Ionizable lipids and uses thereof

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to an ionizable lipid and its application in drug delivery. Background Technology

[0002] In recent years, messenger RNA (mRNA) drugs have become a key treatment for the prevention and treatment of infectious diseases and tumors. The technology of mRNA drugs is recognized in the industry due to its short development cycle, low risk of insertional mutagenesis, and the diversity of encoded proteins; mRNA is particularly suitable for the development of vaccines or therapeutic drugs. However, mRNA itself is very unstable and easily degraded by ubiquitous RNases. Furthermore, due to the inherent negative charge and large molecular weight of mRNA (usually greater than 10⁻⁶), it is also susceptible to degradation. 6 This restriction (Da) limits the entry of mRNA molecules into the cell. Therefore, developing suitable delivery vectors to protect fragile mRNA molecules and deliver them into the cytoplasm is of great importance.

[0003] Various mRNA delivery vectors have been developed, including lipid nanoparticles (LNPs), inorganic nanoparticles, polymer nanoparticles, viral vectors, and exosomes. LNPs are currently widely used as drug delivery vectors, and their main components include ionizable lipids, phospholipids, cholesterol, and lipids containing polyethylene glycol. The most important component of LNPs is the ionizable lipid. Early permanently positively charged cationic lipids exhibited low in vivo circulation time, high toxicity, and severe allergic reactions. This is because their inherent positive charge causes them to adsorb proteins during circulation, making them easily captured and cleared by the reticuloendothelial system; their inherent positive charge interacts with negatively charged cell membranes, leading to cell membrane instability and severe toxicity; and permanently positively charged cationic lipids activate the complement system, causing allergic reactions. Ionizable lipids are uncharged under physiological pH conditions; therefore, LNPs prepared from ionizable lipids have relatively high safety. Ionizable lipids endow LNPs with the ability to escape from lysosomes. Through the proton sponge effect and membrane fusion mechanism, LNPs escape and release mRNA into the cytoplasm, where they bind to the ribosomes that encode the protein for translation.

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

[0005] The purpose of this invention is to provide an ionizable lipid with low toxicity and high delivery efficiency, which can be used as an important component of a drug delivery system.

[0006] In a first aspect, the present invention provides an ionizable lipid, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, said ionizable lipid having the structure of Formula I:

[0007]

[0008] In the formula,

[0009] R1 and R2 are each independently selected from -(CH2)n-, where n is a positive integer from 1 to 14;

[0010] X and Y are each independently -CH- or N;

[0011] L1 and L2 are each independently divalent linker groups or none;

[0012] R3, R4, R5, and R6 are each independently H, CH3, C2-C30 hydrocarbon groups (such as C2-C30 alkyl, C2-C30 alkenyl, C2-C30 alkynyl), or -(CH2)sR a -(CH2)gR b -(CH2)m-CH3, where s and g are each independently selected from positive integers from 1 to 20, and m is selected from integers from 0 to 20, preferably s+g+m is 2-35.

[0013] R a R b Each is independently selected from the functional groups with the structure shown below:

[0014]

[0015] in, Represents a connection key;

[0016] Furthermore, R3 and R4 are not both H, and R5 and R6 are not both H.

[0017] In another preferred embodiment, R3, R4, R5 and R6 are each independently a C4-C30 hydrocarbon group (such as C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl), preferably a C4-C20 hydrocarbon group (such as C4-C20 alkyl, C4-C20 alkenyl, C4-C20 alkynyl).

[0018] In another preferred embodiment, at least two, three, or four of R3, R4, R5, and R6 are C2-C30 hydrocarbon groups (such as C2-C30 alkyl, C2-C30 alkenyl, or C2-C30 alkynyl) or -(CH2)sR a -(CH2)gR b -(CH2)m-CH3, where s, g, m, and R a and Rb As defined above.

[0019] In another preferred embodiment, s+g+m is 3-20, more preferably 4-15.

[0020] In another preferred embodiment, R3 has -R 3a -R 3b -R 3c -R 3d -R 3e The structure,

[0021] Among them, R 3a and R 3c Each is independently -(CH2)n-, where n is a positive integer selected from 1 to 14;

[0022] R 3b and R 3d Each of the following functional groups is independently selected: -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, with preferred groups being -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, and -CH(OH)-;

[0023] R3 is a C2-C20 hydrocarbon group.

[0024] In another preferred embodiment, R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure;

[0025] Among them, R 4a and R 4c Each is independently -(CH2)n-, where n is a positive integer selected from 1 to 14;

[0026] R 4b and R 4dEach of the following functional groups is independently selected: -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, with preferred groups being -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, and -CH(OH)-;

[0027] R 4e It is a C2-C20 hydrocarbon group.

[0028] In another preferred embodiment, R5 has -R 5a -R 5b -R 5c -R 5d -R 5e The structure,

[0029] Among them, R 5a and R 5c Each is independently -(CH2)n-, where n is a positive integer selected from 1 to 14;

[0030] R 5b and R 5d Each of the following functional groups is independently selected: -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, with preferred groups being -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, and -CH(OH)-;

[0031] R 5e It is a C2-C20 hydrocarbon group.

[0032] In another preferred embodiment, R6 has -R 6a -R 6b -R 6c -R 6d -R 6e The structure;

[0033] Among them, R 6a and R 6cEach is independently -(CH2)n-, where n is a positive integer selected from 1 to 14;

[0034] R 6b and R 6d Each of the following functional groups is independently selected: -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, with preferred groups being -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, and -CH(OH)-;

[0035] R 6e It is a C2-C20 hydrocarbon group.

[0036] In another preferred embodiment, L1 has -(L 1a -L 1b -L 1c The structure is )-, where L 1b The expression is -(CH2)n-, where n is a positive integer selected from 1 to 14; L 1a and L 1c Each of the following functional groups is independently selected: -(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)-, with -(C=O)O-, -O(C=O)-, -(SS)-, and -CH(OH)- being preferred.

