Ionizable lipids and their uses

Abstract

The present invention provides ionizable lipids and drug delivery systems containing the ionizable lipids. Specifically, the present invention provides ionizable lipids having the structure of formula (I), or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof. Lipid nanoparticles constructed using the ionizable lipids can enable the safe and efficient delivery of nucleic acid drugs, small molecule drugs, peptide drugs, and protein drugs. JPEG2026521958000055.jpg55124

Description

[Technical Field]

[0001] This invention relates to the field of biopharmaceuticals, and more specifically to ionizable lipids and their use in drug delivery. [Background technology]

[0002] In recent years, messenger RNA (RNA) drugs have become an important therapeutic tool in the prevention and treatment of infectious diseases and tumors. Messenger RNA drug technology is recognized in the industry due to its short development cycle, low insertion mutation risk, and diversity of coding proteins, making mRNA very suitable for vaccine or therapeutic development. However, mRNA itself is very unstable, easily degraded by ubiquitous RNases, and also has inherent negative charge and a large molecular weight (usually 10%). 6 Because of its larger size (Da), mRNA molecules are restricted from entering cells. Therefore, developing appropriate delivery carriers to protect fragile mRNA molecules and deliver them into the cytoplasm is of paramount importance.

[0003] Currently, various mRNA delivery carriers are being developed, including lipid nanoparticles (LNPs), inorganic nanoparticles, polymer nanoparticles, virus-like carriers, and exosomes. LNPs are widely used as drug delivery carriers, and their main components include ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-containing lipids. The most important component in LNPs is ionizable lipids. Early, permanently positively charged cationic lipids exhibited low circulation time, high toxicity, and severe allergic reactions. This is because their inherent positive charge causes them to adsorb proteins during circulation, making them easily captured and removed by the reticular-endothelial system. Their inherent positive charge also interacts with negatively charged cell membranes, destabilizing them and causing severe toxicity. Furthermore, permanently positively charged cationic lipids activate the complement system, triggering allergic reactions. Since ionizable lipids do not become charged under physiological pH conditions, LNPs prepared from ionizable lipids have relatively high safety. Ionizable lipids provide LNP lysosomes with escape capabilities, allowing LNPs to escape via the proton sponge effect and membrane fusion mechanism, releasing mRNA into the cytoplasm. This mRNA then binds to protein-coding ribosomes, translating the encoded protein.

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

[0005] Therefore, developing ionizable lipids with low toxicity and high delivery efficiency is of great importance. [Overview of the project]

[0006] This invention provides ionizable lipids that have low toxicity and high delivery efficiency.

[0007] In a first aspect of the present invention, an ionizable lipid, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein the ionizable lipid has the structure of the following formula I,

[0008] [Chemical] However, R1 and R2 are each independently - (CH2) n - selected from, provided that n is a positive integer from 1 to 14, X and Y are each independently - CH - or N, L1 has a structure of - (L 1a - L 1b ) - from right to left, or not, provided that L 1a is a functional group selected from - O -, - (C = O) O -, - O(C = O) -, - (S - S) -, - 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(NH -, - CH(OH) -, preferably selected from - O -, - (C = O) O -, - O(C = O) -, - CH(OH) -, and L 1b is - (CH2) n -, provided that n is selected from 0, 1, 2, 3 or 4, L2 has a structure of - (L 2a - L 2b ) - from left to right, or not, provided that L 2a is a functional group selected from - O -, - (C = O) O -, - O(C = O) -, - (S - S) -, - 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(NH -, - CH(OH) -, preferably selected from - O -, - (C = O) O -, - O(C = O) -, - CH(OH) -, and L 2b is - (CH2) n -, provided that n is selected from 0, 1, 2, 3 or 4, R3, R4, R5 and R6 are each independently H, CH3, C2 - C 30 hydrocarbon group (e.g., C2 - C 30 alkyl group, C2 - C 30 ]>[end]]alkenyl group, C2 - C 30 alkynyl group), or - (CH2) s - R a - (CH2)g -R b -(CH2) m -CH3, where s and g are independently selected positive integers from 1 to 20, and m is an integer selected from 0 to 20, preferably s+g+m is from 2 to 35. R a , R b Each of these is an independent functional group selected from -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-, and -CH(OH)-, preferably selected from -O-, -(C=O)O-, -O(C=O)-, and -CH(OH)-. Furthermore, R3 and R4 cannot both be H, and R5 and R6 cannot both be H. R7 is a C1-C5 hydrocarbon group or -(CH2). m O(CH2) n The present invention provides an ionizable lipid, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, characterized by being selected from -, wherein m and n are each independently selected from 1, 2, and 3.

[0009] In another preferred example, R3, R4, R5, and R6 are each independently C4~C 30 Hydrocarbon groups (e.g., C4~C) 30 Alkyl alkyl groups, C4-C 30 Alkenyl group, C4~C 30 (alkynyl group), preferably C4-C 20 Hydrocarbon groups (e.g., C4~C) 20 Alkyl alkyl groups, C4-C 20 Alkenyl group, C4~C 20 It is an alkynyl group.

[0010] In other preferred examples, at least two, three, or four of R3, R4, R5, and R6 are C2-C30 hydrocarbon groups (for example, C2-C30 30 Alkyl alkyl groups, C2-C 30 Alkenyl group, C2~C 30Alkynyl group), or -(CH2) s -R a -(CH2) g -R b -(CH2) m -CH3, where s, g, m, R a and R b It is defined as described above.

[0011] In other preferred examples, s+g+m is 3 to 20, and more preferably 4 to 15.

[0012] In another preferred example, R3 is -R 3a -R 3b -R 3c -R 3d -R 3e It has a structure, However, R 3a and R 3c Each is independently -(CH2) n -where n is a positive integer chosen from 1 to 14, R 3b and R 3d Each is independent of the others or -(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-,

[0013] [ka] A functional group selected from, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, and -CH(OH)-, R 3e is C2~C 20 It is a hydrocarbon group.

[0014] In another preferred example, R4 is -R 4a -R 4b -R4c -R 4d -R 4e It has a structure, However, R 4a and R 4c Each is independently -(CH2) n -where n is a positive integer chosen from 1 to 14, R 4b and R 4d Each is independent of the others or -(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-,

[0015] [ka] A functional group selected from, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, and -CH(OH)-, R 4e is C2~C 20 It is a hydrocarbon group.

[0016] In another preferred example, R5 is -R 5a -R 5b -R 5c -R 5d -R 5e It has a structure, However, R 5a and R 5c Each is independently -(CH2) n -where n is a positive integer chosen from 1 to 14, R 5b and R 5dThese are each independent of or 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-,

[0017] [ka] A functional group selected from, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, and -CH(OH)-, R 5e is C2~C 20 It is a hydrocarbon group.

[0018] In another preferred example, R6 is -R 6a -R 6b -R 6c -R 6d -R 6e It has a structure, However, R 6a and R 6c Each is independently -(CH2) n -where n is a positive integer chosen from 1 to 14, R 6b and R 6d These are each independent of or 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-,

[0019] [ka] a functional group selected from, preferably -(C=O)O-, -O(C=O)-, -(S-S)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-, R 6e is a C2-C 20 hydrocarbon group, R7 is a C1-C5 hydrocarbon group or -(CH2) m O(CH2) n selected from -, provided that m and n are each independently selected from 1, 2, 3.

[0020] In another preferred example, X and Y are -CH-.

[0021] In another preferred example, X and Y are N.

[0022] In another preferred example, the ionizable lipid has a structure represented by the following formula:

[0023]

Chemical formula

[0024] In another preferred example, the ionizable lipid has a substructure represented by the following (I-1),

[0025]

Chemical formula

[0026] In another preferred example, R6 is H.

[0027] In other preferred examples, the ionizable lipid has the structure shown in (I-2) below,

[0028] [ka] however, R1 and R2 are independently controlled by -(CH2) n - is chosen from, where n is a positive integer from 1 to 14. L1 moves from right to left - (L 1a -L 1b )- has or does not have the structure, however, L 1a The functional group is selected from -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(NH-, -CH(OH)-, and is preferably selected from -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-, L 1b ha-(CH2) n -where n is selected from 0, 1, 2, 3 or 4, L2 moves from left to right - (L 2a -L 2b )- has or does not have the structure, however, L 2a The functional group is selected from -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(NH-, -CH(OH)-, and is preferably selected from -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-, L 2b ha-(CH2) n -where n is selected from 0, 1, 2, 3 or 4, R3 is -R 3a -R 3b -R 3c -R 3d -R 3e It has the structure, and R4 is -R 4a -R 4b -R 4c -R 4d -R 4e It has the structure, and R5 is -R 5a -R 5b -R 5c -R 5d -R 5e It has a structure, However, R 3a , R 3c , R 4a , R 4c , R 5a , R 5c Each is independently -(CH2) n -where n is a positive integer chosen from 1 to 14, R 3b , R 3d、 R 4b , R 4d , R 5b , R 5d Each is independent of the others or -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-,

[0029] [ka] A functional group selected from, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, and -CH(OH)-, R 3e , R 4e , R 5e Each of these is independently C2~C 20 It is a hydrocarbon group, R7 is a C1-C5 hydrocarbon group or -(CH2). m O(CH2) n - is chosen from, where m and n are independently chosen from 1, 2, and 3, respectively.

[0030] In other preferred examples, the ionizable lipid has the structure shown in (I-3) below,

[0031] [ka] however, R1 and R2 are independently controlled by -(CH2) n - is chosen from, where n is a positive integer from 1 to 14. L1 moves from right to left - (L 1a -L 1b )- has or does not have the structure, however, L 1a The functional group is selected from -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(NH-, -CH(OH)-, and is preferably selected from -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-, L 1b ha-(CH2) n -where n is selected from 0, 1, 2, 3 or 4, L2 moves from left to right - (L 2a -L 2b )- has or does not have the structure, however, L 2a The functional group is selected from -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(NH-, -CH(OH)-, and is preferably selected from -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-, L 2b ha-(CH2) n -where n is selected from 0, 1, 2, 3 or 4, R3 is -R 3a -R 3b -R 3c -R 3d -R 3e It has the structure, and R4 is -R 4a -R 4b -R 4c -R 4d -R 4e It has the structure, and R5 is -R 5a -R 5b -R 5c -R 5d -R 5e It has the structure, and R6 is -R 6a -R6b -R 6c -R 6d -R 6e It has a structure, However, R 3a , R 3c , R 4a , R 4c , R 5a , R 5c、 R 6a and R 6c Each is independently -(CH2) n -where n is a positive integer chosen from 1 to 14, R 3b , R 3d、 R 4b , R 4d , R 5b , R 5d , R 6b and R 6d Each is independent of the others or -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-,

[0032] [ka] A functional group selected from, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, and -CH(OH)-, R 3e , R 4e , R 5e , R 6e Each of these is independently C2~C 20 It is a hydrocarbon group, R7 is a C1-C5 hydrocarbon group or -(CH2). m O(CH2) n - is chosen from, where m and n are independently chosen from 1, 2, and 3, respectively.

