Ionized lipids and their applications

Abstract

The present invention provides ionizable lipids and drug delivery systems comprising said ionizable lipids. Specifically, the present invention provides an ionizable lipid having the structure of formula (I), or a pharmaceutically acceptable salt thereof. The present invention provides salts, tautomers, or stereoisomers of the ionizable lipids. The lipid nanoparticles developed by this method are expected to be useful for the safe and effective delivery of nucleic acid drugs, small molecule drugs, peptide drugs, and protein drugs. Efficient delivery can be achieved. [C47] TIFF2025538515000074.tif37169

Description

[Technical Field]

[0001] The present invention relates to the field of biomedical science, specifically to ionized lipids and drug delivery. Its application in [Background technology]

[0002] In recent years, messenger RNA drugs have become important therapeutic agents in the prevention and treatment of infectious diseases and tumors. Messenger RNA drug technology has been recognized by the industry and is currently being used in research and development cycles. The short length, low risk of insertional mutagenesis, and diversity of the encoded proteins make m RNA is well suited for research and development of vaccines or therapeutics. However, mRNA It itself is sufficiently unstable that it can be easily degraded by ubiquitous RNases. Furthermore, mRNA is inherently negatively charged and has a large molecular weight (usually 10 6 From Da This limits the entry of mRNA molecules into cells. Developing a delivery vehicle to protect fragile mRNA molecules and deliver them to the cytoplasm is , has very important meaning.

[0003] Currently, lipid nanoparticles (LNP), inorganic nanoparticles Various mRNA delivery systems, including particles, polymer nanoparticles, viral vectors, and exosomes, are being developed. Currently, LNPs are used to deliver various drugs (e.g., nucleic acid drugs (mRNA, DNA) A, siRNA, ASO, and other nucleic acid drugs), small molecule drugs, antibodies, polypeptides, etc. It is widely used as a dietary supplement, and its main components are ionized lipids, phospholipids, cholesterol, and The most important component of LNPs is the ionizable lipid. The initial permanently positively charged cationic lipids have a lower in vivo circulation time and a higher It has low toxicity and severe allergic reactions because its inherent positive charge is During the process, proteins are adsorbed and easily captured and removed by the reticulo-endothelial system. This is because their inherent positive charge interacts with the negatively charged cell membrane, causing instability of the membrane. This produces severe toxicity, and the permanently positively charged cationic lipids activate the complement system. Ionized lipids are uncharged under physiological pH conditions and cause allergic reactions. Therefore, LNPs prepared with ionized lipids have a relatively high safety profile. The protein confers on LNPs the ability to escape lysosomes, and is involved in the proton sponge effect and membrane fusion mechanisms. LNPs then escape and release mRNA into the cytoplasm, where it binds to protein-coding ribosomal The ribosomal RNA binds to the genome and translates the encoded protein.

[0004] With the advancement of research and the demand for effective disease treatment, precise target organ delivery of LNPs has emerged. However, the advantage of targeted delivery is primarily the effective accumulation and release of therapeutic drugs in target tissues or cells. This improves the therapeutic effect and reduces the systemic toxicity. Studies on the organs include lymph node targeting, lung targeting, liver targeting, spleen targeting, etc. For NP carrier types, the main target development strategies include designing and screening novel lipid molecules. This includes optimization of LNP component formulation, LNP surface modification, and selection of administration route.

[0005] Recently, Xu Qiaobing and his team independently synthesized the ionized lipid 113-O12B. developed and used to study the targeted delivery of LNP-mRNA to lymph nodes, This is a lipid currently sold by Biotech, ALC-0315 lipid according to the prescription of the lipid. Compared with lymph node targeting, the results showed that the 113-O12B formulation had superior lymph node targeting. However, both still show higher liver accumulation.

[0006] In summary, the research and development of suitable ionizable lipids is essential for achieving high safety and high lysosomal escape. Furthermore, organ-targeted delivery of LNPs is one of the keys to developing LNPs with high efficiency. The exploration and optimization of drug formulations is also an important research and development direction in this field. Summary of the Invention [Problem to be solved by the invention]

[0007] The object of the present invention is to provide ionized lipids with low toxicity and high delivery efficiency. The ionizable lipids can be used as an important component of drug delivery systems.

[0008] Another object of the present invention is to provide an LNP formulation treatment with organ targeting, particularly lymph node targeting. The goal is to provide a way to [Means for solving the problem]

[0009] A first aspect of the present invention relates to an ionizable lipid, or a pharmaceutically acceptable salt, tautomer or derivative thereof. or a stereoisomer thereof, wherein the ionizable lipid has the structure of Formula I: [ka] In the formula: R1 and R2 are each independently -(CH2) n -, where n is selected from: is a positive integer between 1 and 14, X and Y are each independently —CH— or N; L1 and L2 each independently represent a divalent linking group or are absent; R3, R4, R5 and R6 are each independently H, CH3, a C2-C30 hydrocarbon groups (e.g., C2-C30 alkyl groups, C2-C30 chain alkenyl groups, C2-C30 alkyl groups, -alkynyl group), or -(CH2)sR a -(CH2)gR b -(CH2) m -R c in wherein s and g are each independently selected from a positive integer of 1 to 20; and m is is selected from integers of 0 to 20, preferably s+g+m is 2 to 35, R c is CH 3 or C2-C15 hydrocarbon group (e.g., C2-C15 alkyl group, C2-C15 chain alkyl group) alkenyl group, C2-C15 alkynyl group), R a , R b are each independently -CH2-, a C2-C6 alkenyl structure or selected from functional groups as shown [ka] Furthermore, R3 and R4 do not become H at the same time, and R5 and R6 do not become H at the same time. In another preferred embodiment, in formula I: R1 and R2 are each independently -(CH2) n -, where n is selected from: is a positive integer between 1 and 14, X and Y are each independently —CH— or N; L1 and L2 each independently represent a divalent linking group or are absent; R3, R4, R5 and R6 are each independently H, CH3, a C2-C30 hydrocarbon groups (e.g., C2-C30 alkyl groups, C2-C30 chain alkenyl groups, C2-C30 alkyl groups, -alkynyl group), or -(CH2)sR a -(CH2)gR b -(CH2) m -CH3 wherein s and g are each independently selected from a positive integer of 1 to 20, and m is , selected from integers of 0 to 20, preferably s+g+m is 2 to 35; R a , R b are each independently selected from the functional groups shown below: [ka] Furthermore, R3 and R4 do not become H at the same time, and R5 and R6 do not become H at the same time.

[0010] In another preferred example, R3, R4, R5 and R6 are each independently C4 -C30 hydrocarbon group (e.g., C4-C30 alkyl group, C4-C30 chain alkenyl group) , C4-C30 alkynyl group), preferably a C4-C20 hydrocarbon group (e.g., C C4-C20 alkyl group, C4-C20 chain alkenyl group, C4-C20 alkynyl group) be.

[0011] In another preferred embodiment, at least two of R3, R4, R5 and R6 one or four are C2-C30 hydrocarbon groups (e.g., C2-C30 alkyl groups, C2-C3 -0 chain alkenyl group, C2-C30 alkynyl group), or -(CH2)sR a -(CH 2) gR b -(CH2) m -R c where s, g, m, R a , R b and R c teeth In another preferred embodiment, R3, R4, R5 and At least two, three, or four of R6 are C2-C30 hydrocarbon groups (e.g., C2 -C30 alkyl group, C2-C30 chain alkenyl group, C2-C30 alkynyl group), or -(CH2)sR a -(CH2)gR b -(CH2) m -CH3, where s, g, m, R a and R b is as defined above. The above s+g+m is 3 to 20, and more preferably 4 to 15.

[0012] In another preferred embodiment, R3 is -R 3a -R 3b -R 3c -R 3d -R 3e of having a structure, where R 3a and R 3c are each independently -(CH2) n -where: n is selected from a positive integer from 1 to 14; R 3b and R 3d are each independently -CH2-, -(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≡C)-, -CH2-, -(C=O)O-, -O(C=O) -, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)- The functional groups are selected from the following: R 3e is a C2-C20 hydrocarbon group.

[0013] In another preferred embodiment, R4 is -R 4a -R 4b -R 4c -R 4d -R 4e of having a structure, where R 4a and R 4c are each independently -(CH2) n -where: n is selected from a positive integer from 1 to 14; R 4b and R 4d are each independently -CH2-, -(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≡C)-, -CH2-, -(C=O)O-, -O(C=O) -, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)- The functional groups are selected from the following: R 4e is a C2-C20 hydrocarbon group.

[0014] In another preferred embodiment, R5 is -R 5a -R 5b -R 5c -R 5d -R 5e of having a structure, where R 5a and R 5c are each independently -(CH2) n -where: n is selected from a positive integer from 1 to 14; R 5b and R 5dare each independently -CH2-, -(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≡C)-, -CH2-, -(C=O)O-, -O(C=O) -, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)- The functional groups are selected from the following: R 5e is a C2-C20 hydrocarbon group.

[0015] In another preferred embodiment, R6 is -R 6a -R 6b -R 6c -R 6d -R 6e of having a structure, where R 6a and R 6c are each independently -(CH2) n -where: n is selected from a positive integer from 1 to 14; R 6b and R 6d are each independently -CH2-, -(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≡C)-, -CH2-, -(C=O)O-, -O(C=O) -, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)- The functional groups are selected from the following: R 6e is a C2-C20 hydrocarbon group.

[0016] In another preferred embodiment, the L1 is, from left to right, -(L 1a -L 1b -L 1c )-of wherein L 1b is -(CH2) n where n is a positive integer between 1 and 14. is selected from the integers L 1a and L 1c are each independently -CH2-, -NH-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O )S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH -, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, preferably -CH -, -(C=O)O-, -O(C=O)-, -(SS)-, -CH(OH)- The functional groups are selected from:

[0017] In another preferred embodiment, the L2 is, from left to right, -(L 2a -L 2b -L 2c )-of having a structure, where L 2b is -(CH2) n where n is a positive integer from 1 to 14. Selected, L 2a , L 2c are each independently -CH2-, -NH-, 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)-, -(C≡C)-, preferably -CH2-, -(C=O)O- , -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -C The functional group is selected from groups such as H(OH)-.

[0018] In another preferred example, X and Y are —CH—.

[0019] In another preferred embodiment, the ionizable lipid has a substructure represented by the following formula (I-1): and [ka] In the formula: R1 and R2 are each independently -(CH2) n -, where n is selected from: is a positive integer between 1 and 14, L1 is -(L 1a -L 1b -L 1c )-, where L 1b teeth , -(CH2) n where n is selected from a positive integer from 1 to 14; 1a Reach BiL 1c are each independently -CH2-, -(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)N H—, —CH(OH)—, preferably —CH2—, —(C═O)O—, —O(C═O)— , -(SS)-, -CH(OH)-, L2 is -(L 2a -L 2b -L 2c )-, where L 2b teeth , -(CH2) n where n is selected from a positive integer from 1 to 14; 2a Reach BiL 2c are each independently -CH2-, -(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)N H—, —CH(OH)—, preferably —CH2—, —(C═O)O—, —O(C═O)— , -(SS)-, -CH(OH)-, R3, R4, R5 and R6 are each independently a C2-C20 hydrocarbon group.

[0020] In another preferred example, X is N and Y is -CH-. In another preferred example, the L1 is absent. In another preferred example, R5 is H.

[0021] In another preferred embodiment, the ionizable lipid has a substructure represented by the following formula (I-2): and [ka] In the formula: R1 and R2 are each independently -(CH2) n -, where n is selected from: is a positive integer between 1 and 14, L2 is -(L 2a -L 2b -L 2c )-structure, where L 2b is -(CH2) n where n is a positive integer from 1 to 14. Selected, L 2a , L 2care each independently -CH2-, -NH-, 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)—, —(C≡C)—, preferably —CH—, —NH—, —(C =O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C )-, -CH(OH)-, R3 is -R 3a -R 3b -R 3c -R 3d -R 3e and R4 is -R 4a -R 4b -R 4c -R 4d -R 4e having the structure where R 3a , R 3c , R 4a and R 4c are each independently -(CH2) n - where n is selected from a positive integer from 1 to 14; R 3b , R 3d , R 4b and R 4d are each independently -CH2-, -(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)-, -(C≡C)-, preferably -CH2-, -(C=O)O- , -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -C H(OH)-, R 3e and R 4e are each independently a C2-C20 hydrocarbon group, R6 is a C2-C20 hydrocarbon group.

[0022] In another preferred example, X and Y are N. In another preferred example, L1 and L2 are absent.

[0023] In another preferred embodiment, the ionizable lipid has a substructure represented by the following formula (I-3): and [ka] In the formula: R1 and R2 are each independently -(CH2) n -, where n is selected from: is a positive integer between 1 and 14, R3 is -R 3a -R 3b -R 3c -R 3d -R 3e and R4 is -R 4a -R 4b -R 4c -R 4d -R 4e and R5 is -R 5a -R 5b -R 5c - R 5d -R 5e and R6 is -R 6a -R 6b -R 6c -R 6d -R 6e The structure It has a structure, where R 3a , R 3c , R 4a , R4c , R 5a , R 5c , R 6a and R 6c Ha, so Each independently, -(CH2) n where n is a positive integer selected from 1 to 14. And, R 3b , R 3d , R 4b , R 4d , R 5b , R 5d , R 6b and R 6d are each unique In other words, -CH2-, -(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)-, -(C≡C)-, preferred -CH2-, -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C) selected from functional groups such as -(CH2)-(C=C)-, -CH(OH)-, R 3e , R 4e , R 5e , R 6e are each independently a C2-C20 hydrocarbon group. do.

[0024] In another preferred embodiment, the ionizable lipid has the structure shown in formula Ia: [ka] In the formula, each group is as described above.

[0025] In another preferred embodiment, the ionizable lipid has a structure selected from Table 1 below. . [Table 1] TIFF2025538515000010.tif255170TIFF2025538515000011.tif249170TIFF20255385150000 12.tif255170TIFF2025538515000013.tif235170TIFF2025538515000014.tif236170TIFF202 5538515000015.tif251170TIFF2025538515000016.tif244170TIFF2025538515000017.tif24 2170TIFF2025538515000018.tif235170TIFF2025538515000019.tif238170TIFF20255385150 00020.tif231170TIFF2025538515000021.tif243170TIFF2025538515000022.tif237170TIF F2025538515000023.tif247170TIFF2025538515000024.tif241170TIFF2025538515000025.t if242170TIFF2025538515000026.tif248170TIFF2025538515000027.tif248170TIFF2025538 515000028.tif234170TIFF2025538515000029.tif246170TIFF2025538515000030.tif168170

[0026] In another preferred embodiment, the ionizable lipid preferably has the structure shown below: Has. [Table 2]

[0027] In another preferred embodiment, the ionizable lipid is used in the preparation of a drug delivery system. The delivery system may be a lipid nanoparticle (LNP), a liposome, a polymer nanoparticle, or a combination thereof. The present invention is preferably used for preparing lipid nanoparticles, including lipid nanoparticles.