[0037] In another preferred embodiment, L2 has -(L 2a -L 2b -L 2c The structure of )-;

[0038] Among them, L 2b The expression is -(CH2)n-, where n is a positive integer selected from 1 to 14; L 2a L 2cEach of the following functional groups is independently selected: -(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)-, -CH2-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -(C≡C)-, with preferred groups being -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-, and -CH2-.

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

[0040] In another preferred embodiment, the ionizable lipid has the substructure shown in formula (I-1):

[0041]

[0042] In the formula,

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

[0044] L1 has -(L from right to left) 1a -L 1b -L 1c The structure is )-, where L 1b The expression is -(CH2)n-, where n is a positive integer selected from 1 to 14; L 1a and L 1c Each of the following functional groups is independently selected: -(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)-, with -(C=O)O-, -O(C=O)-, -(SS-, -CH(OH)- preferred;

[0045] L2 has -(L from left to right) 2a -L 2b -L 2c The structure of )-; where L 2b The expression is -(CH2)n-, where n is a positive integer selected from 1 to 14; L 2a and L 2cEach of the following functional groups is independently selected: -(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)-, with -(C=O)O-, -O(C=O)-, -(SS-, -CH(OH)- preferred;

[0046] R3, R4, R5, and R6 are each independently a C2-C20 hydrocarbon group.

[0047] In another preferred embodiment, X is N and Y is -CH-.

[0048] In another preferred embodiment, L1 is absent.

[0049] In another preferred embodiment, R5 is H.

[0050] In another preferred embodiment, the ionizable lipid has the substructure shown in formula (I-2):

[0051]

[0052] In the formula,

[0053] R1 and R2 are each independently selected from -(CH2)n-, where n is a positive integer from 1 to 14;

[0054] L2 has -(L from left to right) 2a -L 2b -L 2c The structure of )-;

[0055] Among them, L 2b The expression is -(CH2)n-, where n is a positive integer selected from 1 to 14; L 2a L 2c Each of the following functional groups is independently selected: -(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)-, -CH2-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -(C≡C)-, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-, -CH2-;

[0056] R3 has -R 3a -R 3b -R3c -R 3d -R 3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure;

[0057] Among them, R 3a R 3c R 4a and R 4c Each is independently -(CH2)n-, where n is a positive integer selected from 1 to 14;

[0058] R 3b R 3d R 4b and R 4d Each of the following functional groups is independently selected: -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, with preferred groups being -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, and -CH(OH)-;

[0059] R 3e and R 4e Each is independently a C2-C20 hydrocarbon group;

[0060] R6 is a C2-C20 hydrocarbon group.

[0061] In another preferred embodiment, X and Y are N.

[0062] In another preferred embodiment, L1 and L2 are absent.

[0063] In another preferred embodiment, the ionizable lipid has the substructure shown in formula (I-3):

[0064]

[0065] In the formula,

[0066] R1 and R2 are each independently selected from -(CH2)n-, where n is a positive integer from 1 to 14;

[0067] R3 has -R 3a -R 3b -R3c -R 3d -R 3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure of R5; R5 has -R 5a -R 5b -R 5c -R 5d -R 5e The structure of R6 has -R 6a -R 6b -R 6c -R 6d -R 6e The structure;

[0068] Among them, R 3a R 3c R 4a R 4c R 5a R 5c R 6a and R 6c Each is independently -(CH2)n-, where n is a positive integer selected from 1 to 14;

[0069] R 3b R 3d R 4b R 4d R 5b R 5d R 6b and R 6d Each of the following functional groups is independently selected: -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, with preferred groups being -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, and -CH(OH)-;

[0070] R 3e R 4e R 5e R 6e Each is independently a C2-C20 hydrocarbon group.

[0071] In another preferred embodiment, the ionizable lipid has a structure selected from Table 1 below:

[0072] Table 1

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] In another preferred embodiment, the ionizable lipid preferably has the following structure:

[0097]

[0098] In another preferred embodiment, the ionizable lipid can be used to prepare a drug delivery system, the delivery system including lipid nanoparticles (LNPs), liposomes, polymer nanoparticles, etc., preferably for preparing lipid nanoparticles.

[0099] A second aspect of the present invention provides a method for preparing an ionizable lipid or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof as described in the first aspect of the present invention, the method comprising: method I, method II and method II I;

[0100] Method I includes the following steps:

[0101] (A1) Compounds K2 and K3 are reacted with compound K1 in an inert solvent to obtain compound K4;

[0102] (A2) In an inert solvent, compounds K5 and K6 are reacted with compound K4 to obtain the compound shown in formula (I-1);

[0103]

[0104] R1 and R2 are each independently selected from -(CH2)n-, where n is a positive integer from 1 to 14;

[0105] M and G are each independently and preferably selected from -OH, -COOH, -SH, -NH2, and ethylene oxide;

[0106] L1 has -(L 1a -L 1b -L 1c The structure L2 has -(L)-. 2a -L 2b -L 2c The structure of )-;

[0107] Among them, L 1b and L 2b Each is independently -(CH2)n-, where n is a positive integer selected from 1 to 14;

[0108] L 1a L 1c L 2a and L 2cEach of the following functional groups is independently selected: -(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 -(C=O)O-, -O(C=O)-, -(SS-, -CH(OH)-;

[0109] R3, R4, R5 and R6 are each independently a C2-C20 hydrocarbon group;

[0110] Method II includes the following steps:

[0111] (B1) Compound K7 and K8 are reacted in an inert solvent to obtain compound K9;

[0112] (B2) Deprotects compound K9 to obtain compound K10;

[0113] (B3) In an inert solvent, compounds K11, K12 and K13 are reacted with compound K10 to obtain the compound shown in formula (I-2);

[0114]

[0115] R1 and R2 are each independently selected from -(CH2)n-, where n is a positive integer from 1 to 14;

[0116] L2 has -(L 2a -L 2b -L 2c The structure of )-;

[0117] Among them, L 2b The expression is -(CH2)n-, where n is a positive integer selected from 1 to 14; L 2a L 2c Each of the following functional groups is independently selected: -(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)-, -CH2-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -(C≡C)-, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-, -CH2-;