[0033] In other preferred examples, the ionizable lipid has the structure shown in formula (I-1), and in formula (I-1), R1 and R2 are independently controlled by -(CH2) n - is chosen from, where n is a positive integer between 2 and 8. L1 is a functional group selected from -(C=O)O- and -O(C=O)-. L2 is a functional group selected from -(C=O)O- and -O(C=O)-. R3, R4, R5, and R6 are each independently C5~C 20 It is a hydrocarbon group, R7 is a C1-C5 hydrocarbon group or -(CH2). m O(CH2) n - is chosen from, where m and n are each independently chosen from 1 or 2.

[0034] In other preferred examples, the ionizable lipid has the structure shown in formula (I-2), and in formula (I-2), R1 and R2 are independently controlled by -(CH2) n - is chosen from, where n is a positive integer between 2 and 8. L1 is either absent or -O-(CH2) n -where n is selected from 0, 1, 2, 3 or 4, L2 is a functional group selected from -(C=O)O- and -O(C=O)-. R3 is -R 3a -R 3b -R 3c -R 3d -R 3e It has the structure, and R4 is -R 4a -R 4b -R 4c -R 4d -R 4e It has the structure, and R5 is -R 5a -R 5b -R 5c -R 5d -R 5e It has a structure, However, R 3a , R 3c , R 4a , R 4c , R 5a , R5c Each is independently -(CH2) n -where n is a positive integer chosen from 1 to 3, R 3b and R 4b These are each independently selected from the groups -(C=O)O- and -O(C=O)-, R 3d , R 4d , R 5d These are each independently selected from the groups consisting of -CH2- and -(SS)-, R 5b story, R 3e , R 4e , R 5e Each of these is independently C2~C 15 It is a hydrocarbon group, R7 is a C1-C5 hydrocarbon group or -(CH2). m O(CH2) n - is chosen from, where m and n are each independently chosen from 1 or 2.

[0035] In other preferred examples, the ionizable lipid has the structure shown in formula (I-1), and in formula (I-1), R1 and R2 are independently controlled by -(CH2) n - is chosen from, where n is a positive integer between 2 and 8. L1 is a functional group selected from -NH(C=O)- and -(C=O)NH-. L2 is a functional group selected from -NH(C=O)- and -(C=O)NH-. R3, R4, R5, and R6 are each independently C5~C 20 It is a hydrocarbon group, R7 is a C1-C5 hydrocarbon group or -(CH2). m O(CH2) n - is chosen from, where m and n are each independently chosen from 1 or 2.

[0036] In other preferred examples, the ionizable lipid has the structure shown in formula (I-2), and in formula (I-2), R1 and R2 are independently controlled by -(CH2) n- is chosen from, where n is a positive integer between 2 and 8. L1 is a functional group selected from -(C=O)S- and -S(C=O)-. L2 is a functional group selected from -(C=O)S- and -S(C=O)-. R3, R4, R5, and R6 are each independently C5~C 20 It is a hydrocarbon group, R7 is a C1-C5 hydrocarbon group or -(CH2). m O(CH2) n - is chosen from, where m and n are each independently chosen from 1 or 2.

[0037] In other preferred examples, the ionizable lipid has the structure shown in formula (I-3), and in formula (I-3), R1 and R2 are independently controlled by -(CH2) n - is chosen from, where n is a positive integer between 2 and 8. L1 is either absent or -O-(CH2) n -where n is selected from 0, 1, 2, 3 or 4, L2 is either absent or -O-(CH2). n -where n is selected from 0, 1, 2, 3 or 4, R3 is -R 3a -R 3b -R 3c -R 3d -R 3e It has the structure, and R4 is -R 4a -R 4b -R 4c -R 4d -R 4e It has the structure, and R5 is -R 5a -R 5b -R 5c -R 5d -R 5e It has the structure, and R6 is -R 6a -R 6b -R 6c -R 6d -R 6e It has a structure, However, R 3a , R 3c , R 4a , R 4c , R5a , R 5c、 R 6a , R 6c Each is independently -(CH2) n -where n is a positive integer chosen from 1 to 3, R 3b , R 4b , R 5b and R 6b Each of these is independently selected from the group consisting of -(C=O)O-, -O(C=O)-, and -CH(OH)-. R 3d , R 4d , R 5d and R 6d These are each independently selected from the groups consisting of -CH2- and -(SS)-, R 3e , R 4e , R 5e and R 6e Each of these is independently C2~C 15 It is a hydrocarbon group, R7 is a C1-C5 hydrocarbon group or -(CH2). m O(CH2) n - is chosen from, where m and n are each independently chosen from 1 or 2.

[0038] In another preferred example, R3, R4, R5, and R6 are each independently C5~C 15 alkyl groups, C5~C 15 The alkenyl group. C5~C 15 Selected from the group consisting of alkynyl groups, R7 is selected from C1-C5 alkyl groups, -(CH2)O(CH2)-, or -(CH2)2O(CH2)2-, where m and n are independently selected from 1, 2, and 3, respectively.

[0039] In other preferred examples, the ionizable lipid has a structure selected from Table 1 below,

[0040] Table 1 [Table 1-1]

[0041] (Continued from Table 1) [Table 1-2]

[0042] (Continued from Table 1) [Table 1-3]

[0043] (Continued from Table 1) [Table 1-4]

[0044] (Continued from Table 1) [Table 1-5]

[0045] (Continued from Table 1) [Table 1-6]

[0046] (Continued from Table 1) [Table 1-7]

[0047] (Continued from Table 1) [Table 1-8]

[0048] (Continued from Table 1) [Table 1-9]

[0049] (Continued from Table 1) [Table 1-10]

[0050] (Continued from Table 1) [Table 1-11]

[0051] (Continued from Table 1) [Table 1-12]

[0052] (Continued from Table 1) [Table 1-13]

[0053] (Continued from Table 1) [Table 1-14]

[0054] (Continued from Table 1) [Table 1-15] .

[0055] In another preferred example, R3, R4, R5, and R6 are each independently C5~C 15 It is an alkyl group.

[0056] In other preferred examples, the ionizable lipid has the structure shown in (I-1) below,

[0057] [ka] however, R1 and R2 are independently controlled by -(CH2) n - is chosen from, where n is a positive integer between 4 and 8. L1 is a functional group selected from -O-, -(C=O)O-, -O(C=O)-, and -CH(OH)-. L2 is a functional group selected from -O-, -(C=O)O-, -O(C=O)-, and -CH(OH)-. R3 is -R 3a -R 3b -R 3c -R 3d -R 3e It has the structure, and R4 is -R 4a -R 4b -R 4c -R 4d -R 4e It has the structure, and R5 is -R 5a -R 5b -R 5c -R 5d -R 5e It has the structure, and R6 is -R 6a -R 6b -R 6c -R 6d -R 6e It has a structure, However, R 3a , R 3c , R 4a , R 4c , R 5a , R 5c、 R 6a and R 6c Each is independently -(CH2) n -where n is a positive integer selected from 1 to 14, and R3, R4, R5, and R6 each independently have 4 to 20 CH2 structural fragments. R 3b , R 3d , R 4b , R 4d , R 5b , R 5d , R 6b and R 6d Each of these is an independent functional group selected from the group consisting of -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, and -CH(OH)-. R 3e , R 4e , R 5e , R 6e Each of these is independently C2~C 20is a hydrocarbon group, R7 is a C1-C3 hydrocarbon group or -(CH2)2-O-(CH2)2-.

[0058] In another preferred example, the ionizable lipid has a structure represented by the following formula:

[0059]

Chemical formula

[0060] In a second aspect of the present invention, there is provided a method for preparing the ionizable lipid described in the first aspect of the present invention, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof. The preparation method includes Method I, Method II and Method III. However, Method I is (S1) Under the protection of an inert gas, reacting compound A1 and A2 to obtain compound A3; (S2) Under the protection of an inert gas, removing the tert-butoxycarbonyl group (Boc) from A3 to obtain A4; (S3) Under the protection of an inert gas, reacting A4 with A5 or A6 to obtain A7 (i.e., the compound shown in (I-1)):

[0061]

Chemical formula

[0062] [ka] The step includes obtaining the compound shown, However, R1 and R2 are independently -(CH2) n - is chosen from, where n is a positive integer from 1 to 14. G1 and G2 are each independently selected from active functional groups, preferably from carbonyl groups, halogens (fluorine, chlorine, bromine, iodine, etc.), ethylene oxide, etc. L1 moves from right to left - (L 1a -L 1b )- has or does not have the structure, however, L 1a The functional group is selected from -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(NH-, -CH(OH)-, and is preferably selected from -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-, L 1b ha-(CH2) n -where n is selected from 0, 1, 2, 3 or 4, L2 moves from left to right - (L 2a -L 2b )- has or does not have the structure, however, L 2a The functional group is selected from -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(NH-, -CH(OH)-, and is preferably selected from -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-, L 2b ha-(CH2) n -where n is selected from 0, 1, 2, 3 or 4, R3 is -R 3a -R 3b -R 3c -R 3d -R 3e It has the structure, and R4 is -R 4a -R 4b -R 4c-R 4d -R 4e It has the structure, and R5 is -R 5a -R 5b -R 5c -R 5d -R 5e It has a structure, However, R 3a , R 3c , R 4a , R 4c , R 5a , R 5c Each is independently -(CH2) n -where n is a positive integer chosen from 1 to 14, R 3b , R 3d、 R 4b , R 4d , R 5b , R 5d Each is independent of the others or -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-,

[0063] [ka] A functional group selected from, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, and -CH(OH)-, R 3e , R 4e , R 5e Each of these is independently C2~C 20 It is a hydrocarbon group, R6 is H, R7 is a C1-C5 hydrocarbon group or -(CH2). m O(CH2) n - is chosen from, where m and n are independently chosen from 1, 2, and 3, Method III is, (M1) A step of reacting compounds C1 and C2 under the protection of an inert gas to produce C3, (M2) A step of removing Boc from compound C3 under the protection of an inert gas to produce compound C4, (M3) Under the protection of an inert gas, compound C4 is reacted with C5 or C6 to produce C7, i.e., formula (I-3):

[0064] [ka] The step includes generating the compound shown, However, R1 and R2 are independently -(CH2) n - is chosen from, where n is a positive integer from 1 to 14. G1 and G2 are each independently selected from active functional groups, preferably from carbonyl groups, halogens (fluorine, chlorine, bromine, iodine, etc.), ethylene oxide, etc. L1 moves from right to left - (L 1a -L 1b )- has or does not have the structure, however, L 1a The functional group is selected from -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(NH-, -CH(OH)-, and is preferably selected from -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-, L 1b ha-(CH2) n -where n is selected from 0, 1, 2, 3 or 4, L2 moves from left to right - (L 2a -L 2b )- has or does not have the structure, however, L 2aThe functional group is selected from -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(NH-, -CH(OH)-, and is preferably selected from -O-, -(C=O)O-, -O(C=O)-, -CH(OH)-, L 2b ha-(CH2) n -where n is selected from 0, 1, 2, 3 or 4, R3 is -R 3a -R 3b -R 3c -R 3d -R 3e It has the structure, and R4 is -R 4a -R 4b -R 4c -R 4d -R 4e It has the structure, and R5 is -R 5a -R 5b -R 5c -R 5d -R 5e It has the structure, and R6 is -R 6a -R 6b -R 6c -R 6d -R 6e It has a structure, However, R 3a , R 3c , R 4a , R 4c , R 5a , R 5c、 R 6a and R 6c Each is independently -(CH2) n -where n is a positive integer chosen from 1 to 14, R 3b , R 3d、 R 4b , R 4d , R 5b , R 5d , R 6b and R 6dis each independently none or -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -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-,

[0065]

Chem.

