Two mice are injected for each LNP-hEPO sample.

[0349] 3) Submandibular blood collection of mice: 6h and 24h after injection, submandibular blood was collected. The blood was collected in EDTA anticoagulant tubes and gently mixed, and numbered for later use.

[0350] 4) Serum extraction: After obtaining whole blood, centrifuge at 2000g for 10 minutes. The supernatant is plasma. Store at -80℃ after aliquoting.

[0351] 5) Detect hEPO expression using human Erythropoietin ELISA kit: refer to the instructions for operation, set up two replicate wells for each serum sample. Use an enzyme marker to detect the OD value, calculate the expression level based on the standard curve and dilution ratio, and the conversion unit of IU and μg used in the kit is as follows: 1650 IU = 11 μg.

[0352] The physicochemical property values ​​of the in vivo formulation prepared by AXT-8 and successfully expressed in mice and the in vivo hEPO expression concentration values ​​are shown in Table 2.

[0353] Table 2

[0354]

[0355] C: ≥0.1 and <1;

[0356] D:<0.1

[0357] The results are as follows Figure 6 As shown, 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.

[0358] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

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 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; 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 an inert gas, compound C4 reacts with C5 or C6 to generate C7, i.e., a compound represented by formula (I-3); 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.

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