[0028] A second aspect of the present invention is an ionizable lipid according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof. The present invention provides a method for preparing a salt, tautomer or stereoisomer of the compound of formula (I), which comprises: Method I; Method II; Method II and Method III are included. Here, Method I is (A1) Compounds K2 and K3 are reacted with compound K1 in an inert solvent to obtain compound K 4. (A2) In an inert solvent, compounds K5 and K6 are reacted with compound K4 to obtain a compound of the formula (I- 1) obtaining a compound according to formula (I), [ka] where R1 and R2 are each independently -(CH2) n - is selected from, where , n is a positive integer from 1 to 14; M and G are each independently preferably -OH, -COOH, -SH, -NH2 , ethylene oxide; L1 is -(L 1a -L 1b -L 1c )-, and L2 is -(L 2a -L 2b -L 2c )-structure, where L 1b and L 2b are each independently -(CH2) n -where: n is selected from a positive integer from 1 to 14; L 1a , L 1c , L 2a and L 2c are each independently -CH2-, -(C=O) O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S (C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH( C=S)-, -(C=O)NH-, -CH(OH)-, preferably -CH2-, -(C= -O)O-, -O(C=O)-, -(SS)-, -CH(OH)- Selected, R3, R4, R5, and R6 are each independently a C2-C20 hydrocarbon group; Method II is (B1) reacting compounds K7 and K8 in an inert solvent to obtain compound K9; Floors and (B2) deprotecting compound K9 to obtain compound K10; (B3) Reacting compounds K11, K12 and K13 with compound K10 in an inert solvent. and obtaining a compound represented by formula (I-2), [ka] where R1 and R2 are each independently -(CH2) n - is selected from, where , n is a positive integer from 1 to 14; L2 is -(L 2a -L 2b -L 2c )-structure, where L 2b is -(CH2) n where n is a positive integer from 1 to 14. Selected, L 2a , L 2c are each independently -CH2-, -NH-, 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)—, —(C≡C)—, preferably —CH—, —NH—, —(C =O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C )-, -CH(OH)-, R3 is -R 3a -R 3b -R 3c -R 3d -R 3e and R4 is -R 4a -R 4b -R 4c -R 4d -R 4e having the structure where R 3a , R 3c , R 4a and R 4c are each independently -(CH2) n - where n is selected from a positive integer from 1 to 14; R 3b , R 3d , R 4b and R 4d are each independently -CH2-, -(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)-, -(C≡C)-, preferably -CH2-, -(C=O)O- , -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -C H(OH)-, R 3e and R 4e are each independently a C2-C20 hydrocarbon group, R6 is a C2-C20 hydrocarbon group; Method III is (C1) Reacting compounds K7 and K14 with compound K1 in an inert solvent to obtain compound The stage of obtaining K15 and (C2) Compounds K11, K12, K16, and K17 are reacted with compound K15 in an inert solvent. reacting to obtain a compound of formula (I-3), [ka] where: R1 and R2 are each independently -(CH2) n -, where n is selected from: is a positive integer between 1 and 14, R3 is -R 3a -R 3b -R 3c -R 3d -R 3e and R4 is -R 4a -R 4b -R 4c -R 4d -R 4e and R5 is -R 5a -R 5b -R 5c - R 5d -R 5e and R6 is -R 6a -R 6b -R 6c -R 6d -R 6e The structure It has a structure, where R 3a , R 3c , R 4a , R 4c , R 5a , R 5c , R 6a and R 6c Ha, so Each independently, -(CH2) n where n is a positive integer selected from 1 to 14. And, R 3b , R 3d , R 4b , R 4d , R 5b , R 5d , R 6b and R 6d are each unique In other words, -CH2-, -(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)-, -(C≡C)-, preferred -CH2-, -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C) selected from functional groups such as -(CH2)-(C=C)-, -CH(OH)-, R 3e , R 4e , R 5e , R 6e are each independently a C2-C20 hydrocarbon group. do.

[0029] In another preferred embodiment, the inert solvent is tetrahydrofuran, acetonitrile or is selected from the group consisting of chloroform or combinations thereof. In another preferred example, the reaction temperature in Method I is 0 to 90°C, and the reaction temperature in Method II is The reaction temperature in Method III is 25 to 30°C. In another preferred embodiment, the reaction time of Method I is 1 to 24 hours, and the reaction time of Method II is The reaction time is 1 to 24 hours, and the reaction time for Method III is 1 to 17 hours.

[0030] A third aspect of the present invention provides a lipid nanoparticle (LNP), said lipid nanoparticle comprising: An ionizable lipid according to the first aspect of the present invention, or a pharmaceutically acceptable salt, tautomer or the like thereof This includes stereoisomers. In another preferred embodiment, the lipid nanoparticles further comprise a helper lipid. In another preferred embodiment, the content of the ionizable lipid in the lipid nanoparticles is: It is 30-65% molar ratio of the total lipid content. In another preferred embodiment, the helper lipid is a helper phospholipid, a sterol, a poly mer-conjugated lipids, or combinations thereof. In another preferred embodiment, the helper lipids include helper phospholipids, sterols, and polyphospholipids. It is a combination of a mer-conjugated lipid.

[0031] In another preferred embodiment, the helper phospholipid is preferably 1,2-distearoyl phospholipid. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dioleoyl-sn- Glycero-3-phosphoethanolamine (DOPE), dioleoylphosphatidylcholine DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2- Dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn- Glycero-3-phosphoethanolamine, 1,2-myristoyl-sn-glycero-3- Phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac -(1-glycerin) sodium salt, 1,2-palmitoylphosphatidylglycerol , 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine Distearoylphosphatidylethanolamine, Distearoylphosphatidylethanolamine amine, 1-stearoyl-2-oleoylphosphatidylcholine, 1-stearoyl-2 -oleoyl-phosphatidylethanolamine, or a combination thereof. In another preferred embodiment, the sterol is cholesterol or a cholesterol-derived sterol. Including the body. In another preferred embodiment, the polymer-conjugated lipid is polyethylene glycol. It is a PEG-modified lipid.

[0032] In another preferred embodiment, the polyethylene glycolated lipid is preferably DMG -PEG2000, DSPE-PEG2000, DSG-PEG2000, DSPE-P EG-Mannose, DMG-PEG2000-(polypeptide, tan other active substances such as proteins, amino acids, vitamins, etc.) or combinations thereof will be done. In another preferred embodiment, the lipid nanoparticles contain an ionized lipid, DSPC, cholesterol, and DMG-PEG2000, where ionizable lipid:DSPC:cholesterol The molar ratio of ethanol to DMG-PEG2000 was (30-65):(5-30):(30-5 5):(1-5), preferably (40-50):(10-15):(35-50) :(1~1.5).

[0033] In another preferred embodiment, the lipid nanoparticles contain an ionized lipid, DSPC, cholesterol, DMG-PEG2000, and (1) DOPA (dioleoylphosphatidic acid) or DOPS (dioleoylphosphatidic acid) (2) DMG-PEG 2000 -mannose; or combinations thereof a fifth component selected from the group Here, ionized lipid: DSPC: cholesterol: DMG-PEG2000: fifth The molar ratio of the components is (30-50):(7.5-15):(25-50):(1-1.5). :(0.5~25). In another preferred embodiment, the lipid nanoparticles contain an ionized lipid, DSPC, cholesterol, DSPC, DMG-PEG2000 and DOPA, wherein the ionizable lipid: The molar ratio of cholesterol:DMG-PEG2000:DOPA was (30-50):(7 .5~15):(25~50):(1~1.5):(5~25).

[0034] In another preferred embodiment, the lipid nanoparticles contain an ionized lipid, DSPC, cholesterol, DSPC, DMG-PEG2000, and DOPS, wherein the ionizable lipid: The molar ratio of cholesterol:DMG-PEG2000:DOPS was (30-50):(7 .5~15):(25~50):(1~1.5):(4.5~20).

[0035] In another preferred embodiment, the lipid nanoparticles contain an ionized lipid, DSPC, cholesterol, DMG-PEG2000 and DMG-PEG 2000 -Contains mannose, Here, ionized lipid:DSPC:cholesterol:DMG-PEG2000:DMG The molar ratio of PEG2000-mannose was (30-50):(7.5-15):(25 ~50):(1~1.5):(0.5~2.5). In another preferred embodiment, the ionizable lipid is AL-6 or AL-19.

[0036] In another preferred embodiment, the lipid nanoparticles contain ionized lipids AL-6, DSPC, and cobalt. esterol, DMG-PEG2000 and DOPA, wherein the ionizable lipid AL The molar ratio of DSPC:cholesterol:DMG-PEG2000:DOPA was (3 0~50):(7.5~15):(25~50):(1~1.5):(5~25) , Preferably, (30-45):(7.5-10):(25-35):(1-1.5): (15-25) More preferably, the ratio is (35-45):(7.5-10):(25-35):(1-1.5 ):(20~25).

[0037] In another preferred embodiment, the lipid nanoparticles contain ionized lipids AL-6, DSPC, and cobalt. esterol, DMG-PEG2000 and DOPS, wherein the ionizable lipid AL The molar ratio of DSPC:cholesterol:DMG-PEG2000:DOPS was (3 0~50):(7.5~15):(25~50):(1~1.5):(4.5~20) can be, Preferably, (40-50):(7.5-15):(30-40):(1-1.5): (4.5~20) More preferably, the ratio is (40-50):(7.5-10):(30-35):(1-1.5 ):(8~20).

[0038] In another preferred embodiment, the lipid nanoparticles contain ionized lipids AL-6, DSPC, and cobalt. Contains cholesterol, DMG-PEG2000 and DMG-PEG2000-mannose , Here, ionized lipid:DSPC:cholesterol:DMG-PEG2000:DMG -PEG 2000 -mannose molar ratio is (30-50):(7.5-15):(25 ~50):(1~1.5):(0.5~2.5), Preferably, (40-50):(7.5-15):(35-40):(1-1.5): (0.5~2.5) More preferably, the ratio is (45-50):(7.5-12.5):(35-40):(1-1 .5):(0.5~2.5).

[0039] In another preferred embodiment, the lipid nanoparticles comprise an ionizable lipid AL-19, DSPC, cholesterol and DMG-PEG2000, wherein the ionizable lipid: DSPC: The molar ratio of cholesterol:DMG-PEG2000 was (30-65):(5-30): (30~55):(1~5) Preferably, it is (40-50):(10-15):(35-50):(1-1.5). the law of nature, More preferably, (40-50):(10-15):(40-50):(1-1.5) is. In another preferred embodiment, the lipid nanoparticles comprise an ionizable lipid AL-19, DSPC, Contains cholesterol, DMG-PEG2000 and DOPA, Here, the ionized lipid AL-19:DSPC:cholesterol:DMG-PEG200 The molar ratio of 0:DOPA was (30-50):(7.5-15):(25-50):(1- 1.5):(5~25) Preferably, (30-40):(10-15):(35-45):(1-1.5):( 5-15) More preferably, (35-40):(10-15):(35-45):(1-1.5) :(5~10).

[0040] In another preferred embodiment, the lipid nanoparticles comprise an ionizable lipid AL-19, DSPC, Contains cholesterol, DMG-PEG2000 and DOPS, Here, the ionized lipid AL-19:DSPC:cholesterol:DMG-PEG200 The molar ratio of 0:DOPS was (30-50):(7.5-15):(25-50):(1- 1.5):(4.5~20) Preferably, (30-40):(10-15):(35-45):(1-1.5):( 4.5~20) More preferably, (35-40):(10-15):(35-45):(1-1.5) :(4.5~15).

[0041] In another preferred embodiment, the lipid nanoparticles comprise physiologically active substances encapsulated within the lipid nanoparticles. It further comprises an active agent. In another preferred embodiment, the physiologically active substance is a nucleic acid, a protein, a polypeptide, a small molecule, or a nucleic acid. molecules, or combinations thereof. In another preferred embodiment, the nucleic acid is DNA, a plasmid, a messenger RNA ( mRNA), small interfering RNA (siRNA), antisense oligonucleotides, small molecules RNA, including ribosomal RNA, microRNA and transfer RNA, preferably is mRNA.

[0042] A fourth aspect of the present invention provides a lipid nanoparticle pharmaceutical formulation, the lipid nanoparticle pharmaceutical formulation comprising: The lipid nanoparticles according to the third aspect of the present invention, and a physiologically active substance encapsulated in the lipid nanoparticles. and a pharmaceutically acceptable carrier. In another preferred embodiment, the physiologically active substance is a nucleic acid, a protein, a polypeptide, a small molecule, or a nucleic acid. molecules, or combinations thereof. In another preferred embodiment, the nucleic acid is DNA, a plasmid, a messenger RNA ( mRNA), small interfering RNA (siRNA), antisense oligonucleotides, small molecules RNA, including ribosomal RNA, microRNA and transfer RNA, preferably is mRNA.

[0043] In another preferred embodiment, the physiologically active substance is a nucleic acid, and the lipid nanoparticle In a drug, the molar ratio of ionized N atoms in an ionized lipid molecule to phosphate groups in a nucleic acid molecule is (2 to 10):1, and more preferably (4 to 8):1. In another preferred embodiment, the hydrated particle size of the lipid nanoparticle drug is 50 to 200 nm, The thickness is preferably 70 to 150 nm, and most preferably 75 to 110 nm. In another preferred embodiment, the lipid nanoparticle pharmaceutical formulation is used for the treatment of tumors, infectious diseases and rare diseases. It can be used for treatment and / or prevention. In another preferred embodiment, the lipid nanoparticle pharmaceutical formulation is in the form of an injection, a freeze-dried agent, The drug is selected from the group consisting of a nebulized inhalant and a topical medication. In another preferred embodiment, the lipid nanoparticle pharmaceutical formulation is administered intravenously, intramuscularly, intradermally, or subcutaneously. The drug is administered by injection, such as intrathecal, intraduodenal or intraperitoneal injection. In another preferred embodiment, the lipid nanoparticle pharmaceutical formulation is administered by inhalation, such as intranasal administration. It is administered. In another preferred embodiment, the lipid nanoparticle pharmaceutical preparation is administered by transdermal application or electrode transfer. The compound is administered transdermally, for example by transfection.

[0044] A fifth aspect of the present invention provides a method for preparing a lipid nanoparticle drug according to the fourth aspect of the present invention. The method comprises: (a) an ionizable lipid according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof, a tautomer thereof, The isomer or stereoisomer and optional helper lipid are mixed with an organic solvent to form a lipid organic phase. and (b) mixing a physiologically active substance with an aqueous solvent to obtain an aqueous phase containing a physiologically active substance; , (c) mixing the lipid organic phase of step (a) with the aqueous phase of step (b) to form the lipid organic phase; and obtaining a quality nanoparticle drug.

[0045] In another preferred embodiment, the organic solvent is ethanol, methanol, isopropanol, or the like. acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, tetrahydrofuran and combinations thereof. In another preferred embodiment, the aqueous solvent is a buffer solution. In another preferred example, the aqueous solvent is a buffer solution having a pH range of 3 to 7. In another preferred embodiment, the acidic buffer is a citrate buffer of pH 4.0. In another preferred embodiment, the volume ratio of the lipid organic phase to the physiologically active substance-containing aqueous phase is 1: (2 to 5), preferably 1:(3 to 4). In another preferred embodiment, in step (c), the lipid organic phase and the aqueous phase are separated by a microfluidic Mixed by tip. In another preferred embodiment, the method further comprises purifying the lipid nanoparticle drug obtained in step (c). The method further comprises step (d) of concentrating, filtering and sterilizing the resulting solution.