[0066] In a third aspect of the present invention, lipid nanoparticles (LNP) are provided, and the lipid nanoparticles contain the ionizable lipid described in the first aspect of the present invention, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0067] In other preferred examples, the lipid nanoparticles further contain co-lipids.

[0068] In other preferred examples, in the lipid nanoparticles, the content of the ionizable lipid is in a molar ratio of 30-65% of the total lipid content.

[0069] <000​​​​In other preferred examples, the auxiliary lipids are combinations of auxiliary phospholipids, sterols, and polymer-bound lipids.

[0071] In other preferred examples, the auxiliary phospholipids are preferably 1,2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), dioleoyl lecithin (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphorylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-myristoyl-sn-glycero-3- Selected from phosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoryl-rac-(1-glycero)sodium salt, 1,2-palmitoylphosphatidylglycerol, 1-palmitoyl-2-oleoyllecithin, 1-palmitoyl-2-oleoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, 1-stearoyl-2-oleoylphosphatidylethanolamine, or a combination thereof.

[0072] In other preferred examples, the sterols include cholesterol or cholesterol derivatives.

[0073] In another preferred example, the polymer-bound lipid is polyethylene glycolated (PEG) lipid.

[0074] In other preferred examples, the polyethylene glycolated lipid is preferably selected from the group consisting of DMG-PEG2000, DSPE-PEG2000, DSG-PEG2000, DSPE-PEG-Mannose, DMG-PEG2000-(polypeptides, proteins, amino acids, vitamins, and other active substances) or combinations thereof.

[0075] In other preferred examples, the lipid nanoparticles comprise 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).

[0076] In other preferred examples, the lipid nanoparticles further comprise bioactive substances encapsulated within the lipid nanoparticles.

[0077] In other preferred examples, the bioactive substance is selected from the group consisting of nucleic acids, proteins, polypeptides, small molecules, or combinations thereof.

[0078] In other preferred examples, nucleic acids include DNA, plasmids, messenger RNA (mRNA), small interfering RNs (siRNA), antisense oligonucleotides, small RNAs, ribosomal RNAs, microRNAs, and transfer RNAs, and are preferably mRNA.

[0079] A fourth aspect of the present invention provides a lipid nanoparticle pharmaceutical formulation, the lipid nanoparticle pharmaceutical formulation is i) Lipid nanoparticles according to the third aspect of the present invention, ii) Bioactive substances encapsulated in lipid nanoparticles, iii) comprising a pharmaceutically acceptable carrier.

[0080] In other preferred examples, the bioactive substance is selected from the group consisting of nucleic acids, proteins, polypeptides, small molecules, or combinations thereof.

[0081] In other preferred examples, nucleic acids include DNA, plasmids, messenger RNA (mRNA), small interfering RNs (siRNA), antisense oligonucleotides, small RNAs, ribosomal RNAs, microRNAs, and transfer RNAs, and are preferably mRNA.

[0082] In other preferred examples, the bioactive substance is nucleic acid, and in the lipid nanoparticle pharmaceutical formulation, the molar ratio of ionizable N atoms in the ionizable lipid molecule to phosphate groups in the nucleic acid molecule is (2-10):1, and more preferably (4-8):1.

[0083] In other preferred examples, the hydrated particle size of the lipid nanoparticle pharmaceutical formulation is 50 to 200 nm, preferably 70 to 150 nm, and most preferably 75 to 110 nm.

[0084] In other preferred examples, lipid nanoparticle pharmaceutical formulations can be used for the treatment and / or prevention of tumors, infectious diseases and rare diseases.

[0085] In other preferred examples, the dosage form of the lipid nanoparticle pharmaceutical formulation is selected from the group consisting of injections, lyophilized preparations, nebulizer inhalants, and topical preparations.

[0086] In other preferred examples, the lipid nanoparticle pharmaceutical formulation is administered by injection, i.e., by intravenous, intramuscular, intradermal, subcutaneous, intrasacral, duodenal, or intraperitoneal injection.

[0087] In other preferred examples, the lipid nanoparticle pharmaceutical formulation is administered by inhalation, for example, intranasal administration.

[0088] In other preferred examples, the lipid nanoparticle pharmaceutical formulation is administered transdermally, such as by topical application or electrotherapy.

[0089] A fifth aspect of the present invention provides a method for preparing a lipid nanoparticle pharmaceutical formulation as described in the fourth aspect of the present invention, the method being: (a) A step of obtaining a lipid organic phase by mixing an ionizable lipid described in the first aspect of the present invention, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, and an optional auxiliary lipid with an organic solvent, (b) A step of obtaining an aqueous phase containing a biologically active substance by mixing the biologically active substance with an aqueous solvent, (c) The step of obtaining a lipid nanoparticle pharmaceutical formulation by mixing the lipid organic phase in step (a) with the aqueous phase in step (b).

[0090] In other preferred examples, the organic solvent includes ethanol, methanol, isopropyl alcohol, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, tetrahydrofuran, or a combination thereof.

[0091] In another preferred example, the aqueous solvent is a buffer solution.

[0092] In another preferred example, the aqueous solvent is a buffer solution with a pH range of 3 to 7.

[0093] In another preferred example, the acidic buffer is a citrate buffer at pH 4.0.

[0094] In other preferred examples, the volume ratio of the lipid organic phase to the aqueous phase containing the bioactive substance is 1:(2-5), preferably 1:(3-4).

[0095] In another preferred example, step (c) involves mixing the lipid organic phase and the aqueous phase using a microfluidic chip.

[0096] In other preferred examples, the method further comprises step (d) purifying, concentrating, and filtering the lipid nanoparticle pharmaceutical formulation obtained in step (c).

[0097] A sixth aspect of the present invention provides the use of ionizable lipids, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof, as described in the first aspect of the present invention, in the preparation of drug delivery systems.

[0098] In other preferred examples, the delivery system includes lipid nanoparticles (LNPs), liposomes, polymer nanoparticles, and is preferably used for the production of lipid nanoparticles.

[0099] In another preferred example, a drug delivery system is used to deliver drugs that treat and / or prevent tumors, infectious diseases, and rare diseases.

[0100] A seventh aspect of the present invention provides the use of the lipid nanoparticles 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.

[0101] An eighth aspect of the present invention provides the use of an ionizable lipid or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention in the preparation of a lipid nanoparticle pharmaceutical formulation, wherein the lipid nanoparticle pharmaceutical formulation is used to deliver a bioactive substance to cells in the body of a subject who requires it.

[0102] In other preferred examples, the bioactive substance is selected from the group consisting of DNA, RNA (mRNA, tRNA, rRNA, miRNA), and natural and synthetic oligonucleotides (including antisense oligonucleotides (ASOs), interfering RNA (RNAi), and small interfering RNA (siRNA)).

[0103] In another preferred example, the bioactive substance is mRNA.

[0104] In another preferred example, mRNA is transfected into cells and expressed within those cells.

[0105] In another preferred example, the cells are immune cells.

[0106] In other preferred examples, the cells include T cells, B cells, NK cells, or a combination thereof.

[0107] In other preferred examples, T cells are selected from a group consisting of human primary T cells, JM cells, Jurkat cells, or combinations thereof.

[0108] In other preferred examples, human primary T cells include auxiliary T cells, regulatory T cells, and memory T cells.

[0109] In another preferred example, the ionizable lipid has the structure shown in the following formula:

[0110] [ka]

[0111] Within the scope of the present invention, it should be understood that novel or preferred technical solutions can be formed by combining the above-described technical features of the present invention with the technical features specifically described below (for example, in the examples). Due to space limitations, further details will not be provided here. [Brief explanation of the drawing]

[0112] [Figure 1] The particle size characteristics evaluation results for LNP-mRNA of different formulations consisting of AXT-8 are shown, with a particle size range of 80-130 nm, demonstrating that the physicochemical properties of the LNPs prepared above are similar (Explanation: In the example in the above figure, LNP(AXT-8)-Luc-1 indicates that LNP consisting of the molecule AXT-8 encapsulates Luc mRNA, 1 indicates formulation 1 in a series of LNPs encapsulating Luc mRNA, and the others are similar; LNP(AXT-8)-hEPO-1 indicates that LNP consisting of the molecule AXT-8 encapsulates hEPO mRNA, 1 indicates formulation 1 in a series of LNPs encapsulating hEPO mRNA, and the others are similar; LNP(AXT-8)-eGFP-1 indicates that LNP consisting of the molecule AXT-8 encapsulates eGFP mRNA, 1 indicates formulation 1 in a series of LNPs encapsulating eGFP mRNA; LNP(AXT-8)-mCherry-1 indicates that LNP consisting of the molecule AXT-8 encapsulates mCherry This indicates that mRNA has been encapsulated, and 1 represents formulation 1 in a series of LNPs containing mCherry mRNA. [Figure 2] The results of characterizing the particle size distribution of LNP-mRNAs with different formulations consisting of AXT-8 are shown, and it has been proven that the particle size distribution of nanoparticles consisting of AXT-8 is small, with a PDI < 0.2. [Figure 3] The results of characterizing the inclusion rates of LNP-mRNAs with different formulations consisting of AXT-8 are shown, and all showed inclusion rates of >90%, demonstrating good inclusion results. [Figure 4] The results of transfection of 293T cells with LNP-mRNA in different formulations consisting of AXT-8 are shown in the figure. As shown, with increasing mRNA concentration, the cell expression results for multiple formulations of LNP(AXT-8)-mRNA were all superior to those of lipofectamine. [Figure 5] The cytotoxicity results of LNP(AXT-8)-mRNA, consisting of AXT-8, are shown in the figure. As shown, when the mRNA concentration of the LNP(AL-8) series is lower than 1 ug / mL, the cell suppression rate is almost zero. [Figure 6] The results of in vivo hEPO expression of LNP-mRNA with different formulations of AXT-8 are shown. As is clear from the figure, for each formulation, the in vivo mRNA expression trend increased first and then decreased, and in all cases, it reached a maximum at around 6 hours. [Figure 7] The figure shows the expression results of in vitro transfection of LNP-mRNA containing AXT-8 into JM cells and Jurkat cells. As is clear from the figure, LNP(AXT-8)-mRNA was successfully expressed in both JM cells and Jurkat cells, and the expression levels were superior to those of lipofectamine Max in both cases. [Figure 8] The figure shows the expression results of LNP-mRNA formulated with AXT-8 in vitro transfection into human primary T cells. As is clear from the figure, LNP(AXT-8)-mRNA was successfully expressed in human primary T cells, and the expression levels were all superior to those of lipofectamine Max. [Modes for carrying out the invention]