[0118] R3 has -R 3a -R3b -R 3c -R 3d -R 3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure;

[0119] Among them, R 3a R 3c R 4a and R 4c Each is independently -(CH2)n-, where n is a positive integer selected from 1 to 14;

[0120] R 3b R 3d R 4b and R 4d Each of the following functional groups is independently selected: -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, with preferred groups being -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, and -CH(OH)-;

[0121] R 3e and R 4e Each is independently a C2-C20 hydrocarbon group;

[0122] R6 is a C2-C20 hydrocarbon group;

[0123] Method II includes the following steps:

[0124] (C1) In an inert solvent, compounds K7 and K14 are reacted with compound K1 to obtain compound K15;

[0125] (C2) In an inert solvent, compounds K11, K12, K16, and K17 are reacted with compound K15 to obtain the compound shown in formula (I-3);

[0126]

[0127] in,

[0128] R1 and R2 are each independently selected from -(CH2)n-, where n is a positive integer from 1 to 14;

[0129] R3 has -R 3a -R 3b -R 3c -R 3d -R 3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure of R5; R5 has -R 5a -R 5b -R 5c -R 5d -R 5e The structure of R6 has -R 6a -R 6b -R 6c -R 6d -R 6e The structure;

[0130] Among them, R 3a R 3c R 4a R 4c R 5a R 5c R 6a and R 6c Each is independently -(CH2)n-, where n is a positive integer selected from 1 to 14;

[0131] R 3b R 3d R 4b R 4d R 5b R 5d R 6b and R 6d Each of the following functional groups is independently selected: -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, with preferred groups being -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, and -CH(OH)-;

[0132] R 3e R 4e R 5e R 6e Each is independently a C2-C20 hydrocarbon group.

[0133] In another preferred embodiment, the inert solvent is selected from the group consisting of tetrahydrofuran, acetonitrile, or chloroform, or combinations thereof.

[0134] In another preferred embodiment, the reaction temperature for method I is 0-90℃; the reaction temperature for method II is 0-90℃; and the reaction temperature for method III is 25-30℃.

[0135] In another preferred embodiment, the reaction time for method I is 1-24 h; the reaction time for method II is 1-24 h; and the reaction time for method III is 1-17 h.

[0136] A third aspect of the present invention provides a lipid nanoparticle (LNP) comprising an ionizable lipid as described in the first aspect of the present invention, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

[0137] In another preferred embodiment, the lipid nanoparticles further comprise auxiliary lipids.

[0138] In another preferred embodiment, the content of the ionizable lipid in the lipid nanoparticles is 30-65% molar proportion of the total lipid content.

[0139] In another preferred embodiment, the auxiliary lipids include auxiliary phospholipids, sterols, polymer-conjugated lipids, or combinations thereof.

[0140] In another preferred embodiment, the auxiliary lipid is a combination of auxiliary phospholipids, sterols, and polymer-conjugated lipids.

[0141] In another preferred embodiment, the auxiliary phospholipid is preferably selected from: 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), dioleoyl lecithin (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine, 1,2-Myristoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycerol-3-phospho-rac-(1-glycerol) sodium salt, 1,2-palmitoylphosphatidylglycerol, 1-palmitoyl-2-oleoyllecithin, 1-palmitoyl-2-oleoylphosphatidylethanolamine, distearate phosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, 1-stearoyl-2-oleoylphosphatidylethanolamine, or combinations thereof.

[0142] In another preferred embodiment, the sterol includes cholesterol or a cholesterol derivative.

[0143] In another preferred embodiment, the polymer-conjugated lipid is a polyethylene glycol (PEG) lipid.

[0144] In another preferred embodiment, the PEGylated lipid is preferably selected from the group consisting of DMG-PEG2000, DSPE-PEG2000, DSG-PEG2000, DSPE-PEG-Mannose, DMG-PEG2000 (polypeptides, proteins, amino acids, vitamins, and other active substances) or combinations thereof.

[0145] In another preferred embodiment, the lipid nanoparticles comprise ionizable lipids, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of ionizable lipids:DSPC:cholesterol:DMG-PEG2000 is (30-65):(5-30):(30-55):(1-5), preferably 45:10:43.5:1.5.

[0146] In another preferred embodiment, the lipid nanoparticles further comprise bioactive substances encapsulated within the lipid nanoparticles.

[0147] In another preferred embodiment, the bioactive substance is selected from the group consisting of nucleic acids, proteins, polypeptides, small molecules, or combinations thereof.

[0148] In another preferred embodiment, the nucleic acid includes DNA, plasmid, messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotide, small RNA, ribosomal RNA, microRNA, and transfer RNA, preferably mRNA.

[0149] A fourth aspect of the present invention provides a lipid nanoparticle pharmaceutical formulation comprising lipid nanoparticles as described in the third aspect of the present invention, a bioactive substance encapsulated in the lipid nanoparticles, and a pharmaceutically acceptable carrier.

[0150] In another preferred embodiment, the bioactive substance is selected from the group consisting of nucleic acids, proteins, polypeptides, small molecules, or combinations thereof.

[0151] In another preferred embodiment, the nucleic acid includes DNA, plasmid, messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotide, small RNA, ribosomal RNA, microRNA, and transfer RNA, preferably mRNA.

[0152] In another preferred embodiment, the bioactive substance is nucleic acid, and in the lipid nanoparticle drug, the molar ratio of ionizable N atoms in the ionizable lipid molecule to phosphate groups in the nucleic acid molecule is (2–10):1, more preferably (4–8):1.

[0153] In another preferred embodiment, the hydrated particle size of the lipid nanoparticle drug is 50-200 nm, more preferably 70-150 nm, and most preferably 75-110 nm.

[0154] In another preferred embodiment, the lipid nanoparticle drug formulation can be used for the treatment and / or prevention of tumors, infectious diseases, and rare diseases.

[0155] In another preferred embodiment, the dosage form of the lipid nanoparticle drug formulation is selected from the group consisting of: injections, lyophilized formulations, nebulized inhalers, and topical formulations.