[0046] A sixth aspect of the present invention relates to the preparation of a drug delivery system, comprising the steps of: Use of an ionized lipid of the formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof to provide. In another preferred embodiment, the delivery system is a lipid nanoparticle (LNP), a liposome , polymer nanoparticles, etc., and are preferably used for preparing lipid nanoparticles. In another preferred embodiment, the drug delivery system is used to treat tumors, infectious diseases, and rare diseases. and / or for the delivery of drugs to prevent

[0047] A seventh aspect of the present invention relates to a drug for treating and / or preventing tumors, infectious diseases and rare diseases. The present invention provides a use of the lipid nanoparticles according to the third aspect of the present invention in the preparation of a product. [Effects of the Invention]

[0048] Within the scope of the present invention, the above-mentioned technical features of the present invention and the following (for example, examples) are specifically The technical features described in each of the above mentioned technical features, when combined with each other, are new or preferable. It should be understood that a more suitable technical solution can be constructed. , which will not be repeated here. [Brief explanation of the drawings]

[0049] [Figure 1] The spectrum and results of detecting the molecular weight of AL-6 using LC-MS are shown. The characteristic peaks and their corresponding integrated areas are as shown in the figure, and the results prove that the obtained compound is the target compound. [Figure 2] The spectrum and results of AL-6 purity detection using HPLC-CAD are shown. The peak time of the product is 17.767 minutes, and the purity is over 98%. [Figure 3] The spectrum and results of detecting the molecular weight of AL-17 using LC-MS are shown. The characteristic peaks and their corresponding integrated areas are as shown in the figure, and the results prove that the obtained compound is the target compound. [Figure 4]The spectrum and results of AL-17 purity detection using HPLC-CAD are shown. The peak time of the product is 11.311 minutes, and the purity is over 95%. [Figure 5] The spectrum and results of detecting the molecular weight of AL-18 using LC-MS are shown. The characteristic peaks and their corresponding integrated areas are as shown in the figure, and the results prove that the obtained compound is the target compound. [Figure 6] The spectrum and results of AL-18 purity detection using HPLC-CAD are shown. The peak time of the product is 17.997 minutes, and the purity is over 93%. [Figure 7] The hydrogen spectrum characterization result spectrum of AL-19 is shown. [Figure 8] The molecular weight spectrum of AL-19 detected by LCMS is shown. [Figure 9] The spectrum and results of AL-19 purity detection using HPLC-CAD are shown, and the purity of the product reaches 95.14%. [Figure 10] The hydrogen spectrum characterization result spectrum of AL-20 is shown. [Figure 11] The molecular weight spectrum of AL-20 detected by LCMS is shown. [Figure 12] The spectrum and results of AL-20 purity detection using HPLC-CAD are shown, and the purity of the product reaches 93.54%. [Figure 13] The electrophoresis results of the transcription template are shown. The figure shows three electrophoresis lanes: the first lane contains DNA indicator bands of different lengths, the lane marked 1 contains plasmid DNA, and the lane marked 2 contains linearized transcription template. [Figure 14]The figure shows the transcription template integrity peak graph, where the horizontal axis represents fragment length and the vertical axis represents relative fluorescence units. The left panel shows three peaks, marked LM, 4146, and UM. LM and UM are low- and high-molecular-weight DNA indicator bands, respectively, and 4146 is the target detection band. The right panel shows the fitted electropherogram. [Figure 15] This figure shows the results of RNA electrophoresis after in vitro transcription (IVT). The figure has three electrophoresis lanes. The first lane contains RNA indicator bands of different lengths. The lane marked IVT contains in vitro transcribed RNA, and the lane marked CAP contains enzymatically capped mRNA. [Figure 16] The IVT RNA integrity peak graph is shown, where the horizontal axis represents fragment length and the vertical axis represents relative fluorescence units. The left graph shows two peaks, marked LM and 1795, where LM is the low molecular weight RNA indicator band and 1795 is the target RNA detection band. The right graph shows the fitted electropherogram. [Figure 17] The mRNA integrity peak graph is shown, where the horizontal axis represents fragment length and the vertical axis represents relative fluorescence units. The left graph shows two peaks, marked LM and 1795, where LM is the low molecular weight RNA indicator band and 1795 is the target RNA detection band. The right graph shows the fitted electropherogram. [Figure 18] The characterization results of the physicochemical properties (particle size, PDI, and encapsulation rate) of LNP-mRNA are shown. The particle size range is 70-100 nm, the PDI is <0.2, and the encapsulation rate is 85-100%, which proves that the physicochemical properties are similar to those of the LNP prepared above. [Figure 19] The physicochemical properties of particle size, PDI, and encapsulation efficiency of LNP(AL-20)-Luc in different formulations are shown, and the particle sizes are all less than 100 nm, the PDI is less than 0.2, and the encapsulation efficiency is between 60 and 90%. [Figure 20] The particle size results for lipid nanoparticles AXT-LNP-01 to AXT-LNP-20 (specific formulations are shown in Table 1) are shown. As can be seen from the figure, the particle sizes are all 150 nm or less, and most are around 100 nm. [Figure 21] The particle size distribution results for lipid nanoparticles AXT-LNP-01 to AXT-LNP-20 are shown. As can be seen from the figure, the PDIs are all 0.2 or less, and most are around 0.10. [Figure 22] This is the efficiency with which lipid nanoparticles AXT-LNP-01 to AXT-LNP-20 encapsulate mRNA. As can be seen from the figure, the encapsulation rate exceeds 80%, and is essentially about 90%. [Figure 23] 1 shows the cellular expression and toxicity detection process of LNP-mRNA. [Figure 24] The in vitro cell expression results of LNP(AL-6)-mRNA are shown. As shown in the figure, as the mRNA concentration increases, the cell expression results of multiple formulations of LNP(AL-6) are all superior to those of LNP(SM-102). [Figure 25] The results of cellular expression of LNP(AL-17)-mRNA are shown. As shown in the figure, as the concentration increases, the cellular expression of a series of LNP(AL-17) initially increases and then decreases, and at 1 μg / ml, it is generally lower than that of lipofectamine. [Figure 26] The cytotoxicity results of LNP(AL-6)-mRNA are shown. As can be seen from the figure, the cell inhibition rate of the LNP(AL-6) series products at different mRNA concentrations was almost 0, which was equivalent to that of LNP(SM-102), demonstrating the excellent safety of the LNP(AL-6) molecule. [Figure 27] The cytotoxicity results of LNP(AL-20)-Luc in different formulations are shown. As can be seen from the figure, the cell inhibition rates of LNP(AL-20)-Luc-1 and LNP(AL-20)-Luc-2 formulations at different mRNA concentrations were almost 0, which proves the excellent safety of LNP(AL-20). [Figure 28] 1 shows the in vivo expression detection process of LNP-mRNA. [Figure 29] The in vivo expression results of LNP(AL-6)-hEPO and LNP(SM-102)-hEPO are shown. As can be seen from the figure, the in vivo mRNA expression trends of both LNP(SM-102) and LNP(AL-6) initially increased and then decreased, reaching a maximum at approximately 6 hours. However, the expression of all LNP(AL-6) series products was superior to that of LNP(SM-102). [Figure 30] The in vivo cytotoxicity results of LNP(AL-6)-Luc and LNP(SM-102)-Luc are shown. In the figure, a shows a line graph of the change in mouse body weight over time, and b shows a line graph of the change in in vivo ALT (alanine aminotransferase) and AST (aspartate aminotransferase) content in mice. [Figure 31] The ratio of fluorescence expression values ​​of the living body, organs, and each organ in the saline group and the AXT-LNP-01 formulation is shown. As can be seen from the figure, the expression level of the AXT-LNP-01 formulation in the liver reaches 87.5%. [Figure 32] The figures show the ratios of fluorescence expression values ​​in the living body, organs, and each organ for the AXT-LNP-02, AXT-LNP-03, and AXT-LNP-04 formulations. The above formulations were imaged after adding DOPA at different ratios. As shown in the figures, compared to AXT-LNP-01, the DOPA ratio had a significant effect on organ targeting in mice, and the lymph node targeting rate of AXT-LNP-04 reached 94.47%. [Figure 33] The figures show the ratios of fluorescence expression values ​​in the living body, organs, and each organ for the AXT-LNP-05, AXT-LNP-06, and AXT-LNP-07 formulations. The above formulations were imaged after adding DOPS at different ratios. As shown in the figures, compared to AXT-LNP-01, the DOPS ratio had a significant effect on organ targeting in mice, and the lymph node targeting rate of AXT-LNP-06 reached 95.03%. [Figure 34]The figures show the ratios of fluorescence expression values ​​in the living body, organs, and each organ for the AXT-LNP-08, AXT-LNP-09, AXT-LNP-10, and AXT-LNP-11 formulations. The figures show the imaging results after adding different ratios of DMG-PEG2000-mannose to the formulations. As can be seen from the figures, the ratio of DMG-PEG2000-mannose has a significant effect on organ targeting in mice, and the lymph node targeting rate of AXT-LNP-09 reaches 55.61%. [Figure 35] The figures show the ratios of fluorescence expression values ​​in the living body, organs, and each organ for the AXT-LNP-12, AXT-LNP-13, and AXT-LNP-14 formulations. The above formulations are imaging results obtained by changing the molar ratio of the four components of LNP. As can be seen from the figure, changing the molar ratio of the four components of the AL-19-based ionized cationic lipid can effectively improve its lymph node targeting rate, and the lymph node targeting rate of AXT-LNP-13 reaches 97.06%. [Figure 36] The figures show the ratios of fluorescence expression values ​​in the living body, organs, and each organ for the AXT-LNP-15, AXT-LNP-16, and AXT-LNP-17 formulations. The above formulations are imaging results with different DOPA molar ratios. As can be seen from the figure, changing the molar ratio of the four components of DOPA can effectively improve the lymph node targeting rate, and the lymph node targeting rate of AXT-LNP-15 reaches 91.92%. [Figure 37] The figures show the ratios of fluorescence expression values ​​in the living body, organs, and each organ for the AXT-LNP-18, AXT-LNP-19, and AXT-LNP-20 formulations. The above formulations are imaging results obtained by changing the molar ratio of the four components of DOPS. As can be seen from the figure, changing the DOPS molar ratio can effectively improve the lymph node targeting rate, and the lymph node targeting rate of AXT-LNP-13 reaches 83.42%. DETAILED DESCRIPTION OF THE INVENTION

[0050] The present inventors have unexpectedly discovered ionizable lipids for the first time after extensive and thorough research. The ionized lipid has the advantages of stable physicochemical properties and low toxicity, and is suitable for use in the present invention. Drug delivery systems obtained by encapsulating drug payloads (e.g., mRNA) with ionized lipids The delivery system has high delivery efficiency and low toxicity, and efficiently delivers the drug payload and While improving the expression level of the loaded substance, it also improves the safety of the drug delivery system, The preventive and therapeutic effects of stem become more pronounced.

[0051] Furthermore, the present inventors have developed a novel ionizable lipid-based drug that is effective in targeting lymph nodes. Specifically, the present invention provides a patent for a series of optimized LNP formulations. Targeted to the ionized cationic lipid AL-6, DSPC, Chol, DMG-PEG2 Based on 000, anionic lipids DOPA and DOPS were added, and the formulation By screening multiple formulations with lymph node fluorescence of 90% or more, The highest results were 94.47% and 95.03%, respectively. Based on the above four components, the surface can be coated with DMG-PEG. 2000 -Mannose By modifying the ratio of the modified components, different degrees of lymph node targeting effect can be achieved. For the ionized lipid AL-19, DSPC, Chol, DMG-PEG 2000 Based on this, first, optimize the ratio of the four components and improve the lymph node targeting ratio of the four components. The efficiency can reach 97.06%. Based on these four components, Similarly, when ions DOPA and DOPS were added, a high organ ratio of the lymph nodes was achieved. Appear. Based on this, the present invention was completed.

[0052] term To facilitate understanding of this disclosure, certain terms will first be defined. As such, unless otherwise specified herein, each of the following terms has the meaning indicated below. It should be. The term "alkyl group" refers to a saturated carbon chain having from 1 to 20 carbon atoms. Unless otherwise specified, the chain may be linear or branched or a combination thereof. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s -butyl group, t-butyl group, pentyl group, hexyl group, heptyl group, octyl group, etc. Unless otherwise specified in the specification, alkyl groups are optionally substituted. The term "group" means that the group contains at least one C=C double bond (alkenyl group) or at least one C=C double bond (alkenyl group). Another C≡C triple bond (alkynyl group) refers to an "alkyl group," and "alkenyl group" and "Alkynyl groups" can be collectively referred to as "hydrocarbon groups."

[0053] Ionized lipids As used herein, "ionizable lipids of the present invention" and "ionizable cations of the present invention" refer to The term "lipid" is used interchangeably and refers to a lipid compound having the structure of Formula I, or refers to a pharmaceutically acceptable salt, tautomer or stereoisomer of

[0054] Ionized lipids are protonated and converted to cationic lipids at low pH values, which are essential for normal biogenesis. At physiological pH values, they are converted into helper phospholipids. Helper phospholipids and anions in blood cells This reduces interactions with cell membranes, improving the biocompatibility of lipid nanoparticles. After the molecule is taken up into the cell, the pH value in the endosome becomes low, and the lipids are protonated. The lipid nanoparticles become positively charged and the membrane structure becomes less stable or is destroyed, resulting in endosomal Generally, the pH sensitivity of lipids allows physiologically active ingredients (e.g. For example, this is advantageous for the in vivo delivery of lipid nanoparticles encapsulating mRNA molecules.

[0055] One aspect of the present invention provides an ionizable lipid, which has a structure as shown in Formula I: , [ka] In the formula: R1 and R2 are each independently -(CH2) n -, where n is selected from: is a positive integer between 1 and 14, X and Y are each independently —CH— or N; L1 and L2 each independently represent a divalent linking group or are absent; R3, R4, R5 and R6 are each independently H, CH3, a C2-C30 hydrocarbon groups (e.g., C2-C30 alkyl groups, C2-C30 chain alkenyl groups, C2-C30 alkyl groups, -alkynyl group), or -(CH2)sR a -(CH2)gR b -(CH2) m -R c in wherein s and g are each independently selected from a positive integer of 1 to 20; and m is is selected from integers of 0 to 20, preferably s+g+m is 2 to 35, R c is CH 3 or C2-C15 hydrocarbon group (e.g., C2-C15 alkyl group, C2-C15 chain alkyl group) alkenyl group, C2-C15 alkynyl group), R a , R b are each independently -CH2-, a C2-C6 alkenyl structure or selected from functional groups as shown [ka] Furthermore, R3 and R4 do not become H at the same time, and R5 and R6 do not become H at the same time.

[0056] In a preferred embodiment of the present invention, in formula I: R1 and R2 are each independently -(CH2) n -, where n is selected from: is a positive integer between 1 and 14, X and Y are each independently —CH— or N; L1 and L2 each independently represent a divalent linking group or are absent; R3, R4, R5 and R6 are each independently H, CH3, a C2-C30 hydrocarbon groups (e.g., C2-C30 alkyl groups, C2-C30 chain alkenyl groups, C2-C30 alkyl groups, -alkynyl group), or -(CH2)sR a -(CH2)gR b -(CH2) m -CH3 wherein s and g are each independently selected from a positive integer of 1 to 20, and m is , selected from integers of 0 to 20, preferably s+g+m is 2 to 35; R a , R b are each independently selected from the functional groups shown below: [ka] Furthermore, R3 and R4 do not become H at the same time, and R5 and R6 do not become H at the same time.

[0057] In a preferred embodiment of the present invention, the ionizable lipids each have the following formula (I-1): Substructures shown in (I-2) and (I-3): [ka] When having a substructure shown in formula (I-1), in this case, X and Y in formula (I) are All of the groups are -CH-, and R1 and R2 in the formula are each independently -(CH2) n -mosquito where n is a positive integer from 1 to 14, and L1 is selected from −(L 1a -L 1b -L 1c )-, where L 1b is -(CH2) n - and here where n is selected from a positive integer between 1 and 14, and L 1a and L 1c are each independently -CH2-, -(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)-, preferred -CH2-, -(C=O)O-, -O(C=O)-, -(SS)-, -CH(OH )-, and L2 is selected from functional groups such as -(L 2a -L 2b -L 2c )- where L 2b is -(CH2) n where n is from 1 to 14. is selected from the positive integers, L 2a and L 2c are each independently -CH2-, -(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-, -N H(C=S)-, -(C=O)NH-, -CH(OH)-, preferably -CH2-, -( Functional groups such as -C=O)O-, -O(C=O)-, -(SS)-, and -CH(OH)- R3, R4, R5 and R6 are each independently a C2-C20 hydrocarbon group. is.

[0058] In the case where the compound has a substructure shown in (I-2), in this case, X in formula (I) is N. , Y is -CH-, L1 is absent, L2 is a bivalent linkage gene, and R5 is H, wherein R1 and R2 are each independently —(CH2) n -Select from where n is a positive integer between 1 and 14, and L2 is −(L 2a -L2 b -L 2c )-, where L 2b is -(CH2) n - where n is selected from a positive integer between 1 and 14, and L 2a , L 2c are each independently -CH2 -, -NH-, -(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)-, -(C≡C)-, preferably - CH2-, -NH-, -(C=O)O-, -O(C=O)-, -(SS)-, -(C= R3 is selected from functional groups such as -(C)-(CH2)-(C=C)-, -CH(OH)-, -R 3a -R 3b -R 3c -R 3d -R 3e and R4 is -R 4a -R4 b -R 4c -R 4d -R 4e where R 3a , R 3c , R 4a and R 4c are each independently -(CH2) n -, where n is a positive integer between 1 and 14. Selected from R 3b , R 3d , R 4b and R 4d are each independently -CH2-, - (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)-(CH 2) -(C=C)-, -(C=C)-, -(C≡C)-, preferably -CH2-, -(C =O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C )-, -CH(OH)-, and R 3e and R 4e are each unique R6 is a C2-C20 hydrocarbon group.

[0059] When the compound has a substructure shown in formula (I-3), in this case, X and Y in formula (I) are , N, L1 and L2 are absent, and in the formula, R1 and R2 are each independently Te, -(CH2) n -, where n is a positive integer from 1 to 14, and R3 is , -R 3a -R 3b -R 3c -R 3d -R 3e and R4 is -R 4a -R4b -R 4c -R 4d -R 4e and R5 is -R 5a -R 5b -R 5c -R 5d - R 5e and R6 is -R 6a -R 6b -R 6c -R 6d -R 6e It has the structure , where R 3a , R 3c , R 4a , R 4c , R 5a , R 5c , R 6a and R 6c Ha, so Each independently, -(CH2) n where n is a positive integer selected from 1 to 14. and R 3b , R 3d , R 4b , R 4d , R 5b , R 5d , R 6b and R 6d Yes, respectively Independently, -CH2-, -(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)-, -(C≡C)-, Preferably, -CH2-, -(C=O)O-, -O(C=O)-, -(SS)-, -(C= R3 is selected from functional groups such as -(C)-(CH2)-(C=C)-, -CH(OH)-, e , R 4e , R 5e , R 6eare each independently a C2-C20 hydrocarbon group.