[0113] As a result of diligent research, the inventors unexpectedly discovered an ionizable lipid for the first time. Ionizable lipids have the advantages of stable physicochemical properties and low toxicity. A drug delivery system obtained by encapsulating a drug load (e.g., mRNA) with the ionizable lipid described in the present invention has high delivery efficiency and low toxicity, efficiently delivers the drug load, increases the expression level of the drug load, improves the safety of the drug delivery system, and makes the preventive and therapeutic effects of the drug delivery system more pronounced. Based on this, the present invention was completed.

[0114] term To facilitate understanding of this disclosure, we first define some terms. As used in this application, each of the following terms shall have the meaning set forth below, unless otherwise specified herein.

[0115] The term "alkyl group" refers to a saturated carbon chain having 1 to 20 carbon atoms, and unless otherwise defined, the carbon chain may be linear, branched, or a combination thereof. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, and octyl groups.

[0116] Unless otherwise specified in the specification, alkyl groups may be optionally substituted. The term "unsaturated hydrocarbon group" means that the group has at least one C=C double bond (alkenyl group) or at least one

[0117] [ka] This means that it contains a triple bond (alkynyl group), and the terms "alkyl group," "alkenyl group," and "alkynyl group" may be collectively referred to as "hydrocarbon group."

[0118] In the claims of the present invention, when we describe a "C1-C30 hydrocarbon group," the group may be an alkyl group, alkenyl group, or alkynyl group having 1 to 30 carbon atoms (for example, 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 group, C2~C 30 The term "hydrocarbon group" has a similar meaning.

[0119] When we describe an alkyl group, the group may be a saturated hydrocarbon group having a predetermined number of carbon atoms (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms), may be linear, or may have a branched chain structure, and is usually a linear group without a cyclic structure. Any alkyl group that satisfies the aforementioned number of carbon atoms falls within the scope of this term.

[0120] When we describe an "alkenyl group," the group may be an alkenyl group having a predetermined number of carbon atoms (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms), may be linear, or may have a branched chain structure, and is usually a linear group without a cyclic structure. Any alkenyl group that satisfies the aforementioned number of carbon atoms falls within the scope of this term. In different embodiments of the present invention, the alkenyl group may be a group formed from a monoolefin or a polyhydric olefin (for example, a diolefin).

[0121] "C2~C 30When described as a "linear or branched alkenyl group," the group may be an alkenyl group having a predetermined number of carbon atoms (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms), may be linear, or may have a branched structure, and is usually a linear group without a cyclic structure. Any alkenyl group that satisfies the aforementioned number of carbon atoms falls within the scope of this description. In different embodiments of the present invention, the alkenyl group may be a monoolefin or a polyhydric olefin (for example, a diolefin).

[0122] "C1~C 10 When we describe an alkylene group, the group may be an alkylene group having 1 to 10 carbon atoms (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), and the alkylene group may have a linear or branched chain structure, but is usually a linear group and does not have a cyclic structure.

[0123] In this application, including "none" in the definition of a divalent group means that adjacent structural fragments of the divalent group are directly linked by chemical bonds.

[0124] Ionizable lipids As used herein, the terms “ionizable lipids of the present invention” and “ionizable cationic lipids of the present invention” are interchangeable and both refer to lipid compounds having the structure of formula I, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof.

[0125] Ionizable lipids are protonated and converted to cationic lipids at low pH values, and then converted to auxiliary phospholipids at normal physiological pH values. The interaction between auxiliary phospholipids and the anionic cell membrane of blood cells is reduced, improving the biocompatibility of lipid nanoparticles. After lipid nanoparticles are taken into cells, the low pH in endosomes protonates the lipids, giving them a positive charge, which worsens the stability of the membrane structure and leads to further disruption, favoring the escape of lipid nanoparticles from endosomes. Generally, the pH-sensitive properties of lipids are advantageous for the intracellular delivery of lipid nanoparticles containing bioactive components (e.g., mRNA molecules).

[0126] Supplementary lipids As used herein, the term “auxiliary lipids” in lipid nanoparticles refers to other types of lipids other than ionizable lipids, including auxiliary phospholipids, sterols, polymer-bound lipids, or combinations thereof. Auxiliary lipids are primarily used to improve the performance of lipid nanoparticles, such as stability, delivery efficiency, resistance, and biofractionation.

[0127] In some embodiments, the auxiliary phospholipids are 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), dioleoyl lecithin (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphorylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine, and 1,2-myristoyl-sn-glycero-3-phosphatidyl This includes (but is not limited to) ethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoryl-rac-(1-glycero)sodium salt, 1,2-palmitoylphosphatidylglycerol, 1-palmitoyl-2-oleoyllecithin, 1-palmitoyl-2-oleoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, 1-stearoyl-2-oleoylphosphatidylethanolamine, or combinations thereof.

[0128] In one preferred embodiment of the present invention, the auxiliary phospholipid is DSPC (1,2-distearoyl-sn-glycero-3-phosphorylcholine, also known as distearoylphosphatidylcholine). DSPC is a commonly used phosphatidylcholine, its terminal group is a saturated alkane chain, its melting point is -54°C, it exhibits a cylindrical morphology, and it forms a layered structure in lipid nanoparticles, further stabilizing the structure of lipid nanoparticles.

[0129] In one 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, and its terminal groups consist of two unsaturated alkane chains, with a melting point of -30°C, exhibiting a conical morphology, readily forming an inverted hexagon in lipid nanoparticles, causing instability in the endosomal membrane, and facilitating the escape of lipid nanoparticles from endosomes.

[0130] In some embodiments, the sterol includes (but is not limited to) cholesterol or cholesterol derivatives. Cholesterol can modulate the integrity and stiffness of the lipid membrane and enhance the stability of lipid nanoparticles, and the morphology of the cholesterol derivative can affect the delivery efficiency and biodistribution of lipid nanoparticles, such as the chain length of the hydrophobic end groups of the cholesterol analogue, the flexibility of the sterol ring, and the polarity of the hydroxyl group. Cholesterol also affects the morphology of lipid nanoparticles, with cholesterol derivatives causing the formed lipid nanoparticles to have a multilayer polyhedral structure delimited by lipids rather than a spherical shape. At the same time, cholesterol affects the selectivity of lipid nanoparticles for targets: lipid nanoparticles containing cholesterol oleate have higher selectivity for hepatic endothelial cells than for hepatocytes, and when containing cholesterol with oxidatively modified end groups, the content of lipid nanoparticles in hepatic endothelial cells and Kupffer cells is higher than in hepatocytes.

[0131] In some embodiments, the polymer-bound lipid is a polyethylene glycol (PEG)-bound lipid, also known as polyethylene glycolated lipid or PEGylated lipid. PEGylated lipids have multiple effects on the properties of lipid nanoparticles: the amount of PEGylated lipid used affects the particle size and potential of the lipid nanoparticles, reduces particle aggregation and increases the stability of the lipid nanoparticles, decreases the particle removal rate via the kidney and mononuclear phagocyte system (MPS), extends the particle circulation time, and modifies the surface functional groups with ligands to enhance target delivery capability. Molar mass and lipid length affect the properties of PEGylated lipids; both DMG-PEG2000 and DSG-PEG2000 are neutral phospholipids with saturated alkyl chain lengths of C14, C16, or C18, respectively. However, DMG-PEG2000 can be separated from lipid nanoparticles more quickly, which is advantageous for cellular uptake and exit from endosomes, resulting in superior delivery efficiency of DMG-PEG2000 compared to DSG-PEG2000.

[0132] In one preferred embodiment of the present invention, the auxiliary lipid is a combination of DSPC, cholesterol, and DMG-PEG2000.

[0133] Lipid nanoparticles (LNPs) As used herein, the terms “lipid nanoparticles,” “lipid nanoparticles,” or “LNPs” refer to particles with a diameter of approximately 5 to 500 nm. In some embodiments, lipid nanoparticles contain one or more activators (bioactive substances). In some embodiments, lipid nanoparticles contain nucleic acids. In some embodiments, nucleic acids condense within the nanoparticle with cationic lipids, polymers, or polyvalent small molecules 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 and readily adopt elongated structures. In cells, to cope with volume limitations, DNA can encapsulate itself under suitable solution conditions with the help of ions and other molecules. Typically, DNA condensation is defined as the contraction of an elongated DNA strand into compact, ordered particles containing only one or more molecules. By binding with phosphate groups, cationic lipids can concentrate and densely deposit DNA by neutralizing the charge of the phosphate.

[0134] In some embodiments, the bioactive substance is encapsulated in an LNP. In some embodiments, the bioactive substance may be an anionic compound and includes, but is 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 including DNA, for example, completely or partially defatted viral granules (viral particles), and oligomers and polymeric anionic compounds other than DNA (e.g., acidic polysaccharides and glycoproteins). In some embodiments, the bioactive substance is miscible with an adjuvant.

[0135] In LNP vaccine products, the bioactive substance is typically contained within the LNP. In some embodiments, the bioactive substance includes nucleic acids. Typically, water-soluble nucleic acids condense with cationic lipids or polycationic polymers within the particle, and the particle surface is richly contained with auxiliary phospholipids or PEG lipid derivatives. Excess ionizable cationic lipids may also be present on the surface, and after entering the cell lysosome, these cationic lipids ionize and become positively charged due to the acidic environment of the lysosome, interacting with the lysosomal membrane and facilitating escape from the endosome.