[0156] In another preferred embodiment, the lipid nanoparticle drug formulation is administered by injection, i.e., intravenous, intramuscular, intradermal, subcutaneous, intrathecal, duodenal, or intraperitoneal injection.

[0157] In another preferred embodiment, the lipid nanoparticle drug formulation is administered by inhalation, such as intranasal administration.

[0158] In another preferred embodiment, the lipid nanoparticle drug formulation is administered transdermally, such as by topical application or electrode delivery.

[0159] A fifth aspect of the present invention provides a method for preparing a lipid nanoparticle drug as described in the fourth aspect of the present invention, the method comprising:

[0160] (a) The ionizable lipid as described in the first aspect of the invention or its pharmaceutically acceptable salt, tautomer or stereoisomer and optionally the auxiliary lipid are mixed with an organic solvent to obtain a lipid organic phase;

[0161] (b) The bioactive substance is mixed with an aqueous solvent to obtain an aqueous phase containing the bioactive substance;

[0162] (c) The lipid organic phase from step (a) is mixed with the aqueous phase from step (b) to obtain the lipid nanoparticle drug.

[0163] In another preferred embodiment, the organic solvent includes ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, tetrahydrofuran, or combinations thereof.

[0164] In another preferred embodiment, the aqueous solvent is a buffer solution.

[0165] In another preferred embodiment, the aqueous solvent is a buffer solution with a pH range of 3-7.

[0166] In another preferred embodiment, the acidic buffer is a citrate buffer with a pH of 4.0.

[0167] In another preferred embodiment, the volume ratio of the lipid organic phase to the aqueous phase containing bioactive substances is 1:(2-5), more preferably 1:(3-4).

[0168] In another preferred embodiment, in step (c), the lipid organic phase and the aqueous phase are mixed via a microfluidic chip.

[0169] In another preferred embodiment, the method further includes step (d): purifying, concentrating, and filtering the lipid nanoparticle drug obtained in step (c) to remove bacteria.

[0170] A sixth aspect of the present invention provides the use of an ionizable lipid as described in the first aspect of the present invention, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, for the preparation of a drug delivery system.

[0171] In another preferred embodiment, the delivery system includes lipid nanoparticles (LNPs), liposomes, polymer nanoparticles, etc., and is preferably used to prepare lipid nanoparticles.

[0172] In another preferred embodiment, the drug delivery system is used to deliver drugs for the treatment and / or prevention of tumors, infectious diseases, and rare diseases.

[0173] A seventh aspect of the invention provides the use of lipid nanoparticles as described in the third aspect of the invention in the preparation of medicaments for treating and / or preventing tumors, infectious diseases, and rare diseases.

[0174] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0175] Figure 1 The LC-MS spectra and results of the molecular weight determination of AL-6 are shown. The characteristic peaks and their corresponding integral areas are shown in the figure. The results prove that the obtained compound is the target compound.

[0176] Figure 2 The chromatogram and results of AL-6 purity determination using HPLC-CAD are shown. The product elution time is 17.767 min, and the purity reaches over 98%.

[0177] Figure 3 The LC-MS spectra and results of the molecular weight of AL-17 are shown. The characteristic peaks and their corresponding integral areas are shown in the figure. The results prove that the obtained compound is the target compound.

[0178] Figure 4The chromatogram and results of AL-17 purity determination using HPLC-CAD are shown. The product elution time is 11.311 min, and the purity reaches over 95%.

[0179] Figure 5 The LC-MS spectra and results of the molecular weight determination of AL-18 are shown. The characteristic peaks and their corresponding integral areas are shown in the figure. The results prove that the obtained compound is the target compound.

[0180] Figure 6 The chromatogram and results of AL-18 purity determination using HPLC-CAD are shown. The product elution time is 17.997 min, and the purity reaches over 93%.

[0181] Figure 7 The electrophoresis results of the transcription template are shown: there are 3 electrophoresis lanes in the figure. The first lane is a DNA indicator band of different lengths. Lane 1 is the plasmid DNA, and lane 2 is the linearized transcription template.

[0182] Figure 8 The transcription template integrity peak diagram is shown: the horizontal axis represents fragment length, and the vertical axis represents relative fluorescence units. The left side of the diagram shows three peaks, labeled LM, 4146, and UM. LM and UM are the low- and high-molecular-weight DNA indicator bands, respectively, and 4146 is the target detection band. The right side shows the fitted electrophoresis diagram.

[0183] Figure 9 The results of RNA electrophoresis after in vitro transcription (IVT) are shown: the figure shows three electrophoresis lanes. The first lane is an indicator band of RNA of different lengths. The lane labeled IVT is the RNA transcribed in vitro, and the lane labeled CAP is the mRNA capped by enzymatic method.

[0184] Figure 10 The diagram shows the peaks of IVT RNA integrity: the horizontal axis represents fragment length, and the vertical axis represents relative fluorescence units; there are two peaks on the left, labeled LM and 1795, respectively. LM is the low molecular weight RNA indicator band, and 1795 is the target RNA detection band; the right side is the fitted electrophoresis diagram.

[0185] Figure 11 The graph shows the peaks of mRNA integrity: the horizontal axis represents fragment length, and the vertical axis represents relative fluorescence units; there are two peaks in the left graph, labeled LM and 1795, respectively. LM is the low molecular weight RNA indicator band, and 1795 is the target RNA detection band; the right graph is the fitted electrophoresis diagram.

[0186] Figure 12The physicochemical properties (particle size, PDI, encapsulation efficiency) of LNP-mRNA are shown. The particle size range is 70-100 nm, PDI < 0.2, and encapsulation efficiency is 85-100%, which proves that the physicochemical properties of the LNP prepared above are similar.

[0187] Figure 13 The procedure for detecting LNP-mRNA cell expression and toxicity is shown.

[0188] Figure 14 The in vitro cell expression results of LNP(AL-6)-mRNA are shown in the figure. As the mRNA concentration increases, the cell expression results of multiple formulations of LNP(AL-6) are better than those of LNP(SM-102).