[0060] In a more preferred embodiment of the present invention, the ionizable lipid is one of those shown in Table 1. wherein AL-6, AL-17, AL-18, AL- 19 and AL-20 are preferred.

[0061] Helper lipids As used herein, the term "helper lipid" refers to a lipid nanoparticle that In addition to ionizable lipids, other typical lipids include helper phospholipids, sterols, and polymer components. The term "helper lipid" refers to the inclusion of a helper lipid, a soluble lipid, a soluble lipid, or a combination thereof. To improve the performance of lipid nanoparticles, such as delivery efficiency, tolerability, and bioavailability. Used for.

[0062] In some embodiments, the helper phospholipid is 1,2-distearoyl-s n-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero -3-phosphoethanolamine (DOPE), dioleoylphosphatidylcholine (DO PC), 1,2-dioleoyl-sn-glyceryl-3-phosphocholine, 1,2-dipal Mitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero 1,2-Myristoyl-sn-glycero-3-phosphoethanolamine Ethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1 -glycerol) sodium salt, 1,2-palmitoylphosphatidylglycerol, 1 -Palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine Distearoylphosphatidylethanolamine, Distearoylphosphatidylethanolamine , 1-stearoyl-2-oleoylphosphatidylcholine, 1-stearoyl-2-oleoylphosphatidylcholine including (but not limited to) leioyl-phosphatidylethanolamine, or combinations thereof (Not performed). In a preferred embodiment of the present invention, the helper phospholipid is DSPC(1,2-diphospholipid). Stearoyl-sn-glycero-3-phosphocholine, distearoylphosphatidylcholine DSPC is a commonly used phosphatidylcholine. The terminal groups of PC are saturated alkane chains, the melting point is -54°C, and it has a cylindrical shape. It forms a layered structure within the lipid nanoparticles, making the structure of the lipid nanoparticles more stable.

[0063] In a preferred embodiment of the present invention, the helper phospholipid is DOPE (1,2-dihydroxybenzoate). Oleoyl-sn-glycero-3-phosphoethanolamine, dioleoylphosphatidylinositol DOPE is a commonly used phosphatidylcholinesterase inhibitor. The terminal groups of DOPE are two unsaturated alkane chains, and At -30°C, the nanoparticles were cone-shaped, and hexagonal shapes were easily formed within the nanoparticles. This causes instability in the endosomal membrane and is effective in preventing the escape of lipid nanoparticles from endosomes. Benefit.

[0064] In some embodiments, the sterol is cholesterol or cholesterol Cholesterol is a stimulant for the integrity and rigidity of lipid membranes. The form of cholesterol derivatives can be adjusted to enhance the stability of lipid nanoparticles. is determined by the chain length of the hydrophobic end group of the cholesterol analogue, the flexibility of the sterol ring, and the hydroxyl group. The polarity of the lipid nanoparticles can affect the delivery efficiency and biodistribution of the lipid nanoparticles. The cholesterol derivatives also affected the morphology of the lipid nanoparticles. Lipid nanoparticles are not spherical but have multilayered polyhedral structures separated by lipids. At the same time, cholesterol enhances the selectivity of lipid nanoparticles to target points. Lipid nanoparticles containing cholesterol oleate affected liver endothelial cells more than hepatocytes. When the end groups contain acidified and modified cholesterol, it becomes highly selective for the vesicles. Lipid nanoparticle content in hepatic endothelial cells and Kupffer cells, and in hepatocytes higher than the content.

[0065] In some embodiments, the polymer-conjugated lipid is polyethylene glycol. Polyethylene glycol (PEG) conjugated lipids, also known as PEG PEGylated lipids have various effects on the properties of lipid nanoparticles, The dose of glycated lipids affects the particle size and potential of lipid nanoparticles and reduces particle accumulation. This improves the stability of lipid nanoparticles and enhances their ability to penetrate the kidney and mononuclear phagocyte system. Particle clearance mediated by the clear phagocyte system (MPS) The surface functional groups can be modified with ligands to reduce the dispersion rate and increase the particle circulation time. The molar mass and length of the lipids can be adjusted by adjusting the PEG The properties of the modified lipids were affected by the addition of PEG-2000 and DSG-PEG2000. are neutral phospholipids with saturated alkyl chain lengths of C14, C16, or C18, respectively. However, DMG-PEG2000 can be separated from the lipid nanoparticles more quickly, so the nanoparticles DMG-PEG20 was used to favor cellular uptake and endosomal escape. The delivery efficiency of DSG-PEG2000 is superior to that of DSG-PEG2000. In a preferred embodiment of the present invention, the helper lipid is DSPC, cholesterol and a combination of DMG-PEG2000.

[0066] Lipid nanoparticles (LNPs) As used herein, "lipid nanoparticles," "lipid nanoparticles," or "LNPs" The term refers to granules having a diameter of about 5 to 500 nm. The lipid nanoparticles contain one or more active agents (bioactive substances). In some embodiments, the lipid nanoparticles include nucleic acids. Inside the molecule, cationic lipids, polymers or multivalent small molecules, outside interacting with the biological environment. The nucleic acid is condensed with a lipid coating layer. Due to the repulsion between the phosphate groups, the nucleic acid is condensed with a lipid coating layer. In cells, to cope with volume limitations, DNA is , with the help of ions and other molecules, they can package themselves under the right solution conditions. Usually, DNA condensation occurs when the extended DNA strand contains only one or a few molecules. It is defined as the contraction of proteins into compact, well-ordered particles. By binding to phosphate groups, Cationic lipids neutralize the phosphate charge and allow the DNA to condense and pack.

[0067] In some embodiments, the bioactive agent is encapsulated in the LNP. In terms of form, bioactive substances are DNA, RNA (messenger RNA, transfer RNA) -RNA, ribosomal RNA, microRNA, etc.), natural and synthetic oligonucleotides ( antisense oligonucleotides, interfering RNA and small interfering RNA), nuclear proteins Proteins, peptides, nucleic acids, ribozymes, DNA-containing nucleoproteins, e.g., completely or partially Partially deproteinized virus particles (virions), non-DNA oligomers and Including, but not limited to, polymeric anionic compounds (e.g., acidic polysaccharides and glycoproteins) In some embodiments, the bioactive agent may be an anionic compound, such as It is mixed with an adjuvant.

[0068] In LNP vaccine products, the biologically active agent is usually contained within the LNP. In this embodiment, the biologically active substance comprises a nucleic acid. Typically, the water-soluble nucleic acid is a cationic The particle surface is condensed with a helper phospholipid or P It is rich in EG lipid derivatives. Additional ionized cationic lipids can also be located on the surface. After entering the cell lysosomes, the ionized cationic lipids are absorbed by the acidic environment of the lysosomes. It becomes ionized and positively charged, interacts with the lysosomal membrane, and facilitates escape from the endosome. Promote.

[0069] With respect to LNPs, the ionizable lipids can have different properties or functions. Due to the pKa of the hydroxyl group, when the external pH value is lower than the pKa of the lipid molecule, it becomes protonated. Under these conditions, lipid molecules can electrostatically bind to the phosphate groups of nucleic acids. This allows for the formation and encapsulation of LNPs, and also allows for the formation of LNPs at physiological pH The surface charge of LNPs in biological fluids (e.g., blood) at high LNP surface charge and toxicity, and comfort with both immobilized and free macrophages Rapid clearance circulation, hemolytic toxicity, is associated with immune activation (Filion et al., Biochem. him Biophys Acta. October 23, 1997;1329(2):345 -56).

[0070] In some embodiments, the pKa is sufficiently high that the ionized cationic lipid is They can adopt a positively charged form at the neutral endosomal pH value. By binding to anionic lipids in endosomes, phospholipids can form non-bipolar structures such as hexagonal HII phases. Promotes membrane dissolution of the overlying structure, thereby achieving more efficient intracellular delivery. In this embodiment, the pKa range is between 6.2 and 6.5. For example, the pKa is The unsaturated end may be about 6.2, about 6.3, about 6.4, or about 6.5. It also contributes to the ability of the protein to undergo structural changes (Jayaraman et al., Angew Chem Int E d Engl. 2012 August 20;51(34):8529-33).

[0071] Release of nucleic acids from LNP formulations and other parameters such as liposome clearance rate and circulatory half-life The characteristics include the inclusion of polyethylene glycol and / or sterol (e.g., cholesterol) within the LNP. the presence of any steroids or other potential excipients and the overall chemical structure (as part of the formulation) The ionization of the cationic lipids can vary depending on their molecular weight (including the pKa of the lipid).

[0072] One aspect of the present invention provides lipid nanoparticles (LNPs), the lipid nanoparticles comprising: An ionizable lipid according to the first aspect, or a pharmaceutically acceptable salt, tautomer or Furthermore, the lipid nanoparticles may further contain one or more helper lipids. The helper lipid may be a helper phospholipid, a steroid, a polymer conjugate, or the like. Contains lipids.

[0073] Some specific embodiments of the present invention include: (1) The molar ratio of each component is AL-6:DSPC:cholesterol (Cholesterol l):DMG-PEG 2000 :DOPA(37.5~47.5:7.5~9.5:28 LN containing ionized lipid AL-6 (.9-36.6:1.1-1.4:5-25). P, (2) The molar ratio of each component is AL-6:DSPC:cholesterol:DMG-PEG 200 0:DOPS(41.6~47.6:8.3~9.5:32.1~36.7:1.3~1 LNP containing ionized lipid AL-6, whose solubility is 4:4.8-16.7. (3) The molar ratio of each component is AL-6:DSPC:cholesterol:DMG-PEG 200 0:DMG-PEG 2000 -Mannose (50:10:36~38:1.5:0.5~ 2.5) LNPs containing ionized lipid AL-6; (4) The molar ratio of each component is AL-19:DSPC:cholesterol:DMG-PEG 20 00 Ionized lipid A (40-50:10-15:38.5-48.5:1.5) LNP, including L-19, (5) The molar ratio of each component is AL-19:DSPC:cholesterol:DMG-PEG 20 00 :DOPA(34.0~38.0:12.7~14.2:37.0~41.3:1. LNP containing ionized lipid AL-6, whose solubility is 3-1.4:5.0-15.0; (6) The molar ratio of each component is AL-19:DSPC:cholesterol:DMG-PEG 20 00 :DOPS(33.2~38.1:12.5~14.3:36.3~41.4:1. 3-1.4:4.8-16.7), LNP containing ionized lipid AL-6. The present invention provides an LNP comprising:

[0074] Lipid nanoparticle pharmaceutical formulations Another aspect of the present invention is a lipid nanoparticle pharmaceutical formulation (or lipid nanoparticle drug combination or The lipid nanoparticle pharmaceutical formulation is a lipid nanoparticle composition according to the third aspect of the present invention. a lipid nanoparticle, a physiologically active substance encapsulated in said lipid nanoparticle, and a pharmaceutically acceptable carrier; The lipid nanoparticle pharmaceutical preparation is administered to cells in the body of a subject in need of a physiologically active substance. It is used to deliver.

[0075] In some embodiments, the bioactive agent is encapsulated in the LNP. In terms of form, bioactive substances are DNA, RNA (messenger RNA, transfer RNA) -RNA, ribosomal RNA, microRNA, etc.), natural and synthetic oligonucleotides ( antisense oligonucleotides, interfering RNA and small interfering RNA), nuclear proteins Proteins, peptides, nucleic acids, ribozymes, DNA-containing nucleoproteins, e.g., completely or partially Partially deproteinized virus particles (virions), non-DNA oligomers and Including, but not limited to, polymeric anionic compounds (e.g., acidic polysaccharides and glycoproteins) In some embodiments, the bioactive agent may be an anionic compound, such as It can be mixed with an adjuvant.

[0076] In some embodiments, the LNP composition comprises a nucleic acid, a nucleic acid having the structure shown in formula (I): ionized cationic lipids, helper phospholipids (e.g., DSPC, DOPE, DOPC or or combinations thereof), sterols (e.g., cholesterol or cholesterol derivatives, or plant sterols such as β-sitosterol) and polymer-conjugated lipids (e.g., DMG-PEG2000). In some embodiments, the LNP composition The substance is a nucleic acid, the content of which is 30 to 65% (molar ratio, the same applies below) of the total lipids of the composition. An ionized cationic lipid having the structure shown in Formula I, in an amount of 5 to 30% of the total lipid content of the composition. % of a helper phospholipid (e.g., DSPC, DOPE, DOPC, or a combination thereof) ), sterols (e.g., cholesterol) whose content is 30 to 55% of the total lipids in the composition. or cholesterol derivatives or plant sterols, such as β-sitosterol), and Polymer-conjugated lipids (e.g., DMG) whose content is 1-5% of the total lipids of the composition In addition, in the LNP composition, the ionized lipid molecules are The molar ratio of ionized N atoms to phosphate groups in the nucleic acid molecule is (2-10):1, more preferably Preferably it is (4~8):1.

[0077] In a preferred embodiment of the present invention, the LNP composition comprises a nucleic acid, ionized cationic lipids with structure, helper phospholipids (e.g., DSPC, DOPE, DOPC, etc. or a combination thereof), sterol (e.g., cholesterol or cholesterol sterol derivatives, or plant sterols, e.g., β-sitosterol) and polymer conjugates In a more preferred embodiment of the present invention, the lipids include soluble lipids such as DMG-PEG2000. In one embodiment, the LNP composition comprises a nucleic acid, an ionized cation having the structure shown in formula (I): Ionic lipids, helper phospholipids (e.g., DSPC, DOPE, DOPC, etc., or combinations thereof) sterols (e.g., cholesterol or cholesterol derivatives, or e.g., plant sterols such as β-sitosterol) and polymer-conjugated lipids (e.g., DMG-PEG2000, etc.

[0078] As used herein, the terms "encapsulation" and "encapsulated" refer to mRNA, DNA, siRNA or other nucleic acid drugs are inside the lipid nanoparticles or As used herein, the term "encapsulation" refers to complete encapsulation. For example, lipid nanoparticles containing mRNA. When administered to a subject in need thereof, the composition acts to treat and / or prevent the associated disease. mRNA can be selected.

[0079] As used herein, the term "pharmaceutically acceptable carrier" refers to any Injectants, carriers, excipients, scintillants, sweeteners, diluents, preservatives, dyes / colors, flavors Enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or food and pharmaceutical products emulsifiers approved by the Department for human or livestock use, including, but not limited to: stomach.

[0080] Method for preparing lipid nanoparticle pharmaceutical formulations Another aspect of the present invention provides a method for preparing a lipid nanoparticle pharmaceutical formulation, said method comprising: (a) The ionizable lipid according to the first aspect of the present invention and an optional helper lipid are mixed with an organic solvent. (b) mixing the biologically active substance with an aqueous solvent to obtain a lipid organic phase; (c) obtaining a physiologically active substance-containing aqueous phase; and (b) separating the lipid organic phase from the lipid organic phase of step (a). and mixing the aqueous phase in the aqueous phase of the lipid nanoparticle drug to obtain the lipid nanoparticle drug. The method further comprises the steps of purifying, concentrating, filtering and sterilizing the lipid nanoparticle drug obtained in step (c). It further includes floor (d).

[0081] In some embodiments, the organic solvent is ethanol, methanol, isopropanol, or the like. acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane or tetrahydrofuran Some examples include, but are not limited to, tetrahydrofuran, thiahydrofuran, or combinations thereof. In embodiments, the lipid organic phase contains a small amount of water or a pH buffer. 0% water by volume, e.g., up to about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4% %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% In one embodiment, the lipid organic phase may comprise: Between about 0.05% and 60% water by volume, for example, between about 0.05% and 50% by volume Water, between about 0.05% and 40% water, or between about 5% and 20% water.