[0136] Regarding LNPs, ionizable lipids may have different properties or functions. Due to the pKa of the amino group, if the pH of the environment is lower than the pKa of the lipid molecule, it can be protonated and positively charged. Under these conditions, the lipid molecule can electrostatically bind to the phosphate group of nucleic acid, thereby forming an LNP in which the nucleic acid is encapsulated, and the surface charge of the LNP in a biological fluid (e.g., blood) at the physiological pH is essentially neutral. High LNP surface charge is associated with toxicity, rapid clearance in the circulatory system by fixed and free macrophages, hemolytic toxicity, and immunostimulation (Filion et al. Biochim Biophys Acta. 1997 / 00 / 23; 1329(2):345-56).

[0137] In some embodiments, the pKa can be made sufficiently high so that the ionizable cationic lipid can assume a positively charged form under the pH values ​​of acidic endosomes. Thus, the cationic lipid can bind to endogenous endosomal anionic lipids and facilitate non-duplex membrane cleavage, e.g., hexagonal HII phase membrane cleavage, thereby enabling more efficient intracellular delivery. In some embodiments, the pKa range is between 6.2 and 6.5. For example, the pKa may be approximately 6.2, 6.3, 6.4, or 6.5. The unsaturated end also contributes to the lipid's ability to adopt a non-duplex structure. (Jayaraman et al., Angew Chem Int Ed Engl. 20 Aug. 2012; 51(34):8529-33).

[0138] Other characteristics of LNP formulations, such as nucleic acid release, liposome removal rate, and circulating half-life, can be altered by the presence of polyethylene glycol and / or sterols (e.g., cholesterol) or other potential additives in the LNP and the overall chemical structure (including the pKa of any ionizable cationic lipid as part of the formulation).

[0139] In one aspect of the present invention, lipid nanoparticles (LNPs) are provided, which comprise an ionizable lipid as described in the first aspect of the present invention, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. Furthermore, the lipid nanoparticles further comprise one or more auxiliary lipids, the pre-auxiliary lipids comprising auxiliary phospholipids, steroids, or polymer-conjugated lipids.

[0140] Lipid nanoparticle pharmaceutical formulations In another aspect of the present invention, a lipid nanoparticle pharmaceutical formulation (or lipid nanoparticle pharmaceutical composition or LNP composition) is provided, the lipid nanoparticle pharmaceutical formulation comprising 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 formulation is used to deliver the bioactive substance to cells in the body of a subject who requires it.

[0141] In some embodiments, the bioactive substance is encapsulated in an LNP. In some embodiments, the bioactive substance may be an anionic compound and includes, but is 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 including DNA, for example, completely or partially defatted viral granules (viral particles), and oligomers and polymeric anionic compounds other than DNA (e.g., acidic polysaccharides and glycoproteins). In some embodiments, the bioactive substance is miscible with an adjuvant.

[0142] In some embodiments, the LNP composition comprises nucleic acids, ionizable cationic lipids having the structure shown in formula (I), auxiliary phospholipids (e.g., DSPC, DOPE, DOPC, or a combination thereof), sterols (e.g., cholesterol or cholesterol derivatives, or plant sterols, e.g., β-sitosterol), and polymer-bound lipids (e.g., DMG-PEG2000). In some embodiments, the LNP composition comprises nucleic acids, ionizable cationic lipids having the structure shown in formula I, in a content of 30-65% (molar ratio, the same applies hereafter) of the total lipids of the composition, auxiliary phospholipids (e.g., DSPC, DOPE, DOPC, or a combination thereof) in a content of 5-30% of the total lipids of the composition, sterols (e.g., cholesterol or cholesterol derivatives, or plant sterols, e.g., β-sitosterol) in a content of 30-55% of the total lipids of the composition, and polymer-bound lipids (e.g., DMG-PEG2000) in a content of 1-5% of the total lipids of the composition. Furthermore, in the LNP composition, the molar ratio of ionizable N atoms in the ionizable lipid molecule to phosphate groups in the nucleic acid molecule is (2-10):1, and more preferably (4-8):1.

[0143] In one preferred embodiment of the present invention, the LNP composition comprises nucleic acid, an ionizable cationic lipid having the structure shown in formula (I), auxiliary phospholipids (e.g., DSPC, DOPE, DOPC, etc., or combinations thereof), sterols (e.g., cholesterol or cholesterol derivatives, or plant sterols, e.g., β-sitosterol), and polymer-bound lipids (e.g., DMG-PEG2000). In one more preferred embodiment of the present invention, the LNP composition comprises nucleic acid, an ionizable cationic lipid having the structure shown in formula (I), auxiliary phospholipids (e.g., DSPC, DOPE, DOPC, etc., or combinations thereof), sterols (e.g., cholesterol or cholesterol derivatives, or plant sterols, e.g., β-sitosterol), and polymer-bound lipids (e.g., DMG-PEG2000).

[0144] As used herein, the terms “encapsulation” and “encapsulated” mean that mRNA, DNA, siRNA, or other nucleic acid drugs are encapsulated within or bound to lipid nanoparticles. As used herein, the term “encapsulation” means complete or partial encapsulation. For example, mRNA can be selected and administered to subjects who require it in a lipid nanoparticle composition to treat and / or prevent related diseases.

[0145] As used herein, the term “pharmaceutically acceptable carrier” includes, but is not limited to, any adjuvants, carriers, excipients, scintillators, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, humectants, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that may be used in humans or livestock with the approval of the Food and Drug Administration.

[0146] Method for preparing lipid nanoparticle pharmaceutical formulations In another aspect of the present invention, a method for preparing a lipid nanoparticle pharmaceutical formulation is provided, the method comprising: (a) obtaining a lipid organic phase by mixing an ionizable lipid and an optional auxiliary lipid described in the first aspect of the present invention with an organic solvent; (b) obtaining an aqueous phase containing a bioactive substance by mixing the bioactive substance with an aqueous solvent; and (c) obtaining a lipid nanoparticle pharmaceutical formulation by mixing the lipid organic phase in step (a) with the aqueous phase in step (b). Furthermore, the method further comprises (d) purifying, concentrating, and filtering the lipid nanoparticle pharmaceutical formulation obtained in step (c).

[0147] In some embodiments, the organic solvent includes (but is not limited to) ethanol, methanol, isopropyl alcohol, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, or tetrahydrofuran, or a combination thereof. In some embodiments, the lipid organic phase includes a small amount of water or pH buffer. The lipid organic phase may contain 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 contains 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 of water.

[0148] In some embodiments, the lipid organic phase includes a single type of lipid, such as an ionizable cationic lipid, an auxiliary phospholipid, a sterol, or a polymer-bound lipid. In some embodiments, the lipid organic phase includes multiple types of lipids. In one embodiment of the present invention, the lipid organic phase includes an ionizable cationic lipid having 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, e.g., β-sitosterol), and a polymer-bound lipid (e.g., DMG-PEG2000). In one preferred embodiment of the present invention, the lipid organic phase includes an ionizable cationic lipid having 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, e.g., β-sitosterol), and a polymer-bound lipid (e.g., DMG-PEG2000). In one more preferred embodiment of the present invention, the lipid organic phase comprises an ionizable cationic lipid having 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, e.g., β-sitosterol), and a polymer-bound lipid (e.g., DMG-PEG2000). In one specific embodiment of the present invention, the lipid organic phase comprises an ionizable cationic lipid having the structure shown in formula (I), DSPC, cholesterol, and DMG-PEG2000.

[0149] In some embodiments, the aqueous solvent is water. In some embodiments, the aqueous solvent is an aqueous buffer with a pH between 3 and 8 (for example, pH is about 3, about 4, about 5, or about 6). An aqueous phase containing a bioactive substance, such as nucleic acid (e.g., mRNA), is obtained by dissolving the bioactive substance in the aqueous solvent. The aqueous phase may contain a small amount of water-miscible organic solvent. The aqueous phase may contain at least one organic solvent (e.g., water-miscible organic solvent) in a volume percentage of up to 60% by water, for example, up to 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 percentage between the two. In one embodiment, the aqueous phase contains an organic solvent in a volume of about 0.05% to 60%, for example, an organic solution (e.g., a water-miscible organic solvent) in a volume of about 0.05% to 50%, about 0.05% to 40%, or about 5% to 20%. The aqueous buffer may be a citrate buffer, Tris-HCl buffer, sodium acetate buffer, PBS buffer, or a combination thereof. In some embodiments, the aqueous buffer is a citrate buffer with a pH between 4 and 6 (e.g., pH is about 4, about 5, or about 6). In one embodiment, the aqueous buffer is a citrate buffer with a pH of about 4.

[0150] In some embodiments, a solution of a mixture of a lipid organic phase and an aqueous phase containing a bioactive substance is dilutable, and the mixture includes an LNP suspension. In some embodiments, the pH of the solution of the mixture of the lipid organic phase and the aqueous phase containing a bioactive substance, including the LNP suspension, is adjustable. The pH of the LNP suspension can be diluted or adjusted by adding water, acid, 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 or adjusted.

[0151] In some embodiments, excess reagents, solvents, and unencapsulated nucleic acids can be removed from the LNP suspension by tangential flow filtration (TFF) (e.g., osmosis). 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, TFF is performed on the LNP suspension. In some embodiments, both dialyzing and TFF are performed on the LNP suspension.

[0152] The main advantages of this invention include the following: (a) The ionizable lipids according to the present invention can be combined with other components, such as auxiliary phospholipids (DSPC, DOPE, DOPC, DOPS, etc.), sterols (e.g., cholesterol or cholesterol derivatives), and PEG derivatives (e.g., DMG-PEG lipids, or DMG-PEG substances / or PEG derivatives modified with other groups) to form stable nanoparticles. (b) When the ionizable lipids according to the present invention form nanoparticles with other components and then encapsulate mRNA, the resulting nanoparticles have uniform particle size, high encapsulation efficiency, good stability, enhance the transfection efficiency of mRNA in target tissues or cells, and have low toxicity, thus making the preventive and therapeutic effects of mRNA vaccines / pharmaceuticals more pronounced.

[0153] The present invention will be further described below with reference to specific examples. It should be understood that these examples are merely illustrative and do not limit the scope of the present invention. Experimental methods in the following examples where specific conditions are not explicitly stated generally follow either normal conditions or conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts refer to weight percentages and weight parts.

[0154] Example 1 Preparation of ionizable lipids 1.1 Synthesis Procedure and Characterization of Compound AXT-8

[0155] [ka]

[0156] AXT-8 synthesis flowchart 1.1.1 Synthesis and Characterization of Products 8-31

[0157] [ka]

[0158] tert-butyl N-[(3R)-pyridine-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 overnight at 65°C under the protection of inert nitrogen.

[0159] Subsequently, the reaction mixture was cooled to ambient temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was solvated with 50 mL of water, and the solution obtained by combining it with 3 × 50 mL of ethyl acetate and the organic layer was extracted. The organic phase was then dried and concentrated on anhydrous sodium sulfate.