[0189] Figure 15 The results of LNP(AL-17)-mRNA expression in cells are shown in the figure. As the concentration increases, the expression of LNP(AL-17) in cells first increases and then decreases, and at 1 ug / ml, it is generally lower than that of lipofectamine.

[0190] Figure 16 The results of cytotoxicity of LNP(AL-6)-mRNA were shown. As can be seen from the figure, the cell inhibition rate of LNP(AL-6) series products at different mRNA concentrations was almost 0, which is comparable to that of LNP(SM-102), proving that LNP(AL-6) molecules have good safety.

[0191] Figure 17 The in vivo expression detection procedure for LNP-mRNA is shown.

[0192] Figure 18 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 trend of both LNP(SM-102) and LNP(AL-6) is first increased and then decreased, and both reach their maximum at around 6 hours. However, the expression of LNP(AL-6) series products is better than that of LNP(SM-102).

[0193] Figure 19 The in vivo cytotoxicity results of LNP(AL-6)-Luc and LNP(SM-102)-Luc are shown; Figure a shows the line graph of mouse body weight change over time; Figure b shows the line graph of changes in ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels in mice. Detailed Implementation

[0194] Through extensive and in-depth research, the inventors have unexpectedly discovered for the first time an ionizable lipid. This ionizable lipid has the advantages of stable physicochemical properties and low toxicity. The drug delivery system obtained by encapsulating drug payloads (e.g., mRNA) with the ionizable lipid described in this invention has high delivery efficiency and low toxicity. While efficiently delivering drug payloads and increasing the expression level of drug payloads, it also improves the safety of the drug delivery system, making the preventive and therapeutic effects of the drug delivery system more prominent.

[0195] Based on this, the present invention was completed.

[0196] the term

[0197] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0198] The term "alkyl" refers to a saturated carbon chain having 1 to 20 carbon atoms, which may be straight or branched or a combination thereof, unless otherwise defined. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl and tert-butyl, pentyl, hexyl, heptyl, octyl, etc. Alkyl groups may optionally be substituted unless otherwise specified in the specification. The term "unsaturated hydrocarbon group" means that the group contains at least one C=C double bond (alkenyl) or at least one C≡C triple bond (alkynyl). "alkyl," "alkenyl," and "alkynyl" are collectively referred to as "hydrocarbon groups."

[0199] Ionizable lipids

[0200] As used herein, the terms "ionizable lipids of the present invention" and "ionizable cationic lipids of the present invention" are used interchangeably and both refer to lipid compounds having the structure of Formula I, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0201] Ionizable lipids protonate and transform into cationic lipids at low pH values, while at normal physiological pH values ​​they transform into helper phospholipids. Helper phospholipids interact less with the anionic cell membranes of blood cells, improving the biocompatibility of lipid nanoparticles. When lipid nanoparticles are endocytosed by cells, the pH value within the endosomes is low, causing the lipids to protonate and become positively charged. This reduces or even disrupts the membrane structure, facilitating the escape of lipid nanoparticle endosomes. In summary, the pH-sensitive nature of lipids is beneficial for the in vivo delivery of lipid nanoparticles carrying bioactive components (such as mRNA molecules).

[0202] In one aspect of the invention, an ionizable lipid is provided having a structure as shown in Formula I:

[0203]

[0204] In the formula,

[0205] R1 and R2 are each independently selected from -(CH2)n-, where n is a positive integer from 1 to 14;

[0206] X and Y are each independently -CH- or N;

[0207] L1 and L2 are each independently divalent linker groups or none;

[0208] R3, R4, R5, and R6 are each independently H, CH3, C2-C30 hydrocarbon groups (such as C2-C30 alkyl, C2-C30 alkenyl, C2-C30 alkynyl), or -(CH2)sR a -(CH2)gR b -(CH2)m-CH3, where s and g are each independently selected from positive integers from 1 to 20, and m is selected from integers from 0 to 20, preferably s+g+m is 2-35.

[0209] R a R b Each is independently selected from the functional groups with the structure shown below:

[0210]

[0211] in, Represents a connection key;

[0212] Furthermore, R3 and R4 are not both H, and R5 and R6 are not both H.

[0213] In a preferred embodiment of the present invention, the ionizable lipids have substructures shown in formulas (I-1), (I-2), and (I-3), respectively:

[0214]

[0215] When it has the substructure shown in equation (I-1), X and Y in equation (I) are both -CH-; R1 and R2 are each independently selected from -(CH2)n-, where n is a positive integer from 1 to 14; L1 has -(L 1a -L 1b -L 1c The structure is )-, where L 1b The expression is -(CH2)n-, where n is a positive integer selected from 1 to 14; L 1a and L 1cEach of the following functional groups is independently selected: -(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 -(C=O)O-, -O(C=O)-, -(SS-, -CH(OH)-; L2 has -(L 2a -L 2b -L 2c The structure of )-; where L 2b The expression is -(CH2)n-, where n is a positive integer selected from 1 to 14; L 2a and L 2c Each of the following functional groups is independently selected: -(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 -(C=O)O-, -O(C=O)-, -(SS)-, -CH(OH)-; R3, R4, R5 and R6 are each independently C2-C20 hydrocarbon groups.

[0216] When it has the substructure shown in (I-2), X in equation (I) is N, Y is -CH-, L1 is none, L2 is a bivalent linker, and R5 is H; in the equation, R1 and R2 are each independently selected from -(CH2)n-, where n is a positive integer from 1 to 14; L2 has -(L 2a -L 2b -L 2c The structure of )-; where L 2b The expression is -(CH2)n-, where n is a positive integer selected from 1 to 14; L 2a L 2c Each of the following functional groups is independently selected: -(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)-, -CH2-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -(C≡C)-, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-, -CH2-; R3 has -R 3a -R 3b -R3c -R 3d -R 3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure; where R 3a R 3c R 4a and R 4c Each is independently -(CH2)n-, where n is a positive integer selected from 1 to 14; R 3b R 3d R 4b and R 4d Each of the following functional groups is independently selected: -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-R 3e and R 4e Each is independently a C2-C20 hydrocarbon group; R6 is a C2-C20 hydrocarbon group.