[0082] In some embodiments, the lipid organic phase comprises an ionizable cationic lipid, a helper lipid, These include single types of lipids such as phospholipids, lipids containing sterols or polymer complexes. In some embodiments, the lipid organic phase comprises a variety of lipids. wherein the lipid organic phase comprises an ionizable cationic lipid having the structure shown in Formula I, a helper lipid lipids (e.g., DSPC, DOPE, DOPC or a combination thereof), sterols (e.g., For example, cholesterol or cholesterol derivatives, or, for example, β-sitosterol. Plant sterols) and polymer-conjugated lipids (e.g., DMG-PEG2000) In a preferred embodiment of the present invention, the lipid organic phase comprises the structure shown in formula (I): Ionized cationic lipids, helper phospholipids (e.g., DSPC, DOPE, DOP C or a combination thereof), sterols (e.g., cholesterol or cholesterol-derived or plant sterols, e.g., β-sitosterol) and polymer conjugates In a more preferred embodiment of the present invention, The lipid organic phase comprises an ionizable cationic lipid having the structure shown in formula (I), a helper lipid, lipids (e.g., DSPC, DOPE, DOPC or a combination thereof), sterols (e.g., For example, cholesterol or cholesterol derivatives, or, for example, β-sitosterol. Plant sterols) and polymer-conjugated lipids (e.g., DMG-PEG2000) In a specific embodiment of the present invention, the lipid organic phase comprises the structure shown in formula (I): with ionized cationic lipids, DSPC, cholesterol and DMG-PEG2000 include.

[0083] In some embodiments, the aqueous solvent is water. The aqueous solvent has a pH of 3 to 8 (e.g., a pH of about 3, about 4, about 5, or about 6). It is an aqueous buffer solution containing a physiologically active substance such as a nucleic acid (e.g., mRNA) dissolved in the aqueous solvent. The aqueous phase is then mixed with a small amount of water-miscible organic solvent to obtain an aqueous phase containing the physiologically active substance. The aqueous phase may contain up to 60% by volume of at least one water-miscible organic solvent. Medium, e.g., 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 therebetween of one organic solvent (e.g., In one embodiment, the aqueous phase may comprise about 100% by volume of a water-miscible organic solvent. Between 0.05% and 60% organic solvent, for example, between about 0.05% and 50% by volume, about 0 Between 0.05% and 40%, or between about 5% and 20% of an organic solution (e.g., a water-miscible organic solvent) The aqueous phase buffer includes citrate buffer, Tris-HCl buffer, sodium acetate buffer, In some embodiments, the buffer solution may be a PBS buffer solution, a PBS buffer solution, or a combination thereof. In this case, the aqueous buffer has a pH between 4 and 6 (e.g., a pH of about 4, about 5, or about 6). In one embodiment, the aqueous buffer solution has a pH of about 4. It is a citrate buffer solution.

[0084] In some embodiments, the composition comprises a mixture of a lipid organic phase and a biologically active agent-containing aqueous phase. The solution can be diluted and the mixture comprises an LNP suspension. In the present invention, the pH of the solution containing the mixture of the lipid organic phase and the aqueous phase containing the biologically active substance of the LNP suspension is The LNP suspension can be adjusted by adding water, acid, base, or aqueous buffer. The pH of the suspension can be diluted or adjusted. In some embodiments, the LNP suspension No dilution or adjustment of the pH of the solution is performed. Dilute and adjust H.

[0085] In some embodiments, tangential flow filtration (TFF) (e.g., die The LNP suspension is then filtered to remove excess reagents, solvents, and unencapsulated nucleic acids. Organic solvents (e.g., ethanol) and buffers can also be removed using TFF. In some embodiments, the LNP suspension can be removed by In some embodiments, the LNP suspension is subjected to TFF. In this embodiment, the LNP suspension is subjected to dialysis and TFF.

[0086] The main advantages of the present invention are: (1) The ionizable lipid of the present invention and, for example, a helper phospholipid (DSPC, DOPE) , DOPC, etc.), sterols (e.g., cholesterol or cholesterol derivatives), PEG derivatives (e.g., DMG-PEG lipids, or DMG-P modified with other groups) Other components, such as PEG substances and / or derivatives such as PEG, can form stable nanoparticles. After encapsulating mRNA, the particle size of the nanoparticles is uniform, the encapsulation efficiency is high, and the stability is good. Therefore, it improves the transfection efficiency of mRNA in target tissues or cells. and low toxicity, the preventive and therapeutic effects of mRNA vaccines / drugs are more pronounced. (2) The present invention is based on the design of the ionized cation structure, the addition of modifiers, the fifth component, and the composition of each component. By using strategies such as optimizing the ratio, we were able to target LNP to lymph nodes in mice. This can effectively avoid the expression of LNP in organs such as the liver, spleen, kidney, and heart. The fluorescence distribution in lymph nodes can reach 97.06%. The realization of therapeutic effects is a technical prerequisite for the targeted treatment of diseases, especially tumor vaccines. This will provide a technical prerequisite for the research and development of drug-based systems, and the results will contribute to the field of targeted drug delivery. It has important promotional significance in this field.

[0087] The present invention will now be further described in conjunction with specific examples. These examples are provided to illustrate the present invention. It is understood that the present invention is only used to illustrate the principles of the present invention and is not intended to limit the scope of the present invention. In the following examples, experimental methods for which no specific conditions are given are generally carried out under conventional conditions or manufacturing methods. Subject to the terms proposed by the manufacturer. Unless otherwise specified, percentages and quantities are is calculated in weight percentage and parts by weight.

[0088] Example 1. Preparation of ionizable lipids 1.1. Synthesis steps and characterization of compound AL-6: [ka]

[0089] Synthesis of 1.1.1.6-bromohexyl-2-hexyldecanoate [ka] Add 2-hexylundecanoic acid (55 g, 214.483 mmol) to a 1.0 L three-neck bottle. , 1 equiv), dichloromethane (550 mL), 6-bromo-1-hexanol (4 6.60g, 257.380mmol, 1.2equiv), N,N-dimethylaminopyridinium Lysine (2.62 g, 21.448 mmol, 0.1 equiv) was added and the mixture was stirred at 0°C. Cyclohexylcarbodiimide (53.11 g, 257.380 mmol, 1.2 equiv v) is added. The reaction is stirred at 20°C for 5 hours, the reaction solution is filtered, and the filter cake is Wash with dichloromethane (100 mL x 3). Concentrate the organic phase under reduced pressure. The product was purified by chromatography, eluting with n-heptane:ethyl acetate (10:1). and concentrated to give 6-bromohexyl-2-hexyldecanoate (66g, 73.36%) is obtained as a pale yellow oil. LCMS-PH-AXTER-SDPC-2022-08B-6-3:460[M+C H3CN+1] + 1 H NMR(400MHz,Chloroform-d)δ 4.096(t,J= 6.4Hz,2H),3.437(t,J=6.8Hz,2H),2.398-2.27 7(m,1H),1.949-1.837(m,2H),1.713-1.545(m, 4H), 1.528-1.369(m, 6H), 1.340-1.222(m, 20H) ,0.900(t,J=6.4Hz,6H).

[0090] 1.1.2. (1R,4R)-2,5-Diaza-bicyclo[2.2.1]heptane hydrochloride Salt synthesis [ka] A 50 mL single-serving bottle is charged with t-butyl(1R,4R)-2,5-diaza-bicyclo[2. 2.1]Heptane (1.0 g, 5.044 mmol, 1 equiv), dioxane hydrochloride Add ethanol (10 mL, 4 M) and stir the reaction at room temperature for 2-4 hours. Concentrate to dryness and 1.1 g of (1R,4R)-2,5-diaza-bicyclo[2.2.1]heptane hydrochloride Obtain crude product as an off-white solid. LCMS-PH-AXTER-SDPC-2022-08B-6-5:99[M+1] + 1 H NMR(400MHz,DMSO-d6)δ 4.422(s,2H),3.5 50(d,J=13.6Hz,2H),3.329-3.318(m,2H),2.06 4(s,2H).

[0091] 1.1.3. Synthesis of the final product [ka] (1R,4R)-2,5-diaza-bicyclo[2.2.1]hexyl ether in a 20 mL single-portion bottle Butane hydrochloride (0.6 g, 4.457 mmol, 1 equiv), acetonitrile (6 m L), N,N-dimethylethylamine (3.46 g, 26.742 mmol, 6.0 eq uiv), 6-bromohexyl-2-hexyldecanoate (4.11g, 9.805m mol, 2.2 equiv) is added. The reaction mixture is stirred at 70°C for 12 hours. Cool to room temperature, dilute with ethyl acetate (120 mL), wash with three 30 mL portions of water, and The mixture is dried over sodium sulfate, filtered, and then concentrated to dryness under reduced pressure. The product was purified by methanol / ethyl acetate = (5 / 95) and concentrated to dryness. , 1.2 g (HPLC: 94.7%) of product is obtained. The product was diluted with n-heptane (120 mL) and then methanol / water (3 / 1, 36 mL) Wash once with acetonitrile / water (3 / 1, 36 mL), water (36 mL), and add anhydrous sulfuric acid. The mixture was dried over sodium hydroxide, filtered, and concentrated to dryness under reduced pressure to give 980 mg (HPLC: 9 980 mg (HPLC: 95.4%) of the product was purified by reverse phase preparative separation: Prep-HPLC (IntelFlash-1, Column: C18 silica gel; m obile phase A:water(0.5%TFA),phase B:Ace tonitrile;40% to 95%gradient in 25min,95 The collected solution was purified by HPLC (Electron Microscope, ELSD) and saturated bicarbonate. The mixture was adjusted to pH 8 with aqueous sodium chloride solution and extracted twice with 60 mL of n-heptane. The organic phase was washed once with 30 mL of water, dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The final product (HPLC: 99.3%) of 6-[(1R,4R)-5-{ 6-[(2-hexyldecyl)oxy]hexyl}-2,5-diazabicyclo[2.2. 1]heptan-2-yl]hexyl 2-hexyldecanoate was obtained, 0.5344 g of product The item is delivered. LCMS-PH-AXTER-SDPC-2022-08B-6-0:775.7[M +1] + 1 H NMR(300MHz,Chloroform-d)δ 4.059(t,J= 6.6Hz,4H),3.282(s,2H)2.682(s,4H),2.632-2 .513(m,2H),2.501-2.392(m,2H),2.380-2.261 (m,2H),1.720(s,2H),1.714-1.541(m,8H),1.5 13-1.196(m,56H),0.876(t,J=7.2Hz,12H). The hydrogen spectrum of AL-6 is shown in FIG. 1 and the purity characterization diagram is shown in FIG.

[0092] 1.2. Synthesis steps and characterization of compound AL-17: [ka]

[0093] 1.2.1.(1R,4R)-2,5-diazabicyclo[2.2.1]heptane dihydrochloride Salt synthesis [ka] t-Butyl (1R,4R)-2,5-diazabiphenyl in a 250 mL three-neck bottle at room temperature. Cyclo[2.2.1]heptane-2-carboxylate (9 g, 45.394 mmol, (1 equiv) was dissolved in a hydrogen chloride dioxane solution (90 mL) and stirred at room temperature for 2 hours. The final reaction mixture was directly spin-evaporated and spin-dried under reduced pressure to give (1R,4R) -2,5-diazabicyclo[2.2.1]heptane dihydrochloride (7.4g, 95.29%) is obtained as an off-white solid. LCMS-PH-AXTER-SDPC-2022-07B-17-1:99[M+H ] + 1 H NMR(300MHz,DMSO-d6,ppm)δ 10.038(s,4H ),4.477-4.363(m,2H),3.542(d,J=12.391Hz,2 H),3.269(dd,J=12.394,2.741Hz,2H),2.058(s ,2H).

[0094] 1.2.2. t-Butyl N-{2-[(1R,4R)-5-{2-[(t-butylcarbamate (bornyl)aminoethyl}-2,5-diazabicyclo[2.2.1]heptan-2-yl] Synthesis of {ethyl}carbamate [ka] At room temperature, in a 100 mL three-necked bottle, add (1R,4R)-2,5-diazabicyclo[2. 2.1]Heptane dihydrochloride (3 g, 17.537 mmol, 1 equiv), methanol (30 mL), N-Boc-2-diaminoacetaldehyde (8.37 g, 52.611 mmol, 3equiv), NaBH(OAc)3(18.58g, 87.685mmo l, 5 equiv) and glacial acetic acid (3.16 g, 52.611 mmol, 3 equiv) The reaction mixture is stirred at room temperature for 10 hours and the reaction is complete as determined by LCMS. The reaction was quenched by pouring into saturated sodium carbonate solution (90 mL) and adding DCM (3 × 10 0 mL), the organic phases were combined, dried over MgSO4, filtered, and spun dry. The crude product was purified by silica gel column (PE / EA (1:1)) to give t-butyl N- {2-[(1R,4R)-5-{2-[(t-butylcarbonyl)aminoethyl}-2, 5-diazabicyclo[2.2.1]heptan-2-yl]ethyl}carbamate (3.9 g, 52.11%). LCMS-PH-AXTER-SDPC-2022-07B-17-2:385[M+ H] + 1 H NMR(300MHz,Chloroform-d,ppm)δ 5.100( s,2H),3.276(d,J=2.315Hz,2H),3.161(t,J=5. 385Hz,4H),2.686(qd,J=9.556,4.317Hz,6H),2 .553(dt,J=12.223,6.321Hz,2H),1.682(s,2H) ,1.461(s,18H).

[0095] 1.2.3.2-[(1R,4R)-5-(2-aminoethyl)-2,5-diazabis ... Synthesis of chloro[2.2.1]heptan-2-yl]acetamide [ka] In a 100 mL single-mouth bottle, add t-butyl N-{2-[(1R,4R)-5-{ 2-[(t-butylcarbonyl)aminoethyl}-2,5-diazabicyclo[2.2.1 ]heptan-2-yl]ethyl}carbamate (3.5 g, 9.102 mmol, 1 eq Add uiv) and hydrogen chloride dioxane solution (35 mL) in sequence. Let the reaction proceed at room temperature for 2 hours. The completion of the reaction was detected by LCMS. The crude product was spin-dried and then transferred to an ion exchange resin ( PL-HCO3MP SPE 500mg / 6mL, 50 / pk,eluted wit ACN / HO (1:4)) and 2-[(1R,4R)-5-(2- Aminoethyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl]acetate Obtain mido (2g, crude). LCMS-PH-AXTER-SDPC-2022-07B-17-3:185[M+ H] + 1 H NMR(300MHz,DMSO-d6,ppm)δ 7.607-7.097 (m,4H),3.272(s,2H),2.728(h,J=7.149,6.463 Hz,6H),2.620(s,6H),1.585(s,2H).

[0096] 1.2.4. Synthesis of the final product [ka] At room temperature, in a 40 mL reaction bottle, add 2-[(1R,4R)-5-(2-aminoethyl) -2,5-diazabicyclo[2.2.1]heptan-2-yl]acetamide (800m g, 4.341 mmol, 1 equiv), i-PrOH (8 mL) and 2-(octane disulfanyl)ethyl acrylate (6000.42 mg, 21.705 mmol, 5 (equiv) are added in order and reacted at 70°C for 60 hours. The final reaction mixture is concentrated under reduced pressure. The crude product was purified by Prep-HPLC (column XB-Phenylgel; mob ile phase,i-PrOH in Water(0.1%TFA),50% t o 90%gradient in 20min;detector,UV 200nm The resulting product was purified by spin evaporation. The organic solution was removed, the remaining aqueous phase was adjusted to pH 9 with sodium carbonate, and the basic aqueous phase was diluted with EtOAc. Extract with c (2 x 20 mL), combine the organic phases, and wash once with saturated brine (1 x 20 mL). The extract was then dried over anhydrous sodium sulfate, filtered, spun dry, and dissolved in n-heptane (20 mL ), decolorized by adding activated carbon (200 mg), filtered, spin-dried, and finally The final product 7 (1.0039 g, 17.32%) is obtained as a yellow oil. LCMS-PH-AXTER-SDPC-2022-07B-17-0:1289.7 [M+H] + 1 H NMR(300MHz,Chloroform-d,ppm)δ 4.342( t,J=6.697Hz,8H),3.281(s,2H),2.909(t,J=6. 743Hz,8H),2.825(t,J=7.184Hz,8H),2.752-2. 672(m,12H),2.550(p,J=4.122Hz,6H),2.480(t ,J=7.123Hz,8H),1.763-1.629(m,10H),1.426- 1.254(m,42H),0.932-0.869(m,12H). The hydrogen spectrum of AL-17 is shown in Figure 3, and the HPLC purity characterization diagram is shown in Figure 4. will be done.