[0160] As a result, the obtained tert-butyl N-[(3R)-1-(2-hydroxyethyl)pyrrolidine-3-yl]carbamate (0.72 g, yield 48.52%) is a colorless oil. The mass spectrum and nuclear magnetic data are shown below. LC-MS-8-31:(ES,m / z):231[M+H]+; 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).

[0161] 1.1.2 Synthesis and Characterization of Products 8-32

[0162] [ka]

[0163] tert-butyl N-[(3R)-1-(2-hydroxyethyl)pyridine-3-yl]carbamate (0.72 g, 3.126 mmol, 1 equiv) and DCM (7.2 mL) were placed in a 100 mL three-necked round-bottom bottle and protected with inert nitrogen gas.

[0164] Next, trifluoroacetaldehyde (1.45 mL) was added dropwise at 0°C, and the resulting solution was stirred at 0°C for 30 minutes and detected by LC-MS. The resulting mixture was concentrated.

[0165] As a result, the obtained 2-[(3R)-3-aminopyrrolidine-1-yl]ethanol, trifluoroacetaldehyde (0.5g, yield: 70.08%) is a pale yellow oil. The mass spectrum and nuclear magnetic property evaluation results are shown below. LC-MS-8-32:(ES,m / z):131[M+H]+; 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).

[0166] 1.1.3 Synthesis and Characterization of Final Products

[0167] [ka]

[0168] Regarding the synthesis procedure, 1) Place 2-[(3R)-3-aminopyridine-1-yl]ethanol in a 500 mL three-necked round-bottom flask, protect with inert nitrogen gas, and sequentially add trifluoroacetaldehyde (300 mg, 1.315 mmol, 1 equiv), 6-oxyhexyl-2-hexyldecanoate (1025.43 mg, 2.893 mmol, 2.2 equiv), DCM (15 mL), and THF (15 mL). Stir the resulting solution at room temperature for 0.5 hours. 2) At 0°C, add diacetyl peroxide and sodium acetate borate (835.82 mg, 3.945 mmol, 3 equiv) one drop at a time, controlling the drop-off time to 5 minutes. 3) Stir continuously at 0°C for 0.5 hours, then stir overnight at room temperature. 4) Add 50 mL of saturated NH4Cl to quench the reaction. 5) Extract the obtained solution with 3 × 50 mL of DCM, combine with the organic layer, 6) The organic phase is dried and concentrated on anhydrous sodium sulfate. 7) Under the following conditions (IntelFlash-1), the residue was further purified by flash-prepare-HPLC: 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 minutes, detector, ultraviolet 220 nm, and 8) The acceptable fractions were combined and concentrated, the residue was dissolved in DCM (50 mL), the organic phase was washed with 2 × 50 mL of NaHCO3 (aq, 2 M), 1 × 50 mL of water, and brine (50 mL), and dried on anhydrous Na2SO4.

[0169] The filtered filtrate was concentrated under reduced pressure. The resulting 6-({6-[(2-hexyldecyl)oxy]hexyl}[(3R)-1-(2-hydroxyethyl)pyrrolidine-3-yl]amino)hexylhexyldecanoate (0.2321 g, yield 21.87%) is a yellow oil. The mass spectrum, nuclear magnetic and HPLC characterization results are shown below. LC-MS:(ES,m / z):807.8[M+H] + ; H-NMR:1H NMR(300MHz,Chloroform-d,ppm)δ4.062(t,J=6.6Hz,4H),3.639(t,J=5.1Hz,2H),3.419(t,J=6.9Hz,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); Purity detection of AXT-8 using HPLC-CAD revealed a product peak appearance time of 18.039 min, indicating a purity of over 94%.

[0170] Example 2 mRNA was encapsulated using ionizable lipids and assembled as LNP-mRNA. The present inventors have determined that the step of assembling LNP-mRNA by encapsulating mRNA using an ionizable lipid is, 1) A step of thoroughly mixing ionizable lipids (AXT-8), DSPC (distearoylphosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphorylcholine), cholesterol, and DMG-PEG2000 in different proportions in an ethanol phase to form an organic phase, 2) Dissolve mRNA in sodium citrate solution (pH=4.0) and mix thoroughly as an aqueous phase, 3) Transfer the aqueous phase and organic phase to appropriate BD syringes, and remove as many air bubbles as possible. 4) Using a PNI nanometer, the aqueous phase and organic phase are mixed under conditions of a volume ratio of 3:1 and a flow rate of 12 mL / min, and then sequentially purified, concentrated, and filtered to obtain a lipid nanoparticle (LNP-mRNA) product containing mRNA. 5) The step of performing physicochemical quality control on the final LNP-mRNA product.

[0171] Methods and results of physicochemical quality control of LNP-mRNA 1) Particle size and PDI (distribution): The particle size and distribution of LNPs were detected using a nanoparticle size analyzer, and the results are shown in detail in Figures 1 and 2. 2) Inclusion Rate (EE%): After staining total mRNA and free mRNA using RiboGreen, they were detected with a microplate reader, and the inclusion rate was calculated. The results are shown in detail in Figure 3. 3) pH: The pH of the final product detected using a pH meter was 7.2-7.4. 4) Osmotic pressure: The osmotic pressure of LNP-mRNA was detected using a freezing point depression osmometer, and the osmotic pressure at a detection concentration of 100 μg / mL was 280-310 mOsmol / kg. 5) mRNA integrity: The integrity of the mRNA after encapsulation (i.e., mRNA purity) was detected using Agilent Fragment Analyzers, and the purity was measured to be greater than 90% using an RNA kit (15nt).

[0172] Example 3 In vitro expression detection of LNP-mRNA This invention further performs LNP-mRNA cell expression screening experiments, and the specific procedure is as follows. 1) Cell seeding: After digesting 293T cells, the cell density is increased to 2 × 10⁻⁶. 4 The cells were adjusted to one well and seeded at 100 μL / well in a 96-well plate, then placed in a cell incubator overnight. 2) Test grouping: LNP(AXT-8)-Luc series products were used as the sample group, Lipofectamine Messenger MAX as the positive control group, and a mixture of culture medium and cells (100 μL) was added to create the negative control group. 3) Cell transfection: LNP-Luc does not require transfection. Each well was filled with 100 ng of mRNA as the maximum concentration, and three dilutions were performed for a total of six dilutions. Three duplicate wells were provided for each sample. 10 μL of each LNP-Luciferase at different dilution levels was uniformly placed in a 96-well plate, mixed, and then cultured in an incubator. 4) Kit-based detection: After 48 hours of transfection, the luminescence intensity of the luciferase was detected using a BioTek SYNERGY microplate reader, following the instructions in the ONE-Glo® EX Luciferase Assay System kit manual, and the OD values ​​were averaged and compared.

[0173] As shown in Figure 4, all LNP(AXT-8)-Luc samples with different formulations were able to be normally expressed in cells, and their expression levels increased with increasing mRNA concentration. This indicates that LNP-mRNA prepared from AXT-8 is efficiently expressed in vitro.

[0174] Example 4 In vitro toxicity detection of LNP-mRNA The specific procedure for the cytotoxicity experiment is as follows: 1) Cell seeding: After digesting 293T cells, the cell density was adjusted to 2 × 10⁴ / well, and the cells were seeded at 100 μL / well in a 96-well plate and placed in a cell incubator overnight. 2) Grouping: LNP(AXT-8)-Luc was used as the sample group. A blank control group was created by adding only 100 μL of culture medium, a negative control group was created by adding only 100 μL of a mixture of culture medium and cells, and a positive control group was created by adding 100 μL of a mixture of culture medium and cells to the apoptosis inducer Staurosporine (STS). 3) Cell transfection: LNP-Luc does not require transfection. Each well is filled with 100 ng of mRNA as the maximum concentration, and three dilutions (buffer dilutions) are performed, resulting in a total of four dilutions. 10 μL of each LNP-Luc at different dilution ratios was uniformly placed in a 96-well plate, with three duplicate wells for each sample. After mixing, the samples were placed in an incubator for culture. 10 μL of 100 μM STS (PBS dilution) was placed in a 96-well plate, with six duplicate wells. After mixing, the samples were placed in an incubator for culture. 4) Kit-based detection: Cells were cultured for 48 hours, and the procedure described in the instructions for the CellTiter-Glo® Luminescent Cell V(I)bility Assay kit was followed. Luminescence intensity was detected using a BioTek SYNERGY microplate reader, and the OD values ​​were averaged and compared. Cell suppression rates were calculated according to the formula "(Negative control group - Sample group / Positive control group) ÷ (Negative control group - Blank control group) × 100%" and dose-response-inhibition curves were plotted.

[0175] As shown in Figure 5, the results suggest that the series of LNP-mRNA products consisting of AXT-8 have no significant effect on cell proliferation at concentrations lower than 1 μg / mL, and that there is no apparent toxicity within the detection range.

[0176] Example 5 Detection of LNP-mRNA expression in vivo To clarify the in vivo expression capacity and toxicity of LNP-mRNA assembled with AXT-8, we further designed hEPO ELISA expression detection experiments and toxicity experiments, and measured their expression levels and toxicity in mice with healthy innate immunity. The procedure is as follows: 1) Inclusion of LNP-mRNA: hEPO mRNA was dissolved in an aqueous buffer and mixed to form the aqueous phase. Ionizable lipids (AXT-8), DSPC, cholesterol, and DMG-PEG2000 were each dissolved in anhydrous ethanol and thoroughly mixed in a fixed ratio to form the organic phase. The aqueous and organic phases were transferred to syringes, and parameters (volume ratio of aqueous to organic phase: 3:1, flow rate: 12 mL / min) were set using a PNI microflow controlled nanometer to prepare hEPO-LNP. LNP-hEPO was concentrated, purified, filtered to remove bacteria, and injected into mice after passing quality control. 2) Mouse tail vein injection: Mice were fixed, and injections were administered to the selected tail vein. The injection volume of LNP-hEPO was 5 μg. LNP(AXT-8)-hEPO was used as the sample group, and the group injected with only the solvent was used as the negative control group. Each LNP-hEPO sample was injected into two mice. 3) Submandibular blood collection from mice: Submandibular blood was collected 6 and 24 hours after injection. The blood was collected in EDTA anticoagulant tubes, gently mixed, numbered, and set aside for later use. 4) Serum extraction: After obtaining whole blood, 2000g was centrifuged for 10 minutes, and the supernatant was plasma. After dispensing, it was stored at -80°C. 5) Detection of hEPO expression using a human erythropoietin ELISA kit: Follow the instructions and prepare two duplicate wells for each serum sample. The OD value was detected using a microplate reader, and the expression level was calculated from the calibration curve and dilution ratio. The conversion units between IU and μg used in the kit are as follows: 1650 IU = 11 μg.