[0217] When it has the substructure shown in equation (I-3), X and Y in equation (I) are both N, and L1 and L2 are none; in the equation, R1 and R2 are each independently selected from -(CH2)n-, where n is a positive integer from 1 to 14; R3 has -R 3a -R 3b -R 3c -R 3d -R 3e The structure of R4 has -R 4a -R 4b -R 4c -R 4d -R 4e The structure of R5; R5 has -R 5a -R 5b -R 5c -R 5d -R 5e The structure of R6 has -R 6a -R 6b -R 6c -R 6d -R 6e The structure; where R3a R 3c R 4a R 4c R 5a R 5c R 6a and R 6c Each is independently -(CH2)n-, where n is a positive integer selected from 1 to 14; R 3b R 3d R 4b R 4d R 5b R 5d R 6b and R 6d Each is independently selected from the following functional groups: -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, -(C=C)-(CH2)-(C=C)-, -(C=C)-, -CH2-, -(C≡C)-, preferably -(C=O)O-, -O(C=O)-, -(SS)-, -(C=C)-(CH2)-(C=C)-, -CH(OH)-; R 3e R 4e R 5e R 6e Each is independently a C2-C20 hydrocarbon group.

[0218] In a more preferred embodiment of the invention, the ionizable lipid has a structure selected from those shown in Table 1, wherein AL-6, AL-17 and AL-18 are preferred in Table 1.

[0219] assist lipids

[0220] As used herein, the term "accessory lipid" refers to other types of lipids in lipid nanoparticles besides ionizable lipids, including accessory phospholipids, sterols, polymer-conjugated lipids, or combinations thereof. Accessory lipids are primarily used to improve the properties of lipid nanoparticles, such as stability, delivery efficiency, tolerability, and biocompatibility.

[0221] In some embodiments, the auxiliary phospholipids include (but are not limited to) 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), dioleoyl lecithin (DOPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine. Amines, 1,2-myristoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycerol-3-phospho-rac-(1-glycerol) sodium salt, 1,2-palmitoylphosphatidylglycerol, 1-palmitoyl-2-oleoyllecithin, 1-palmitoyl-2-oleoylphosphatidylethanolamine, distearate phosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, 1-stearoyl-2-oleoylphosphatidylethanolamine, or combinations thereof.

[0222] In a preferred embodiment of the present invention, the auxiliary phospholipid is DSPC (1,2-distearyl-sn-glycerol-3-phosphocholine, also known as distearylphosphatidylcholine). DSPC is a commonly used phosphatidylcholine. The tail group of DSPC is a saturated alkane chain, with a melting point of -54°C and a cylindrical shape. It forms a layered structure in lipid nanoparticles, making the structure of lipid nanoparticles more stable.

[0223] In a preferred embodiment of the present invention, the auxiliary phospholipid is DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, also known as dioleoylphosphatidylethanolamine). DOPE is a commonly used phosphatidylethanolamine. The tail group of DOPE consists of two unsaturated alkane chains, has a melting point of -30°C, and is conical in shape. In lipid nanoparticles, it easily forms an inverted hexagon, causing instability in the endosome membrane and facilitating the escape of lipid nanoparticle endosomes.

[0224] In some embodiments, the sterols include (but are not limited to) cholesterol or cholesterol derivatives. Cholesterol can modulate the integrity and stiffness of lipid membranes, enhancing the stability of lipid nanoparticles, while the morphology of cholesterol derivatives can affect the delivery efficiency and biodistribution of lipid nanoparticles, such as the chain length of the hydrophobic tail group of cholesterol analogs, the flexibility of the sterol ring, and the polarity of the hydroxyl group. Cholesterol also affects the morphology of lipid nanoparticles; cholesterol derivatives result in lipid nanoparticles with a multilayered polyhedral structure defined by lipids rather than a spherical shape. Simultaneously, cholesterol affects the selectivity of lipid nanoparticles for their target sites: lipid nanoparticles containing cholesterol oleate are more selective for hepatic endothelial cells than for hepatocytes; when containing cholesterol with oxidized tail groups, the content of lipid nanoparticles in hepatic endothelial cells and Kupffer cells is higher than that in hepatocytes.

[0225] In some embodiments, the polymer-conjugated lipids are polyethylene glycol (PEG)-conjugated lipids, also known as PEGylated lipids or PEGylated lipids. PEGylated lipids have multiple effects on the properties of lipid nanoparticles: the amount of PEGylated lipids affects the particle size and potential of lipid nanoparticles; it reduces particle aggregation and improves the stability of lipid nanoparticles; it reduces the renal and mononuclear phagocyte system (MPS)-mediated particle clearance rate and prolongs particle circulation time; the surface functional groups can be modified with ligands to improve targeted delivery capability. Molar mass and lipid length affect the properties of PEGylated lipids. DMG-PEG2000 and DSG-PEG2000 are both neutral phospholipids with saturated alkyl chain lengths of C14, C16, or C18, respectively. However, DMG-PEG2000 can separate from lipid nanoparticles more quickly, facilitating cellular uptake of nanoparticles and endosome escape. Therefore, the delivery efficiency of DMG-PEG2000 is superior to that of DSG-PEG2000.

[0226] In a preferred embodiment of the invention, the auxiliary lipid is a combination of DSPC, cholesterol, and DMG-PEG2000.

[0227] Lipid nanoparticles (LNP)

[0228] As used herein, the terms "lipid nanoparticles," "lipid nanoparticles," or "LNP" refer to particles with a diameter of approximately 5 to 500 nm. In some embodiments, the lipid nanoparticles contain one or more active agents (bioactive substances). In some embodiments, the lipid nanoparticles include nucleic acids. In some embodiments, the nucleic acids are condensed within the nanoparticles with cationic lipids, polymers, or multivalent small molecules, and an external lipid coating that interacts with the biological environment. Nucleic acids are naturally rigid polymers and tend to have elongated configurations due to the repulsive forces between phosphate groups. In cells, to cope with volume constraints, DNA can package itself under appropriate solution conditions with the help of ions and other molecules. Typically, DNA condensation is defined as the collapse of an elongated DNA strand into a compact, ordered particle containing only one or a few molecules. By binding to phosphate groups, cationic lipids can concentrate DNA and cause it to pack tightly together by neutralizing the phosphate charge.