[0097] 1.3. Synthesis steps and characterization of compound AL-18: [ka]

[0098] 1.3.1.3-(acetylsulfonyl)-2-[(acetylsulfonyl)methyl]propion Synthesis of propanoic acid [ka] In a 250 ml round-bottom flask, add 3-bromo-2-(bromomethyl)propanoic acid (20 g , 81.335mmol, 1equiv), 1-(potassium sulfonyl)ethanone (23 0.22g, 203.337mmol, 2.5equiv) and NaOH (1equiv, The resulting mixture was stirred at room temperature for 16 hours. Stir. Acidify with 1M HCl until a white emulsion forms. Extract three times with ethyl acetate. The combined organic layers are washed with brine (pH ≈ 1) and then dried over anhydrous Na2SO4. The solution was filtered and concentrated under reduced pressure to give 3-(acetylsulfonyl)-2-[(acetylsulfonyl) The acid was obtained as a yellow oil and carried on to the next step without further purification. Used directly in the reaction of the next step.

[0099] LCMS-PH-AXTER-SDPC-2022-07B-18-3:235[M- 1]- 1.3.2.Synthesis of 3-sulfonyl-2-(sulfonylmethyl)propanoic acid [ka] In a three-necked round-bottom flask, add 3-(acetylsulfonyl)-2-[(acetylsulfonyl)methyl] ethyl]propanoic acid (13 g, 55.085 mmol, 1 equiv) and NaOH (1 M , 10V) was added, the temperature was maintained at 0°C, and NaOH (13 g, 55.085 mmol, 1 equiv) is added in batches. The resulting mixture is stirred at room temperature for 16 hours, then 0 Cool to 0°C and acidify with HCl (6M) until a white emulsion forms. Extract three times with ethyl acetate. The combined organic phases were dried over anhydrous Na2SO4. The filtrate was filtered and then concentrated under reduced pressure. This gives 3-sulfonyl-2-(sulfonylmethyl)propanoic acid as a yellow oil. LCMS-PH-AXTER-SDPC-2022-07B-18-3:151[M- 1]-

[0100] 1.3.3.3-(Octyldisulfonyl)-2-[(octyldisulfonyl)methyl ]Synthesis of propanoic acid [ka] In a round-bottom flask, 2-(octyldisulfonyl)pyridine (37.2 g, 140.05 3mmol, 3equiv), AcOH(584mg, 9.737mmol, 0.2eq) uiv) and MeOH (37 ml, 5V) were added, and the mixture was stirred at room temperature for 1 hour. (Hydroxymethyl)propanoic acid (7.4 g, 48.684 mmol, 1 equiv) (5VM The resulting mixture was stirred at room temperature for 16 hours and then cooled to room temperature under reduced pressure. Spin evaporate. Purify by silica gel column chromatography and distill off n-heptane. / EA (20:1) and 3-(octyldisulfonyl)-2-[(octyl (I)(disulfonyl)methyl]propanoic acid is obtained as a yellow oil. LCMS-PH-AXTER-SDPC-2022-07B-18-4:439[M- 1]- 1 H NMR(400MHz,Chloroform-d)δ 3.354-3.28 7(m,1H),3.125-3.074(m,2H),3.034-2.984(m, 2H),2.746(t,J=7.6Hz,4H),1.735-1.680(m,4 H),1.561-1.270(m,20H),0.922-0.877(m,6H) .

[0101] 1.3.4.4-Bromobutyl 3-(octyldisulfonyl)-2-[(octyldisulfonyl) Synthesis of [(sulfonyl)methyl]propionate [ka] At room temperature, a round-bottom flask was charged with 3-(octyldisulfonyl)-2-[(octyldisulfonyl) (methyl)propanoic acid (4.7g, 10.656mmol, 1equiv), 4-bromo Mobutan-1-ol (4.9 g, 31.973 mmol, 3 equiv) and DMAP (390 mg, 3.197 mmol, 0.3 equiv) and DCM (25 mL, 5 V) To the above mixture, add EDCI (3.1 g, 15.986 mmol, 1.5 equiv. v) (dissolved in 25 mL of DCM) was added dropwise, and the resulting mixture was stirred at room temperature for 16 hours. The resulting mixture was filtered, the filter cake was washed with DCM, and the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography and dissolved in n-heptane. 4-Bromobutyl 3-(octyldisulfonyl)-2-[(octyldisulfonyl) (methyl)propionate as a yellow oil. 1 H NMR(400MHz,Chloroform-d)δ 4.198(t,J= 6.4Hz,2H),3.464(t,J=6.4Hz,2H),3.278-3.24 3(m,1H),3.067-2.792(m,4H),2.730(t,J=7.2H z,4H),2.024-1.954(m,2H),1.875-1.821(m,2H ),1.741-1.651(m,4H),1.417-1.264(m,20H),0 .917-0.883(m,6H).

[0102] 1.3.5.Synthesis of 2-(octyldisulfonyl)pyridine [ka] 2,2′-Bipyridyl disulfide (60 g, 273.972 mmol, 2 equiv ) was dissolved in ethanol (100 ml, 5V) and HOAc (1.6 g, 27.3 97 mmol, 0.2 equiv) was added, and then 1-octanethiol was added under nitrogen gas protection. ol (20 g, 136.986 mmol, 1 equiv) (diluted with 100 ml EtOH The reaction mixture was concentrated under reduced pressure and purified by column chromatography. The 2-(octyl)-2- octyl ... (Iodosulfonyl)pyridine is obtained as a yellow oil. LCMS:256[M+1] + 1 H NMR (400 MHz, Chloroform-d) δ 1 H NMR (400 MHz,DMSO-d6)δ 8.560-8.371(m,1H),7.832(td ,J=7.7,1.9Hz,1H),7.764(d,J=8.0Hz,1H),7.2 77-7.191(m,1H),2.835(t,J=7.2Hz,2H),1.616 (p,J=7.3Hz,2H),1.345(dq,J=12.9,6.7Hz,2H) ,1.221(s,8H),0.846(t,J=6.7Hz,3H).

[0103] 1.3.6. Synthesis of the final product [ka] In a 50 ml round-bottom flask, add 4-bromobutyl 3-(octyldisulfonyl)-2-[ (Octyldisulfonyl)methyl]propanoic acid (3.5 g, 6.076 mmol, 3 eq uiv), (1R,4R)-2,5-diazabicyclo[2.2.1]heptane (269m g, 2.025 mmol, 1 equiv) and K2CO3 (838 mg, 6.076 mm ol, 3 equiv) was added, and MeCN (30 mL, 10 V) was added, and the resulting mixture The mixture was stirred at room temperature for 16 hours, extracted three times with ethyl acetate, and the combined organic layers were washed with anhydrous Na2S Dry over O4. After filtration, the filtrate is concentrated under reduced pressure. The crude product is purified by preparative HPLC. The preparation conditions were as follows: chromatography column, xb-phenyl gel, mobile phase, i-Pr OH / MeCN (1 / 1) and water (with 0.1% TFA added), 50% to 90% gradient (Detector: UV 200 nm, flow rate: 90 mL / min) The obtained fraction was purified under vacuum. Concentrate to remove the organic solvent and extract with heptane (2 x 40 mL). Combine the organic layers and Wash with brine (1 x 40 mL) and dry over anhydrous Na2SO4. Concentrate the filtrate under reduced pressure. The crude product (600 mg, purity 82.8%) was obtained by high-pressure preparative chromatography. Therefore, the preparation conditions (chromatography column, xb-phenyl gel, mobile phase , i-PrOH / MeCN (1 / 1) and water (without additives), 50% to 90% gradient (Detector: UV 200 nm, flow rate: 90 mL / min). The resulting solution was placed under vacuum. Concentrate to remove the organic solvent. Extract twice with n-heptane (2 x 40 mL). The combined extracts are washed with brine (1 x 40 mL) and dried over anhydrous Na2SO4. After filtration, the mixture was concentrated under reduced pressure to give the final product 4-[(1R,4R)-5-(4-{[3-( octyldisulfonyl)-2-[(octyldisulfonyl)methyl]propyl]oxy}butan 3-(octyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl]butyl Octyldisulfonyl)-2-[(octyldisulfonyl)methyl]propanoate (130m g, purity 63.4%) as a yellow oil. LCMS-PH-AXTER-SDPC-2022-07B-18-3:1087[M +1] + 1 H NMR(400MHz,Chloroform-d)δ 4.197-4.12 1(m,4H),3.454-3.339(m,2H),3.245(p,J=6.8H z,2H),3.304-2.944(m,8H),2.796-2.481(m,14 H),2.592-2.494(m,2H),1.760-1.652(m,16H), 1.445-1.202(m,42H),0.90(d,J=6.9Hz,12H). The hydrogen spectrum of AL-18 is shown in Figure 5, and the HPLC purity characterization diagram is shown in Figure 6. will be done.

[0104] 1.4. Synthesis steps and characterization of compound AL-19: The synthesis process of AL-19 is as follows: [ka]

[0105] Synthesis of 1.4.1.19-3 [ka] 7-Bromoheptanoic acid (21 g, 100.47 mmol, 1 equiv) was dissolved in THF (1 25 mL) and heated at 0°C under a nitrogen gas atmosphere in Li2CuCl4 (128 mL, 0. Addition of 2M, 0.2 equiv) was completed within 10 minutes, and then isobutylmagnesium bromide was added at 0°C. Add sodium (193 mL, 1 M, 1.5 equiv) dropwise. Stir the reaction mixture at room temperature for 2 hours. The reaction is monitored by thin layer chromatography. The reaction solution is diluted with hydrochloric acid (1M) The mixture is quenched by stirring with ethyl acetate (3×100 mL) and extracted with ethyl acetate (3×100 mL). The combined organic layers are washed with water (2 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The product was purified by silica gel column chromatography and dissolved in PE / EA (10:1). Thus eluted to give product 19-3 (18 g, 81.2%) as a yellow oil.

[0106] The hydrogen spectrum characterization results for 19-3 are as follows: 1 H NMR (300 MHz,Chloroform-d)δ3.639(t,J=6.6Hz,2H),1. 603-1.459(m,3H),1.367-1.238(m,12H),0.862 (d,J=6.6Hz,6H).

[0107] Synthesis of 1.4.2.19-5 [ka] At room temperature, a round-bottom flask was charged with product 19-3 (10.656 mmol, 1 equiv), 4- Bromobutan-1-ol (4.9 g, 31.973 mmol, 3 equiv) and DM AP (390 mg, 3.197 mmol, 0.3 equiv) and DCM (25 mL, 5 V) to the above mixture. uiv) (dissolved in 25 mL of DCM) was added. The resulting mixture was stirred at room temperature for 16 h. The resulting mixture was filtered, the filter cake was washed with DCM, and the filtrate was concentrated under reduced pressure. The mixture was purified by silica gel column chromatography and extracted with n-heptane. The 19-5 product is obtained by elution.

[0108] The hydrogen spectrum characterization of 19-5 is as follows: 1 H NMR (300 M z,Chloroform-d)δ 6.396(dd,J=17.3,1.6Hz,1 H),6.119(dd,J=17.3,10.4Hz,1H),5.808(dd,J =10.4,1.6Hz,1H),4.150(t,J=6.8Hz,2H),1.75 4-1.605(m,2H),1.568-1.474(m,1H),1.403-1. 134(m,13H),0.862(d,J=6.6Hz,6H).

[0109] Synthesis of 1.4.3.19-7 [ka] At room temperature, a 250 mL three-necked round-bottom flask was charged with t-butyl (1R,4R)-2,5-diazonium nitrate. Zacyclo[2.2.1]n-heptane-2-carboxylate (5g, 25.219mm ol, 1 equiv) and dioxane hydrochloride solution (50 mL) were added and stirred at room temperature for 3 hours. The resulting mixture was concentrated under reduced pressure to give (1R,4R)-2,5-diazacyclo[ 2.2.1] Heptane is obtained as a white solid. The crude product is carried on to the next step without further purification. Use directly on the floor.

[0110] The hydrogen spectrum characterization of 19-7 is as follows: 1 H NMR (300 M z,DMSO-d6)δ 9.835(s,3H),4.423(d,J=2.6Hz, 2H), 3.588-3.477(m, 2H), 3.299(s, 2H).

[0111] Synthesis of 1.4.4.19-9 [ka] At room temperature, add (1R,4R)-2,5-diazabicyclo[2.2 .1]heptane (2.1 g, 12.35 mmol, 1 equiv) and MeOH (21. 00mL), benzyl (3-oxopropyl)carbamate (8.2g, 39.53mm ol, 3.2equiv), STAB(13.0g, 61.76mmol, 5equiv ) is added. The reaction is stirred at room temperature for 12 hours. The resulting mixture is concentrated under reduced pressure. The mixture was purified by silica gel column chromatography and eluted in CH2Cl2 / MeOH (5:1 ) to give the product 19-9 (3.3 g, 55.7%) as a yellow solid. The hydrogen spectrum characterization of 19-9 is as follows: 1 H NMR (400MH z,Chloroform-d)δ 7.395-7.273(m,10H),5.54 1(s,2H),5.085(s,4H),3.626(s,2H),3.268(s, 4H), 3.250-2.321(m, 8H), 1.979-1.501(m, 6H).

[0112] Synthesis of 1.4.5.19-10 [ka] N-{3-[(1R,4R)-5-(3-{[(benzyloxy)carbonyl]amino }propyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl]propyl} Carbamate (3.3 g, 6.87 mmol, 1 equiv) and Pd / C (1.65 g , 50% w / w) was dissolved in MeOH (33 mL) and heated at room temperature under a hydrogen gas atmosphere for 2 hours. Stir. Filter and wash the filter cake with MeOH (2 x 100 mL). Concentrate under reduced pressure to give 3-[(1R,4R)-5-(3-aminopropyl)- 2,5-Diazabicyclo[2.2.1]heptan-2-yl]propan-1-amine (1 0.33g, 91.7%) as an off-white oil.

[0113] The hydrogen spectrum characterization of 19-10 is as follows: 1 H NMR (400M Hz,Methanol-d4)δ 2.874(t,J=6.9Hz,4H),2.7 97(d,J=10.5Hz,3H),2.763-2.693(m,3H),2.68 4-2.598(m,4H),1.713(dd,J=16.0,9.0Hz,10H) .

[0114] 1.4.6. Synthesis of the final product AL-19 [ka] In a 20 mL bottle, product 19-10 (4.457 mmol, 1 equiv), acetonitrile Trinitrile (6 mL), N,N-dimethylethylamine (3.46 g, 26.742 mm ol, 6.0 equiv), product 19-5 (43.142 mmol, 4.4 equiv) The reaction mixture is stirred at 70°C for 12 hours. The reaction mixture is cooled to room temperature and ethyl acetate ( Dilute with 120 mL of water, wash with 30 mL of water three times, dry with anhydrous sodium sulfate, After filtration, the mixture was concentrated to dryness under reduced pressure. The mixture was purified by silica gel column chromatography using methanol / acetic acid. When the ethyl acetate ratio was 5 / 95, the product was generated. After concentrating and drying, 1.2 g (HPLC: 94 1.2 g (HPLC: 94.7%) of the product was obtained by eluting with n-heptane (12 0 mL), followed by methanol / water (3 / 1, 36 mL), acetonitrile / water ( 3 / 1, 36 mL), washed once with water (36 mL), dried over anhydrous sodium sulfate, and filtered. After filtration, the mixture was concentrated to dryness under reduced pressure to give 980 mg (HPLC: 95.4%) of the product. 80 mg (HPLC: 95.4%) of the product was purified by reverse phase preparative HPLC (Int elFlash-1, Column: C18 silica gel, mobile phase A :water(0.5%TFA), phase B:Acetonitrile, 40% to 95%gradient in 25min, 95%in 5min, Dete The collected solution was purified by ethanol (ELSD). The pH was adjusted to 0.5 with saturated aqueous sodium bicarbonate. The temperature was adjusted to 8°C and extracted twice with 60 mL of n-heptane. The combined organic phase was diluted with 30 mL of water. The product AL was obtained by washing the cellulose acetate solution twice, drying it over anhydrous sodium sulfate, filtering it, and concentrating it to dryness under reduced pressure. You get -19.