[0177] Table 2 shows the numerical values ​​of the physicochemical properties and in vivo hEPO expression concentrations of the in vivo formulation successfully expressed in mice, prepared from AXT-8.

[0178] Table 2 [Table 2] C: ≥ 0.1 and < 1, D:<0.1

[0179] As shown in Figure 6, the LNP(AXT-8)-hEPO series prepared from AXT-8 was successfully expressed in mice, suggesting that LNPs formed with AXT-8 can be efficiently expressed in vivo via mRNA.

[0180] Example 6 Detection of LNP-mRNA expression in the T cell line To demonstrate that LNP-mRNA assembled with AXT-8 has the ability to transfect human T cell lines in vitro, we designed experiments to transfect JM cells and Jurkat cells with LNP-eGFP. Simultaneously, we used the transfection reagent Lipofectamine® MessengerMAX® (Liposome MessengerMAX, Thermo, catalog number: LMRNA001) as a positive control group and detected the expression level of eGFP in the cells. The procedure is as follows: 1) Cell seeding: AO / PI cells were counted and the culture medium contained only FBS, without adding an antibiotic mixture (penicillin / streptomycin). JM cells or Jurkat cells were then seeded. * 10 6 The solution was adjusted to / ml, and 200 ul of cells were seeded into each well of a 24-well plate (i.e., 2 * 10 5 1) Cell transfection: Add 6 ug / well of mRNA encapsulated in AXT-8, replenish with medium to 1 ml, and add only FBS to the medium without antibiotic mixture (penicillin / streptomycin). 3) Detection by flow cytometry: Cells were cultured at 37°C for 24 hours and then detected by flow cytometry. Each cell was collected in a 1.5 ml EP tube, marked with the sample name, centrifuged with 400 g for 5 minutes, and the supernatant was aspirated. The cells were washed once with 1 ml of 2% FBS (PBS prepared), centrifuged with 400 g for 5 minutes, and the supernatant was aspirated. The corresponding antibody was added to the cell suspension and stained for 20 minutes. The cells were washed once with 1 ml of 2% FBS (PBS prepared), centrifuged with 400 g for 5 minutes, and the supernatant was aspirated. The cells were resuspended in 50 µl of 2% FBS (PBS prepared) and detected by flow cytometry. As shown in Figure 7, LNP-eGFP prepared from AXT-8 was successfully expressed in JM cells and Jurkat cells.

[0181] Example 7 Detection of LNP-mRNA expression in human primary T cells To demonstrate that LNP-mRNA assembled with AXT-8 has the ability to transfect human T cell lines in vitro, we designed an experiment in which LNP-mCherry transfects human primary T cells. Simultaneously, we used the transfection reagent Lipofectamine® MessengerMAX® (Liposome MessengerMAX) as a positive control group and detected the expression level of mCherry in the cells. The procedure is as follows: 1) Isolation of primary T cells from human PBMCs: Magnetic bead markers (hereinafter, 10 7 (Taking cells as an example, the corresponding reagent was increased in proportion to the number of cells.) 10 7 Resuspend the cells in 80 μL of buffer for each total number of cells, add 20 μL of CD3 MicroBeads, incubate in the refrigerator for 15 minutes (2-8°C), wash the cells with buffer, and rinse with 500 μL of buffer up to 10 8 The cells were resuspended. The MS column was placed on a magnetic holder, and T cells were sorted using the MS column. 2) Transfection of primary T cells with AXT-8: Transfection of primary T cells * 10 6 The solution was adjusted to / ml, and 200 ul of cells were seeded into each well of a 24-well plate (i.e., 2 * 10 5 ( / well). AXT-8 was added at 6 ug / ml, and X-vivo medium was replenished to 1 ml. IL-2 at a final concentration of 100 IU / ml was added to each well. After incubation at 37°C for 24 hours, flow detection was performed. 3) Detection by flow cytometry: Each cell was collected in a 1.5 ml EP tube, labeled, and centrifuged with 400 g for 5 minutes, discarding the supernatant. The cells were washed once with 1 ml of 2% FBS (PBS preparation), centrifuged with 400 g for 5 minutes, and discarded the supernatant. The corresponding antibody was added to the cell suspension and stained for 20 minutes. The cells were washed once with 1 ml of 2% FBS (PBS preparation), centrifuged with 400 g for 5 minutes, and discarded the supernatant. The cells were resuspended in 50 µl of 2% FBS (PBS preparation) and detected by flow cytometry.

[0182] As shown in Figure 8, LNP-mCherry prepared from AXT-8 was successfully expressed in human primary T cells.

[0183] All documents referenced in this invention are cited by reference in this application, just as each document is cited by reference on its own. Furthermore, after reading the above explanation of this invention, those skilled in the art should understand that various changes or modifications can be made to the invention, and that these equivalent forms are also included within the scope limited by the claims appended to this application.

Claims

1. Ionizable lipids, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof, The ionizable lipid has the structure of formula I shown below, 【Chemistry 1】 however, R １ and R ２ Each is independently - (CH ２ ) ｎ - is selected from, where n is a positive integer from 1 to 14. X and Y are independently -CH- or N, L １ has a structure of -(L １ａ -L １ｂ )- or not, provided that L １ａ is a functional group selected from -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -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)-, and L １ｂ is -(CH ２ ) ｎ -, provided that n is selected from 0, 1, 2, 3 or 4, L ２ From left to right - (L ２ａ -L ２ｂ ) - has or does not have the structure, however, L ２ａ L is a functional group selected from -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, and -CH(OH)-. ２ｂ ha- (CH ２ ) ｎ - where n is selected from 0, 1, 2, 3 or 4, R ３ , R ４ , R ５ and R ６ H and CH are independent of each other. ３ , C ２ ~C ３０ hydrocarbon group, or -(CH ２ ) ｓ -R ａ - (CH ２ ) ｇ -R ｂ - (CH ２ ) ｍ -CH ３ Herein, s and g are independently selected positive integers from 1 to 20, and m is an integer selected from 0 to 20. R ａ , R ｂ Each of these is an independent functional group selected from -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, and -CH(OH)-. And R ３ and R ４ Both are not H, and R ５ and R ６ They cannot both be H, R ７ is C １ ~C ５ hydrocarbon group or -(CH ２ ) ｍ O(CH ２ ) ｎ An ionizable lipid, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, characterized by being selected from -, wherein m and n are each independently selected from 1, 2, and 3.

2. The ionizable lipid has the structure shown in the following formula (I-1), 【Chemistry 2】 however, R １ and R ２ Each is independently - (CH ２ ) ｎ - is selected from, where n is a positive integer from 1 to 14. L １ From right to left - (L １ａ -L １ｂ ) - has or does not have the structure, however, L １ａ L is a functional group selected from -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, and -CH(OH)-. １ｂ ha- (CH ２ ) ｎ - where n is selected from 0, 1, 2, 3 or 4, L ２ From left to right - (L ２ａ -L ２ｂ ) - has or does not have the structure, however, L ２ａ L is a functional group selected from -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, and -CH(OH)-. ２ｂ ha- (CH ２ ) ｎ - where n is selected from 0, 1, 2, 3 or 4, R ３ , R ４ , R ５ and R ６ Each is independently C ２ ~C ２０ It is a hydrocarbon group, R ７ is C １ ~C ５ hydrocarbon group or -(CH ２ ) ｍ O(CH ２ ) ｎ An ionizable lipid according to claim 1, characterized in that m and n are selected from -, wherein m and n are each independently selected from 1, 2, and 3, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

3. The ionizable lipid has the structure shown in the following formula (I-2): 【Transformation 3】 however, R １ and R ２ Each is independently - (CH ２ ) ｎ - is selected from, where n is a positive integer from 1 to 14. L １ From right to left - (L １ａ -L １ｂ ) - has or does not have the structure, however, L １ａ L is a functional group selected from -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, and -CH(OH)-. １ｂ ha- (CH ２ ) ｎ - where n is selected from 0, 1, 2, 3 or 4, L ２ has a structure of -(L ２ａ -L ２ｂ )- or not, provided that L ２ａ is a functional group selected from -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -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)-, and L ２ｂ is -(CH ２ ) ｎ -, provided that n is selected from 0, 1, 2, 3 or 4, R ３ is -R ３ａ -R ３ｂ -R ３ｃ -R ３ｄ -R ３ｅ has the structure of, R ４ is -R ４ａ -R ４ｂ -R ４ｃ -R ４ｄ -R ４ｅ has the structure of, R ５ is -R ５ａ -R ５ｂ -R ５ｃ -R ５ｄ -R ５ｅ has the structure of, However, R ３ａ , R ３ｃ , R ４ａ , R ４ｃ , R ５ａ , R ５ｃ Each is independently - (CH ２ ) ｎ - where n is a positive integer chosen from 1 to 14, R ３ｂ ,R ３ｄ ,R ４ｂ ,R ４ｄ ,R ５ｂ ,R ５ｄ Each independently has or -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -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)-(CH ２ )-(C=C)-、-(C=C)-、-CH ２ -、 【Chemistry 4】 A functional group selected from among, R ３ｅ , R ４ｅ , R ５ｅ Each is independently C ２ ~C ２０ It is a hydrocarbon group, R ７ is C １ ~C ５ hydrocarbon group or -(CH ２ ) ｍ O(CH ２ ) ｎ An ionizable lipid according to claim 1, characterized in that m and n are selected from -, wherein m and n are each independently selected from 1, 2, and 3, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

4. The ionizable lipid has the structure shown in the following formula (I-3): 【Transformation 5】 however, R １ and R ２ Each is independently - (CH ２ ) ｎ - is selected from, where n is a positive integer from 1 to 14. L １ From right to left - (L １ａ -L １ｂ ) - has or does not have the structure, however, L １ａ L is a functional group selected from -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, and -CH(OH)-. １ｂ ha- (CH ２ ) ｎ - where n is selected from 0, 1, 2, 3 or 4, L ２ From left to right - (L ２ａ -L ２ｂ ) - has or does not have the structure, however, L ２ａ L is a functional group selected from -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, and -CH(OH)-. ２ｂ ha- (CH ２ ) ｎ - where n is selected from 0, 1, 2, 3 or 4, R ３ Ha-R ３ａ -R ３ｂ -R ３ｃ -R ３ｄ -R ３ｅ It has a structure, R ４ Ha-R ４ａ -R ４ｂ -R ４ｃ -R ４ｄ -R ４ｅ It has a structure, R ５ Ha-R ５ａ -R ５ｂ -R ５ｃ -R ５ｄ -R ５ｅ It has a structure, R ６ Ha-R ６ａ -R ６ｂ -R ６ｃ -R ６ｄ -R ６ｅ Having a structure, However, R ３ａ , R ３ｃ , R ４ａ , R ４ｃ , R ５ａ , R ５ｃ、 R ６ａ and R ６ｃ Each is independently - (CH ２ ) ｎ - where n is a positive integer chosen from 1 to 14, R ３ｂ ,R ３ｄ ,R ４ｂ ,R ４ｄ ,R ５ｂ ,R ５ｄ ,R ６ｂ and ６ｄ Each independently has or -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -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)-(CH ２ )-(C=C)-、-(C=C)-、-CH ２ -、 【Transformation 6】 A functional group selected from among, R ３ｅ , R ４ｅ , R ５ｅ , R ６ｅ Each is independently C ２ ~C ２０ It is a hydrocarbon group, R ７ is C １ ~C ５ hydrocarbon group or -(CH ２ ) ｍ O(CH ２ ) ｎ An ionizable lipid according to claim 1, characterized in that m and n are selected from -, wherein m and n are each independently selected from 1, 2, and 3, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