[0229] In some embodiments, the bioactive substance is encapsulated in an LNP. In some embodiments, the bioactive substance can be anionic compounds, including but not limited to DNA, RNA (messenger RNA, transfer RNA, ribosomal RNA, microRNA, etc.), natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNA, and small interfering RNA), nucleoproteins, peptides, nucleic acids, ribozymes, DNA-containing nucleoproteins such as intact or partially deproteinized viral particles (viral particles), and oligomeric and polymeric anionic compounds other than DNA (e.g., acidic polysaccharides and glycoproteins). In some embodiments, the bioactive substance can be mixed with an adjuvant.

[0230] In LNP vaccine products, the bioactive substance is typically contained within the LNP itself. In some embodiments, the bioactive substance includes nucleic acids. Typically, water-soluble nucleic acids are condensed within the particle with cationic lipids or polycationic polymers, and the particle surface is enriched with accessory phospholipids or PEG lipid derivatives. Additional ionizable cationic lipids may also be located on the surface; upon entering the lysosome, these ionizable cationic lipids become positively charged due to the acidic environment of the lysosome, interacting with the lysosomal membrane and facilitating endosome escape.

[0231] Regarding LNPs, ionizable lipids can have different properties or functions. Due to the pKa of the amino group, when the external pH is lower than the pKa of the lipid molecule, it can be protonated and become positively charged. Under these conditions, the lipid molecule can electrostatically bind to the phosphate group of nucleic acid, which leads to LNP formation and nucleic acid encapsulation, and the surface charge of LNPs in biological fluids (e.g., blood) at physiological pH is essentially neutral. High LNP surface charge is associated with toxicity, rapid clearance of circulating LNPs by fixed and free macrophages, hemolytic toxicity, including immune activation (Filion et al., Biochim Biophys Acta. 1997 Oct 23; 1329(2):345-56).

[0232] In some embodiments, the pKa can be high enough that ionizable cationic lipids can take a positively charged form at acidic endosomal pH. This allows the cationic lipids to bind to endogenous endosomal anionic lipids to promote membrane lysis of non-bilayer structures, such as the hexagonal HII phase, resulting in more efficient intracellular delivery. In some embodiments, the pKa ranges from 6.2 to 6.5. For example, the pKa can be about 6.2, about 6.3, about 6.4, or about 6.5. Unsaturated tails also contribute to the lipids' ability to take a non-bilayer form. (Jayaraman et al., Angew Chem Int Ed Engl. 20 Aug 2012; 51(34):8529-33).

[0233] The release of nucleic acids in LNP formulations, as well as other characteristics such as liposome clearance and circulating half-life, can be altered by the presence of polyethylene glycol and / or sterols (e.g., cholesterol) or other potential additives in the LNP, and by the overall chemical structure (including the pKa of any ionizable cationic lipids that are part of the formulation).

[0234] In one aspect of the invention, a lipid nanoparticle (LNP) is provided, the lipid nanoparticle comprising the ionizable lipid described in the first aspect of the invention, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. Further, the lipid nanoparticle also comprises one or more auxiliary lipids, including auxiliary phospholipids, steroids, and polymer-conjugated lipids.

[0235] Lipid nanoparticle drug formulation

[0236] In another aspect of the invention, a lipid nanoparticle pharmaceutical formulation (or lipid nanoparticle pharmaceutical combination or LNP composition) is provided, the lipid nanoparticle pharmaceutical formulation comprising lipid nanoparticles as described in the third aspect of the invention, a bioactive substance encapsulated within the lipid nanoparticles, and a pharmaceutically acceptable carrier. The lipid nanoparticle pharmaceutical formulation is used to deliver the bioactive substance to cells in a subject in need.

[0237] In some embodiments, the bioactive substance is encapsulated in an LNP. In some embodiments, the bioactive substance can be anionic compounds, including but not limited to DNA, RNA (messenger RNA, transfer RNA, ribosomal RNA, microRNA, etc.), natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNA, and small interfering RNA), nucleoproteins, peptides, nucleic acids, ribozymes, DNA-containing nucleoproteins such as intact or partially deproteinized viral particles (viral particles), and oligomeric and polymeric anionic compounds other than DNA (e.g., acidic polysaccharides and glycoproteins). In some embodiments, the bioactive substance can be mixed with an adjuvant.

[0238] In some embodiments, the LNP composition comprises: nucleic acid; an ionizable cationic lipid having the structure shown in Formula (I); a cofactor phospholipid (e.g., DSPC, DOPE, DOPC, or combinations thereof); a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol); and a polymer-conjugated lipid (e.g., DMG-PEG2000). In some embodiments, the LNP composition comprises: nucleic acid; an ionizable cationic lipid having the structure shown in Formula I, comprising 30-65% (molar percentage, the same below) of the total lipids of the composition; a cofactor phospholipid (e.g., DSPC, DOPE, DOPC, or combinations thereof) comprising 5-30% of the total lipids of the composition; a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol) comprising 30-55% of the total lipids of the composition; and a polymer-conjugated lipid (e.g., DMG-PEG2000) comprising 1-5% of the total lipids of the composition. Furthermore, in the LNP composition, the molar ratio of ionizable N atoms in the ionizable lipid molecule to phosphate groups in the nucleic acid molecule is (2-10):1, more preferably (4-8):1.