[0115] The hydrogen spectral characterization of AL-19 is as follows: 1 H NMR (400M Hz,Chloroform-d)δ 4.048(t,J=6.8Hz,8H),3. 247(s,2H),2.761(t,J=7.3Hz,8H),2.649(s,4H ),2.446(dt,J=14.6,7.1Hz,16H),1.598-1.501 (m,18H),1.362-1.209(m,40H),1.186-1.108(m ,8H),0.863(d,J=6.6Hz,24H).

[0116] LCMS: 1118 [M+H] + is the target molecular weight and the purity reaches 95.14%. do. The hydrogen spectrum characterization results of AL-19 are shown in Figure 7 and are shown in LCMS The results are shown in Figure 8, and the HPLC purity detection results are shown in Figure 9. is.

[0117] 1.5. Synthesis and Characterization of AL-20 Compound The synthesis process of AL-20 is as follows: [ka] 3-[(1R,4R)-5-(3-aminopropyl)-2,5-dimethyl-2-(2-aminopropyl) ... -diazabicyclo[2.2.1]heptan-2-yl]propan-1-amine (19-1 0) (4.457 mmol, 1 equiv), acetonitrile (6 mL), N,N-dimethylformamide (1 mL) ethylamine (3.46 g, 26.742 mmol, 6.0 equiv), product 19 -5 (11.14 mmol, 2.5 equiv) was added, and the reaction mixture was heated at 70°C for 10 hours. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (120 mL), and diluted with water (30 mL each). The silica is washed three times with HCl, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The mixture was purified by gel column chromatography using methanol / ethyl acetate (5 / 95) as the mobile phase. Concentration to dryness gives 1.2 g (HPLC: 94.7%) of product. C: The product (94.7%) was diluted with n-heptane (120 mL) and then with methanol / water (3 / 1, 36 mL), acetonitrile / water (3 / 1, 36 mL), water (36 mL) The mixture was washed twice, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to give 980 ml g (HPLC: 95.4%) of product. 980 mg (HPLC: 95.4%) of product Reverse phase preparative purification: Prep-HPLC (IntelFlash-1, Column: C1 8 Silica gel, mobile phase A: water (0.5% TFA), pha se B: Acetonitrile, 40% to 95% gradient in Purify by 25 min, 95% in 5 min, Detector, ELSD). The collected solution was adjusted to pH 8 with saturated aqueous sodium bicarbonate and diluted with 60 mL of n-heptane. The combined organic phase was washed once with 30 mL of water, dried over anhydrous sodium sulfate, and After filtration and concentration to dryness under reduced pressure, the final product AL-20 is obtained.

[0118] LCMS: 892 [M+H] + is the target molecular weight. The hydrogen spectral characterization of AL-20 is as follows: 1 H NMR (400M Hz,Chloroform-d,ppm)δ 4.157-3.974(m,6H), 3.343-3.307(m,2H),2.902(t,J=6.5Hz,2H),2. 792-2.663(m,10H),2.550(t,J=6.4Hz,4H),2.4 99-2.426(m,8H),1.771-1.451(m,15H),1.413- 1.221(m,30H),1.193-1.143(m,6H),0.882(d,J =6.5Hz,18H). The hydrogen spectrum characterization results of AL-20 are shown in Figure 10. The S results are shown in Figure 11, and the HPLC purity detection results are shown in Figure 12. That's right.

[0119] Example 2. mRNA synthesis process, quality control methods and results mRNA preparation for delivery: The plasmid containing the transcription template was incubated in a shaker at 30°C for 24 hours, and then the plasmid was The plasmid was extracted and purified using a Smid extraction kit, digested with Bsa I, and then lysed on beads. The linearized transcription template is obtained by purifying the template using Nanodrop One. The concentration was measured using an Agilent Fragment Analyzer. Analyze template purity and confirm the quality using the dsDNA 915 Reagent kit , transcription template purity is measured to be 98-100%. mRNA is prepared in a two-step process. The IVT system is reacted at 37°C for 3 hours, followed by DNase The DNA template was digested with I and purified with RNA Clean Beads. Measure the concentration with Microdrop One and adjust the concentration to 1 mg / mL. The RNA was treated at 65°C for 10 minutes and then immediately placed on ice to open the secondary structure of the RNA and remove the capping system. The reaction was allowed to proceed at 37°C for 1 hour, and the DNA was purified using RNA Clean Beads. Measure the concentration in one tube, adjust the concentration to 1 mg / mL, aliquot and store at -20 °C.

[0120] The Agilent Fragment Analyzer was used to purify the transcription template. Analyze the purity and ensure that the transcription template purity is 95% or higher using an RNA kit (15 nt). Measured to be 100%. A260 / A280 using Nanodrop One The ratio is measured to be approximately 1.9. Using the ELISA method, the dsRNA residues are measured to be 1. The DNA residue was detected using a fluorescent quantitative PCR method. It is detected at 3-30pg / μg. The transfer template electropherogram, transfer template integrity peak graph, in vitro Electropherogram after transcription reaction (IVT), IVT RNA integrity peak graph and mRNA The completeness peak graphs are shown in Figures 13, 14, 15, 16 and 17, respectively.

[0121] Example 3. Assembly into LNP-mRNA by encapsulation of mRNA using ionizable lipids hand 3.1. Preparation example 1 of lipid nanoparticles (LNP-mRNA) ionized lipids (SM-102, AL-6, AL-17, AL-19, and AL-20), DSPC (distearoylphosphatidylcholine, 1,2-distearoyl-sn-glycol) Cholesterol, DMG-PEG2000 were mixed at different ratios. The mRNA was mixed into the ethanol phase and mixed thoroughly as an organic phase. The aqueous phase and organic phase were dissolved in a HCl (H=4.0) solution and mixed thoroughly. Transfer the solution to a suitable BD syringe, remove any air bubbles, and then use the PNI nanopreparation device to separate the solution into the aqueous phase and the active ingredient. The volume ratio of the aqueous phase to the aqueous phase was 3:1, and the mixture was mixed at a flow rate of 12 mL / min. The sample is allowed to stand, purified, concentrated, filtered, and sterilized, and the resulting product contains mRNA. The final product, LNP-mRNA, is subjected to physicochemical quality control.

[0122] 3.2. Preparation example 2 of lipid nanoparticles (LNP-mRNA) 1) Ionized lipid AL-6 (or AL-19), DSPC, Chol, DMG-PEG 2000, DOPA (or DOPS), DMG-PEG 2000 -mannose) at different ratios Mix the ethanol phase at the same rate and mix thoroughly with a vortex mixer. 2) m The RNA was dissolved in 50 mM sodium acetate (pH 4.0) solution and used as the aqueous phase. Mix thoroughly with 3) Transfer the aqueous and organic phases into appropriate BD syringes, removing any air bubbles. 4) Using a PNI nanopreparation device, the volume ratio of the aqueous phase to the organic phase was 3:1, and the flow rate was 1 Mix at 2 mL / min and then purify, concentrate, filter, and sterilize the mixture to obtain mRNA. Lipid nanoparticles encapsulating AXT-LNP-x (x stands for different series such as 01, 02, and 03) The rear number is shown in Table 1 below. 5) Perform physicochemical quality control on the final product LNP-mRNA.

[0123] [Table 3] TIFF2025538515000064.tif80170

[0124] 3.3. Physicochemical quality control methods and results of LNP-mRNA: 1) Particle size and PDI (distribution): The particle size and distribution of LNPs are measured using a nanoparticle analyzer. Detect. 2) Encapsulation rate (EE%): Total mRNA and fr with RiboGreen, respectively After staining, the ee mRNA is detected by an ELISA reader and the encapsulation rate is calculated. 3) pH: Use a pH meter to detect the pH of the final product = 7.2-7.4. 4) Osmolarity: Use a freezing point osmometer to detect the osmolarity of LNP-mRNA, and determine the detected concentration. When the concentration is 100 μg / mL, the osmotic pressure is 280 to 310 mOsmol / kg. 5) mRNA integrity: using Agilent Fragment Analyzers The integrity of the mRNA after packaging (i.e., mRNA purity) was detected using an RNA kit (15 nt) to determine purity greater than 90%. Some of the detection results are shown in FIGS.

[0125] Example 4. In vitro expression detection of LNP-mRNA According to the process shown in Figure 23, the LNP-mRNA prepared in Example 3.1 was Then, a cell expression screening experiment was carried out, and the specific steps are as follows: 1) Cell plating: After digesting 293T cells, the cell density was adjusted to 2 × 10 4 / well The solution was adjusted to 100 μL / well and plated in a 96-well plate. Place in the oven overnight. 2) Test grouping: Sample groups were LNP(AL-6)-Luc, LNP(AL -17)-Luc series products were used, and LNP(SM-102)-Luc was used as a positive control. As a negative control, add 100 μL of the mixture of medium and cells alone. do. 3) Cell transfection: LNP-Luc requires transfection. The highest concentration was 100 ng of mRNA per well, diluted 3-fold, for a total of six wells. The gradient is set up in three duplicate wells for each sample. Add 10 μL of LNP-Luciferase evenly to a 96-well cell plate. After mixing uniformly, place in an incubator and culture. 4) Kit detection: 48 hours after transfection, ONE-Glo TM EXLu Follow the instructions in the Ciferase Assay System kit step by step. Luciferase luminescence assay using the BioTek SYNERGY ELISA reader The intensity is detected and compared with the average OD value. The results are shown in Figure 24. The LNP(AL-6)-Luc product It is normally expressed in the nucleus, and the expression level increases with increasing mRNA concentration. When the concentration of AL-6 cells was 1 μg / mL, the cell expression level of the AL-6 group was 1 μg / mL, and the cell expression level of the positive control group (LNP(SM-102 )-Luc) cell expression level is significantly higher than that of the AL-6 preparation. The resulting LNP-mRNA is efficiently expressed in vitro. Figure 25 shows the expression of LNP(AL-17)-Luc series products in 293T cells. Shows.

[0126] Example 5. In vitro toxicity detection of LNP-mRNA Cytotoxicity experiments were carried out according to the process shown in Figure 23, with the specific steps being as follows: It is. 1) Cell plating: After digesting 293T cells, the cell density was adjusted to 2 × 10 4 / well The solution was adjusted to 100 μL / well and plated in a 96-well plate. Place in the oven overnight. 2) Test grouping: LNP(AL-6)-Luc and LNP(S) were used as sample groups. M-102)-Luc was used as a blank control, and 100 μL of medium alone was added. As a control, only 100 μL of the mixture of medium and cells was added. Staurosporine (STS)-induced apoptosis was also administered as a positive control to the mixture of cells. An inducer is added. 3) Cell transfection: LNP-Luc requires transfection. The highest concentration was 100 ng of mRNA per well, diluted 3-fold with buffer A total of four gradients were prepared by adding 10 μL of each of the different dilutions of LNP-Luc to a 96-well plate. Add the samples evenly to the wells of the cell plate and set up three duplicate wells for each sample. After mixing evenly, place in an incubator and culture. STS (diluted in PBS) was added to a 96-well cell plate, and six duplicate wells were added. After uniformly mixing, the mixture is placed in an incubator and cultured. 4) Kit detection: After 48 hours of culture, CellTiter-Glo® Lumi Follow the instructions of the Native Cell V(I)bility Assay kit. Stepwise operation and luminescence intensity were measured using the BioTek SYNERGY ELISA reader. Detect and compare the average OD values ​​using the formula: (negative control group - sample group / positive control group) ÷ The cell inhibition rate was calculated according to the formula: (negative control group - blank control group) x 100%. -Create a response-inhibition curve. The results are shown in Figures 26 and 27, and are consistent with those obtained by AL-6 in Example 3.1. When the concentration of the synthesized LNP-mRNA product was less than 1 μg / mL, cell proliferation was not observed. There was no significant effect (Fig. 26), which indicates that there is no obvious toxicity within the detection concentration range and that AL A series of LNP-mRNA products formed by -20 were also clearly evident within the detection concentration range. This demonstrates the absence of toxicity (Figure 27).

[0127] Example 6. Detection of in vivo expression of LNP-mRNA The in vivo expression capacity and toxicity of LNP-mRNA assembled in AL-6 were clearly explained. To clarify this, we will measure expression and toxicity in vivo in mice with intact innate immunity. Further hEPO ELISA expression detection experiments and toxicity experiments were designed, and the steps were as follows: As shown. The experimental process is shown in FIG. 1) Encapsulation of LNP-mRNA: hEPO mRNA is dissolved in an aqueous buffer. The ionized lipid (SM-102 or AL-6), DSPC, and cholesterol were mixed uniformly as a phase. Dissolve terol and DMG-PEG2000 in absolute ethanol and mix in a fixed ratio. After thoroughly and uniformly mixing, transfer the aqueous phase and organic phase into syringes. Set the parameters using the PNI microfluidic nanopreparation device (aqueous and organic phases) The volume ratio was 3:1 and the flow rate was 12 mL / min. LNP-hEPO is concentrated, purified, sterilized, filtered, and passes quality control before being marketed. It is injected into the mouse. 2) Mouse tail vein injection: Immobilize the mouse and inject it into the tail vein. The injection amount of PO was 5 μg. LNP(AL-6)-hEPO was set as the sample group. LNP(SM-102)-hEPO was used as a positive control group, and only the vehicle was injected. Each LNP-hEPO sample was injected into two mice, which served as a negative control group. 3) Submandibular blood collection: Blood was collected from the submandibular area 6, 24, and 48 hours after injection. Blood is collected in EDTA anticoagulant tubes, gently mixed to homogenize, numbered, and stored. 4) Serum extraction: After collecting whole blood, it was centrifuged at 2000 g for 10 minutes, and the supernatant was separated into plasma and serum. Aliquot and store at -80°C. 5) hEPO expression level measured using a human erythropoietin ELISA kit Detect the markers, follow the instructions, and prepare two duplicates for each serum sample. Set up wells. Use an ELISA reader to detect OD values ​​and calculate standard curves and dilution ratios. Calculate the expression level according to The results are shown in Figure 29. The LNP-hEPO series prepared in AL-6 Our products were successfully expressed in vivo in mice, and the overall expression levels (AUC) were This experiment demonstrated that the LNs formed by AL-6 were significantly higher than those of the positive control (SM-102). We demonstrate that P can efficiently mediate mRNA expression in vivo.

[0128] Example 7. In vivo toxicity detection of LNP-mRNA The in vivo toxicity detection process is shown in Figure 28, and the specific steps are as follows: . 1) Encapsulation of LNP-mRNA: Luciferase mRNA is placed in the aqueous buffer. The aqueous phase is dissolved and mixed uniformly, and the ionized lipid (SM-102 or AL-6), DS PC, cholesterol, and DMG-PEG2000 were each dissolved in absolute ethanol. The aqueous phase and the organic phase are mixed thoroughly and uniformly at a certain ratio and then separated into an organic phase. Transfer into a syringe and use the PNI microfluidic nanopreparation device to prepare LNP-Luciferas LNP-Luciferase is concentrated, purified, sterilized, filtered, and After passing quality control, the mice are injected. 2) Mouse tail vein injection: Immobilize the mouse and inject into the tail vein. The injection doses of ciferase were 5, 15, and 30 μg, respectively. -Luc was set as the sample group, and LNP(SM-102)-Luc was set as the positive control group. Each LNP-Luc group was injected with 30 μg of LNP-Luc and the vehicle alone was injected as a negative control group. Inject the IFERASE sample into two mice. 3) Weighing of mice: Mice were weighed daily and weight data were recorded continuously for 7 days. The weight gain or loss is recorded and calculated as a percentage. 4) Blood collection from the submandibular region of mice: before injection (0 hours), and 24 and 96 hours after injection. Blood is collected from the bottom into EDTA anticoagulant tubes, gently mixed uniformly, numbered, and stored. 5) Extraction of serum: After collecting whole blood, it was centrifuged at 2000 g for 10 minutes, and the supernatant was separated into plasma and serum. Aliquot and store at -80°C. 6) Biochemistry detection: ALT using the IUBIO iChem340 blood biochemistry analyzer (alanine aminotransferase) and AST (aspartate aminotransferase) The concentration values ​​were statistically analyzed and the average values ​​were calculated, after which the groups were divided into groups. The difference between them is compared and a curve is generated. The results are shown in Figure 30. The results of the mouse toxicity experiment showed that different doses of LN After injection of P(AL-6)-Luc, the mice were in a relatively normal state and no deaths occurred. After injection, the body weight of the mice was significantly reduced, and the degree of reduction was not significantly related to the injection dose. There was a significant correlation between the weight of the mice and the AL-6 levels, which gradually returned to normal after 2 days. This indicates that the adverse effects are reversible.