5. R ３ , R ４ , R ５ and R ６ Each is independently C ５ ~C １５ An ionizable lipid according to claim 1, characterized in that it is an alkyl group, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

6. The ionizable lipid has the structure shown in the following formula (I-1), 【Transformation 7】 however, R １ and R ２ Each is independently - (CH ２ ) ｎ - is selected from, where n is a positive integer between 4 and 8. L １ The functional group is selected from -O-, -(C=O)O-, -O(C=O)-, and -CH(OH)-. L ２ The functional group is selected from -O-, -(C=O)O-, -O(C=O)-, and -CH(OH)-. R ３ Ha-R ３ａ -R ３ｂ -R ３ｃ -R ３ｄ -R ３ｅ It has a structure, R ４ Ha-R ４ａ -R ４ｂ -R ４ｃ -R ４ｄ -R ４ｅ It has a structure, R ５ Ha-R ５ａ -R ５ｂ -R ５ｃ -R ５ｄ -R ５ｅ It has a structure, R ６ Ha-R ６ａ -R ６ｂ -R ６ｃ -R ６ｄ -R ６ｅ Having a structure, However, R ３ａ , R ３ｃ , R ４ａ , R ４ｃ , R ５ａ , R ５ｃ、 R ６ａ and R ６ｃ Each is independently - (CH ２ ) ｎ - where n is a positive integer selected from 1 to 14, and R ３ , R ４ , R ５ and R ６ Each of these consists of 4 to 20 CH groups independently. ２ Having structural fragments, R ３ｂ , R ３ｄ , R ４ｂ , R ４ｄ , R ５ｂ , R ５ｄ , R ６ｂ and R ６ｄ These are each independent of -(C=O)O-, -O(C=O)-, -(S-S)-, -(C=C)-(CH ２ A functional group selected from the group consisting of )-(C=C)- and -CH(OH)-, R ３ｅ , R ４ｅ , R ５ｅ , R ６ｅ Each is independently C ２ ~C ２０ It is a hydrocarbon group, R ７ is C １ ~C ３ hydrocarbon group or -(CH ２ ) ２ -O-(CH ２ ) ２ - The ionizable lipid according to claim 1, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

7. The ionizable lipid described above has the structure shown in the following formula, characterized by the ionizable lipid described in claim 1, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof: 【Transformation 8】

8. A method for preparing an ionizable lipid according to claim 1, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, The preparation method includes Method I, Method II, and Method III. However, Method I is, (S1) A step of reacting compounds A1 and A2 under the protection of an inert gas to obtain compound A3, (S2) Under the protection of an inert gas, the tert-butoxycarbonyl group (Boc) is removed from A3 to obtain A4, (S3) Under the protection of an inert gas, A4 and A5 or A6 are reacted to form A7 (i.e., the compound shown in (I-1)): 【Chemistry 9】 This includes the step of obtaining However, R １ and R ２ Each is independently - (CH ２ ) ｎ - is selected from, where n is a positive integer from 1 to 14. G １ and G ２ Each is independently selected from the active functional groups, L １ From right to left - (L １ａ -L １ｂ ) - has or does not have the structure, however, L １ａ L is a functional group selected from -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, and -CH(OH)-. １ｂ ha- (CH ２ ) ｎ - where n is selected from 0, 1, 2, 3 or 4, L ２ From left to right - (L ２ａ -L ２ｂ ) - has or does not have the structure, however, L ２ａ L is a functional group selected from O, -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, and -CH(OH)-. ２ｂ ha- (CH ２ ) ｎ - where n is selected from 0, 1, 2, 3 or 4, R ３ , R ４ , R ５ and R ６ Each is independently C ２ ~C ２０ It is a hydrocarbon group, R ７ is C １ ~C ５ hydrocarbon group or -(CH ２ ) ｍ O(CH ２ ) ｎ - is selected from, however m and n are each independently selected from 1, 2, and 3. Method II is, (D1) A step of obtaining compound B3 by reacting compounds B1 and B2 under the protection of an inert gas, (D2) A step of obtaining compound B5 by reacting compounds B3 and B4 under the protection of an inert gas, (D3) Under the protection of an inert gas, compounds B5 and B6 are reacted to produce B7, i.e., (I-2): 【Chemistry 10】 The step includes obtaining the compound shown, However, R １ and R ２ Each is independently - (CH ２ ) ｎ - is selected from, where n is a positive integer from 1 to 14. G １ and G ２ Each is independently selected from the active functional groups, L １ From right to left - (L １ａ -L １ｂ ) - has or does not have the structure, however, L １ａ L is a functional group selected from -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, and -CH(OH)-. １ｂ ha- (CH ２ ) ｎ - where n is selected from 0, 1, 2, 3 or 4, L ２ From left to right - (L ２ａ -L ２ｂ ) - has or does not have the structure, however, L ２ａ L is a functional group selected from -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, and -CH(OH)-. ２ｂ ha- (CH ２ ) ｎ - where n is selected from 0, 1, 2, 3 or 4, R ３ Ha-R ３ａ -R ３ｂ -R ３ｃ -R ３ｄ -R ３ｅ It has a structure, R ４ Ha-R ４ａ -R ４ｂ -R ４ｃ -R ４ｄ -R ４ｅ It has a structure, R ５ Ha-R ５ａ -R ５ｂ -R ５ｃ -R ５ｄ -R ５ｅ Having a structure, However, R ３ａ , R ３ｃ , R ４ａ , R ４ｃ , R ５ａ , R ５ｃ Each is independently - (CH ２ ) ｎ - where n is a positive integer chosen from 1 to 14, R ３ｂ ,R ３ｄ ,R ４ｂ ,R ４ｄ ,R ５ｂ ,R ５ｄ Each independently has or -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -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)-(CH ２ )-(C=C)-、-(C=C)-、-CH ２ -、 【Chemistry 11】 A functional group selected from among, R ３ｅ , R ４ｅ , R ５ｅ Each is independently C ２ ~C ２０ It is a hydrocarbon group, R ６ H is, R ７ is C １ ~C ５ hydrocarbon group or -(CH ２ ) ｍ O(CH ２ ) ｎ - is selected from, however m and n are each independently selected from 1, 2, and 3. Method III is, (M1) A step of reacting compounds C1 and C2 under the protection of an inert gas to produce C3, (M2) A step of removing Boc from compound C3 under the protection of an inert gas to produce compound C4, (M3) Under the protection of an inert gas, compound C4 is reacted with C5 or C6 to produce C7, i.e., formula (I-3): 【Chemistry 12】 The step includes generating the compound shown, However, R １ and R ２ Each is independently - (CH ２ ) ｎ - is selected from, where n is a positive integer from 1 to 14. G １ and G ２ Each is independently selected from the active functional groups, L １ From right to left - (L １ａ -L １ｂ ) - has or does not have the structure, however, L １ａ L is a functional group selected from -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, and -CH(OH)-. １ｂ ha- (CH ２ ) ｎ - where n is selected from 0, 1, 2, 3 or 4, L ２ From left to right - (L ２ａ -L ２ｂ ) - has or does not have the structure, however, L ２ａ L is a functional group selected from -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, and -CH(OH)-. ２ｂ ha- (CH ２ ) ｎ - where n is selected from 0, 1, 2, 3 or 4, R ３ Ha-R ３ａ -R ３ｂ -R ３ｃ -R ３ｄ -R ３ｅ It has a structure, R ４ Ha-R ４ａ -R ４ｂ -R ４ｃ -R ４ｄ -R ４ｅ It has a structure, R ５ Ha-R ５ａ -R ５ｂ -R ５ｃ -R ５ｄ -R ５ｅ It has a structure, R ６ Ha-R ６ａ -R ６ｂ -R ６ｃ -R ６ｄ -R ６ｅ Having a structure, However, R ３ａ , R ３ｃ , R ４ａ , R ４ｃ , R ５ａ , R ５ｃ、 R ６ａ and R ６ｃ Each is independently - (CH ２ ) ｎ - where n is a positive integer chosen from 1 to 14, R ３ｂ ,R ３ｄ ,R ４ｂ ,R ４ｄ ,R ５ｂ ,R ５ｄ ,R ６ｂ and ６ｄ Each independently has or -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -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)-(CH ２ )-(C=C)-、-(C=C)-、-CH ２ -、 【Chemistry 13】 A functional group selected from among, R ３ｅ , R ４ｅ , R ５ｅ , R ６ｅ Each is independently C ２ ~C ２０ It is a hydrocarbon group, R ７ is C １ ~C ５ hydrocarbon group or -(CH ２ ) ｍ O(CH ２ ) ｎ A preparation method characterized by being selected from -, wherein m and n are each independently selected from 1, 2, and 3.

9. Lipid nanoparticles (LNPs) characterized by comprising an ionizable lipid as described in claim 1, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

10. i) Lipid nanoparticles according to claim 9, ii) The bioactive substance encapsulated in the lipid nanoparticles, iii) A lipid nanoparticle pharmaceutical formulation characterized by comprising a pharmaceutically acceptable carrier.

11. (a) A step of obtaining a lipid organic phase by mixing an ionizable lipid according to claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, and an optional auxiliary lipid with an organic solvent, (b) A step of obtaining an aqueous phase containing a biologically active substance by mixing the biologically active substance with an aqueous solvent, A method for preparing a lipid nanoparticle pharmaceutical formulation according to claim 10, comprising the step of (c) mixing the lipid organic phase in step (a) with the aqueous phase in step (b) to obtain the lipid nanoparticle pharmaceutical formulation.

12. Use of the ionizable lipid described in claim 1, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, in the preparation of 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. Use of the ionizable lipid described in claim 1, or a pharmaceutically acceptable salt thereof, in the preparation of a lipid nanoparticle pharmaceutical formulation, wherein the lipid nanoparticle pharmaceutical formulation is used to deliver a bioactive substance to cells in the body of a subject who requires it.

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