[0129] The day after injection, ALT and AST levels in the mice increased, and the degree of increase was significantly correlated with the injection dose. After 4 days, liver enzymes returned to pre-injection levels, indicating that AL-6 has a beneficial effect on liver function. This shows that the effects are reversible. In this experiment, a high dose of LNP(SM-102)-Luc was injected simultaneously, and the mice body weight Its effects on steroid and ALT / AST levels are comparable to those of AL-6 at the same dose. In the experimental system, the high dose group of LNP(AL-6)-Luc and LNP(SM-102) There was no significant difference in the effects of AL-6 and SM-Luc on mice, which may be indirectly related to the effects of AL-6 and SM-Luc on mice. This indicates that the toxicity of -102 is equivalent.

[0130] Example 8. In vivo imaging experiments in mice In this example, different AXT-LNP-x test articles (e.g., as shown in Table 1 above) Each compound was administered subcutaneously to the back of a BALB / c mouse in a single dose (10 μg / mouse). (There were three mice in each group), and 6 hours after injection, D-Luciferi nSubstrate imaging was used and the hair on each animal, particularly on the head, chest and back, was removed prior to imaging. Shave and administer 0.2 mL of D-Lucine at a concentration of 15 mg / mL to each animal prior to imaging. A ferin fluorescent substrate solution was injected intraperitoneally, and the animals were anesthetized with isoflurane and subjected to intravital imaging and Immediate autopsy is performed to image organs such as the heart, liver, spleen, lungs, kidneys, and lymph nodes. Live imaging, organ imaging, and fluorescence expression ratio of each organ in mice treated with each formulation The results are shown in FIGS. The results show that all of the test compounds have potent organ targeting properties. The mechanism of this is mainly the addition of the fifth component (anion DOPA, DOPS) and the modifying component DM. Three variations of G-PEG2000-mannose and targeted ionizable lipids were tested, and the results were As can be seen from the results, the anion in the formulation, the modifying component DMG-PEG2000-manno Adjusting the ratio of lipids and ionized lipids also alters the ratio of target lymph nodes to various degrees. The distribution of most LNPs in lymph nodes can reach over 90%. and significantly reduces the accumulation and expression of LNP in organs such as the liver, spleen, kidney, and heart. It is possible.

[0131] All documents referred to in this application are incorporated herein by reference as if each were individually incorporated by reference. As such, the present application is hereby incorporated by reference. After reading this specification, those skilled in the art will be able to make various changes or modifications to the present invention and to acquire equivalent forms thereof. Any such modifications are within the scope defined by the appended claims of this application.

Claims

1. An ionized lipid, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. hand, The ionizable lipid has the structure of Formula I: 【Chemistry 39】 In the formula: R １ and R ２ are each independently -(CH ２ ) ｎ -, where n is selected from: is a positive integer between 1 and 14, X and Y are each independently —CH— or N; L １ and L ２ are each independently a divalent linking group or are absent, R ３ , R ４ , R ５ and R ６ are each independently H, CH ３ , C2-C30 hydrocarbons groups (e.g., C2-C30 alkyl groups, C2-C30 chain alkenyl groups, C2-C30 alkyl groups, -alkynyl group), or -(CH ２ ) s-R ａ - (CH ２ ) g-R ｂ - (CH ２ ) ｍ -R ｃ in wherein s and g are each independently selected from a positive integer of 1 to 20; and m is is selected from integers from 0 to 20, preferably s+g+m is 2 to 35, R ｃ is CH ３ or a C2-C15 hydrocarbon group (e.g., a C2-C15 alkyl group, a C2-C15 chain alkyl group, a C2-C15 alkynyl group, a C2-C15 alkenyl group, R ａ , R ｂ are each independently —CH ２ -, C2-C6 alkenyl structure or selected from functional groups as shown 【Chemistry 40】 Also, R ３ and R ４ does not become H at the same time, and R ５ and R ６ At the same time, it should not become H The ionizable lipid, or a pharmaceutically acceptable salt, tautomer, or Stereoisomers.

2. The ionizable lipid has a substructure shown in formula (I-1): 【Chemistry 41】 In the formula: R １ and R ２ are each independently -(CH ２ ) ｎ -, where n is selected from: is a positive integer between 1 and 14, L １ From left to right, １ａ -L １ｂ -L １ｃ )-, wherein L １ｂ teeth , -(CH ２ ) ｎ -, where n is selected from a positive integer from 1 to 14; １ａ Reach BiL １ｃ are each independently —CH ２ -, -(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)N H—, —CH(OH)—, preferably —CH ２ -, -(C=O)O-, -O(C=O)- , —(S—S)—, —CH(OH)—; L ２ From left to right, ２ａ -L ２ｂ -L ２ｃ )-, wherein L ２ｂ teeth , -(CH ２ ) ｎ -, where n is selected from a positive integer from 1 to 14; ２ａ Reach BiL ２ｃ are each independently —CH ２ -, -(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)N H—, —CH(OH)—, preferably —CH ２ -, -(C=O)O-, -O(C=O)- , —(S—S)—, —CH(OH)—; R ３ , R ４ , R ５ and R ６ are each independently a C2-C20 hydrocarbon group. Characterized by The ionizable lipid according to claim 1, or a pharmaceutically acceptable salt, tautomer or Stereoisomers.

3. The ionizable lipid has a substructure shown in formula (I-2): 【Chemistry 42】 In the formula: R １ and R ２ are each independently -(CH ２ ) ｎ -, where n is selected from: is a positive integer between 1 and 14, L ２ From left to right, ２ａ -L ２ｂ -L ２ｃ )-, Here, L ２ｂ is -(CH ２ ) ｎ -, where n is a positive integer from 1 to 14. Selected, L ２ａ , L ２ｃ are each independently —CH ２ -, -NH-, -(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)—, —(C≡C)—, preferably —CH ２ -, -NH-, -(C =O)O-、-O(C=O)-、-(S-S)-、-(C=C)-(CH ２ )-(C=C )-, —CH(OH)—, R ３ is -R ３ａ -R ３ｂ -R ３ｃ -R ３ｄ -R ３ｅ and R ４ is -R ４ａ -R ４ｂ -R ４ｃ -R ４ｄ -R ４ｅ having the structure Here, R ３ａ , R ３ｃ , R ４ａ and R ４ｃ are each independently -(CH ２ ) ｎ - where n is selected from a positive integer from 1 to 14; R ３ｂ , R ３ｄ , R ４ｂ and R ４ｄ are each independently —CH ２ -, -(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)—, —(C≡C)—, preferably —CH ２ -, -(C=O)O- 、-O(C=O)-、-(S-S)-、-(C=C)-(CH ２ )-(C=C)-、-C selected from functional groups such as H(OH)-; R ３ｅ and R ４ｅ are each independently a C2-C20 hydrocarbon group; R ６ is a C2-C20 hydrocarbon group The ionizable lipid according to claim 1, or a pharmaceutically acceptable salt, tautomer or Stereoisomers.

4. The ionizable lipid has a substructure shown in formula (I-3): 【Chemistry 43】 In the formula: R １ and R ２ are each independently -(CH ２ ) ｎ -, where n is selected from: is a positive integer between 1 and 14, R ３ is -R ３ａ -R ３ｂ -R ３ｃ -R ３ｄ -R ３ｅ and R ４ is -R ４ａ -R ４ｂ -R ４ｃ -R ４ｄ -R ４ｅ and R ５ is -R ５ａ -R ５ｂ -R ５ｃ - R ５ｄ -R ５ｅ and R ６ is -R ６ａ -R ６ｂ -R ６ｃ -R ６ｄ -R ６ｅ The structure It has a structure, Here, R ３ａ , R ３ｃ , R ４ａ , R ４ｃ , R ５ａ , R ５ｃ , R ６ａ and R ６ｃ Ha, so Each independently represents -(CH ２ ) ｎ -, where n is a positive integer selected from 1 to 14. And, R ３ｂ , R ３ｄ , R ４ｂ , R ４ｄ , R ５ｂ , R ５ｄ , R ６ｂ and R ６ｄ are each unique Stand up, -CH ２ -, -(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)--(C≡C)-, preferably は-CH ２ -、-(C=O)O-、-O(C=O)-、-(S-S)-、-(C=C)- (CH ２ )-(C=C)-, -CH(OH)-, R ３ｅ , R ４ｅ , R ５ｅ , R ６ｅ are each independently a C2-C20 hydrocarbon group. It is characterized by The ionizable lipid according to claim 1, or a pharmaceutically acceptable salt, tautomer or Stereoisomers.

5. The preparation methods include Method I, Method II and Method III; wherein Method I is (A1) In an inert solvent, compound K ２ and K3 are reacted with compound K1 to obtain compound K obtaining a value of 4; (A2) In an inert solvent, compounds K5 and K6 are reacted with compound K4 to obtain a compound of the formula (I- obtaining a compound according to formula (1), 【Chemistry 44】 Here, R １ and R ２ are each independently -(CH ２ ) ｎ - is selected from, where , n is a positive integer from 1 to 14; M and G are each independently preferably —OH, —COOH, —SH, or —NH ２ , ethylene oxide; L １ Is -(L １ａ -L １ｂ -L １ｃ )-structure, and L ２ Is -(L ２ａ -L ２ｂ -L ２ｃ )-, Here, L １ｂ and L ２ｂ are each independently -(CH ２ ) ｎ -, where: n is selected from a positive integer from 1 to 14; L １ａ , L １ｃ , L ２ａ and L ２ｃ are each independently —CH ２ -, -(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)—, preferably —CH ２ -, -(C= -O)O-, -O(C=O)-, -(S-S)-, -CH(OH)-, Selected, R ３ , R ４ , R ５ and R ６ are each independently a C2-C20 hydrocarbon group; Method II is (B1) reacting compounds K7 and K8 in an inert solvent to obtain compound K9; Floors and (B2) deprotecting compound K9 to obtain compound K10; (B3) Reacting compounds K11, K12 and K13 with compound K10 in an inert solvent. and obtaining a compound represented by formula (I-2), 【Chemistry 45】 Here, R １ and R ２ are each independently -(CH ２ ) ｎ - is selected from, where , n is a positive integer from 1 to 14; L ２ Is -(L ２ａ -L ２ｂ -L ２ｃ )-, Here, L ２ｂ is -(CH ２ ) ｎ -, where n is a positive integer from 1 to 14. Selected, L ２ａ , L ２ｃ are each independently —CH ２ -, -NH-, -(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)—, —(C≡C)—, preferably —CH ２ -, -NH-, -(C =O)O-、-O(C=O)-、-(S-S)-、-(C=C)-(CH ２ )-(C=C )-, —CH(OH)—, R ３ is -R ３ａ -R ３ｂ -R ３ｃ -R ３ｄ -R ３ｅ and R ４ is -R ４ａ -R ４ｂ -R ４ｃ -R ４ｄ -R ４ｅ having the structure Here, R ３ａ , R ３ｃ , R ４ａ and R ４ｃ are each independently -(CH ２ ) ｎ - where n is selected from a positive integer from 1 to 14; R ３ｂ , R ３ｄ , R ４ｂ and R ４ｄ are each independently —CH ２ -, -(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)—, —(C≡C)—, preferably —CH ２ -, -(C=O)O- 、-O(C=O)-、-(S-S)-、-(C=C)-(CH ２ )-(C=C)-、-C selected from functional groups such as H(OH)-; R ３ｅ and R ４ｅ are each independently a C2-C20 hydrocarbon group; R ６ is a C2-C20 hydrocarbon group, Method III comprises: (C1) Reacting compounds K7 and K14 with compound K1 in an inert solvent to obtain compound K1. obtaining K15; (C2) Compounds K11, K12, K16, and K17 are reacted with compound K15 in an inert solvent. reacting to obtain a compound of formula (I-3), 【Chemistry 46】 where: R １ and R ２ are each independently -(CH ２ ) ｎ -, where n is selected from: is a positive integer between 1 and 14, R ３ is -R ３ａ -R ３ｂ -R ３ｃ -R ３ｄ -R ３ｅ and R ４ is -R ４ａ -R ４ｂ -R ４ｃ -R ４ｄ -R ４ｅ and R ５ is -R ５ａ -R ５ｂ -R ５ｃ - R ５ｄ -R ５ｅ and R ６ is -R ６ａ -R ６ｂ -R ６ｃ -R ６ｄ -R ６ｅ The structure It has a structure, Here, R ３ａ , R ３ｃ , R ４ａ , R ４ｃ , R ５ａ , R ５ｃ , R ６ａ and R ６ｃ Ha, so Each independently represents -(CH ２ ) ｎ -, where n is a positive integer selected from 1 to 14. And, R ３ｂ , R ３ｄ , R ４ｂ , R ４ｄ , R ５ｂ , R ５ｄ , R ６ｂ and R ６ｄ are each unique Stand up, -CH ２ -, -(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)-, -(C≡C)-, preferred くは-CH ２ -、-(C=O)O-、-O(C=O)-、-(H-S)-、-(C=C) - (CH ２ )-(C=C)-, -CH(OH)-, R ３ｅ , R ４ｅ , R ５ｅ , R ６ｅ are each independently a C2-C20 hydrocarbon group. The ionized lipid according to claim 1, or a pharmaceutically acceptable salt thereof, Methods for preparing salts, tautomers or stereoisomers.

6. A lipid nanoparticle (LNP), The lipid nanoparticles are ionized lipids according to any one of claims 1 to 4, or pharmaceutical compositions thereof. The lipid nanoparticles are characterized in that they contain a salt, tautomer or stereoisomer that is acceptable to the particle.

7. The lipid nanoparticles further comprise a helper lipid, and the helper lipid is a helper lipid. lipids, sterols, polymer-conjugated lipids, or combinations thereof; Here, the lipid nanoparticles are capable of preferentially delivering the physiologically active substance encapsulated therein to lymph nodes. and delivers to organs or tissues other than lymph nodes (e.g., heart, liver, spleen, lung, kidney, brain, etc.). etc.) The lipid nanoparticle of claim 6.

8. The lipid nanoparticles comprise an ionizable lipid, DSPC, cholesterol, and DMG-PEG2 000, wherein the ionizable lipid:DSPC:cholesterol:DMG-PEG20 The molar ratio of 00 is (30-65):(5-30):(30-55):(1-5). It is characterized by The lipid nanoparticle of claim 7.

9. The lipid nanoparticles contain an ionized lipid, DSPC, cholesterol, DMG-PEG20 00, and (1) DOPA (dioleoylphosphatidic acid) or DOPS (dioleoylphosphatidic acid) (2) DMG-PEG ２０００ - mannose, contains a fifth component selected from the group of combinations thereof; Here, ionized lipid: DSPC: cholesterol: DMG-PEG2000: fifth The molar ratio of the components is (30-50): (7.5-15): (25-50): (1-1.5). : (0.5 to 25) The lipid nanoparticle of claim 7.

10. The ionizable lipid is characterized by having the structure shown below: The lipid nanoparticle of claim 6. Table 4

11. A lipid nanoparticle pharmaceutical formulation, comprising: The lipid nanoparticle pharmaceutical preparation comprises the lipid nanoparticles according to claim 6, The lipid composition is characterized by comprising a physiologically active substance incorporated therein, and a pharmaceutically acceptable carrier. High-quality nanoparticle pharmaceutical formulations.

12. The bioactive substance may be a nucleic acid, a protein, a polypeptide, a small molecule, or a combination thereof. characterized in that the compound is selected from the group consisting of The lipid nanoparticle pharmaceutical formulation according to claim 11.

13. 12. A method for preparing the lipid nanoparticle pharmaceutical formulation according to claim 11, comprising: The method comprises: (a) an ionized lipid according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof; The tautomer or stereoisomer and optional helper lipid are mixed with an organic solvent to form a lipid-containing The stage of acquiring the machine phase; (b) mixing a physiologically active substance with an aqueous solvent to obtain an aqueous phase containing a physiologically active substance; 、 (c) mixing the lipid organic phase of step (a) with the aqueous phase of step (b) to form the lipid organic phase; and obtaining a nanoparticle drug. Methods for preparing nanoparticle pharmaceutical formulations.

14. 10. The ionized lipid according to claim 1, or or the use of a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

15. Claims in the preparation of drugs for treating and / or preventing tumors, infectious diseases and rare diseases Item 7. Use of the lipid nanoparticles described in item 6.

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