[0239] In a preferred embodiment of the invention, the LNP composition comprises: nucleic acid, an ionizable cationic lipid having the structure shown in formula (I), a cofactor phospholipid (e.g., DSPC, DOPE, DOPC, etc., or combinations thereof), a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol), and a polymer-conjugated lipid (e.g., DMG-PEG2000, etc.). In a more preferred embodiment of the invention, the LNP composition comprises: nucleic acid, an ionizable cationic lipid having the structure shown in formula (I), a cofactor phospholipid (e.g., DSPC, DOPE, DOPC, etc., or combinations thereof), a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol), and a polymer-conjugated lipid (e.g., DMG-PEG2000, etc.).

[0240] As used herein, the terms “encapsulation” and “encapsulated” refer to mRNA, DNA, siRNA, or other nucleic acid drugs being contained within or bound to lipid nanoparticles. As used herein, the term “encapsulation” refers to complete or partial encapsulation. For example, mRNA may be selected to treat and / or prevent associated diseases when administered to a subject in need of a lipid nanoparticle composition comprising mRNA.

[0241] As used herein, the term “pharmaceuticalally acceptable carrier” includes, but is not limited to, any adjuvant, carrier, excipient, scintillation agent, sweetener, diluent, preservative, dye / coloring agent, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the Food and Drug Administration for use in humans or livestock.

[0242] Preparation method of lipid nanoparticle drug formulation

[0243] In another aspect of the present invention, a method for preparing a lipid nanoparticle drug formulation is provided, the method comprising: (a) mixing the ionizable lipid and optionally an auxiliary lipid described in the first aspect of the present invention with an organic solvent to obtain a lipid organic phase; (b) mixing a bioactive substance with an aqueous solvent to obtain an aqueous phase containing the bioactive substance; and (c) mixing the lipid organic phase from step (a) with the aqueous phase from step (b) to obtain the lipid nanoparticle drug. Further, the method further comprises step (d): purifying, concentrating, and sterilizing the lipid nanoparticle drug obtained in step (c).

[0244] In some embodiments, the organic solvent includes (but is not limited to) ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, or tetrahydrofuran, or combinations thereof. In some embodiments, the lipid organic phase includes a small percentage of water or a pH buffer. The lipid organic phase may contain up to 60% by volume of water, for example, up to about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% by volume of water. In one embodiment, the lipid organic phase contains between about 0.05% and 60% by volume of water, for example, between about 0.05% and 50%, between about 0.05% and 40%, or between about 5% and 20% by volume of water.

[0245] In some embodiments, the lipid organic phase comprises a single type of lipid, such as an ionizable cationic lipid, a cofactor phospholipid, a sterol, or a polymer-conjugated lipid. In some embodiments, the lipid organic phase comprises multiple lipids. In one embodiment of the invention, the lipid organic phase comprises an ionizable cationic lipid having the structure shown in Formula (I), a cofactor phospholipid (e.g., DSPC, DOPE, DOPC, or combinations thereof), a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol), and a polymer-conjugated lipid (e.g., DMG-PEG2000). In a preferred embodiment of the invention, the lipid organic phase comprises an ionizable cationic lipid having the structure shown in Formula (I), a cofactor phospholipid (e.g., DSPC, DOPE, DOPC, or combinations thereof), a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol), and a polymer-conjugated lipid (e.g., DMG-PEG2000). In a more preferred embodiment of the invention, the lipid organic phase comprises an ionizable cationic lipid having the structure shown in Formula (I), an accessory phospholipid (e.g., DSPC, DOPE, DOPC, or combinations thereof), a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol), and a polymer-conjugated lipid (e.g., DMG-PEG2000). In a specific embodiment of the invention, the lipid organic phase comprises an ionizable cationic lipid having the structure shown in Formula (I), DSPC, cholesterol, and DMG-PEG2000.

[0246] In some embodiments, the aqueous solvent is water. In some embodiments, the aqueous solvent is an aqueous buffer solution with a pH between 3 and 8 (e.g., pH of about 3, about 4, about 5, or about 6, etc.). A bioactive substance, such as a nucleic acid (e.g., mRNA), is dissolved in the aqueous solvent to obtain an aqueous phase containing the bioactive substance. The aqueous phase may contain a small percentage of a water-miscible organic solvent. The aqueous phase may contain up to 60% by volume of at least one water-miscible organic solvent, such as up to about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or any volume percentage between both of an organic solvent (e.g., a water-miscible organic solvent). In one embodiment, the aqueous phase comprises between about 0.05% and 60% by volume an organic solvent, such as an organic solution (e.g., a water-miscible organic solvent) between about 0.05% and 50%, about 0.05% and 40%, or about 5% and 20% by volume. The aqueous buffer may be a citrate buffer, Tris-HCl buffer, sodium acetate buffer, PBS buffer, or a combination thereof. In some embodiments, the aqueous buffer is a citrate buffer with a pH between 4 and 6 (e.g., a pH of about 4, about 5, or about 6). In one embodiment, the aqueous buffer solution is a citrate buffer with a pH of about 4.

[0247] In some embodiments, a solution comprising a mixture of a lipid organic phase and an aqueous phase containing a bioactive substance, including an LNP suspension, may be diluted. In some embodiments, the pH of the solution comprising the lipid organic phase and the aqueous phase containing the bioactive substance of the LNP suspension may be adjusted. The pH of the LNP suspension may be diluted or adjusted by adding water, acid, base, or an aqueous buffer. In some embodiments, the pH of the LNP suspension is not diluted or adjusted. In some embodiments, the pH of the LNP suspension is diluted and adjusted.

[0248] In some embodiments, excess reagents, solvents, and unencapsulated nucleic acids can be removed from the LNP suspension by tangential flow filtration (TFF) (e.g., percolation). Organic solvents (e.g., ethanol) and buffers can also be removed from the LNP suspension by TFF. In some embodiments, the LNP suspension is dialyzed. In some embodiments, the LNP suspension is subjected to TFF. In some embodiments, the LNP suspension is subjected to both dialyzed and TFF.

[0249] The main advantages of this invention are:

[0250] The ionizable lipids in this invention, along with other components such as helper phospholipids (DSPC, DOPE, DOPC, etc.), sterols (such as cholesterol or cholesterol derivatives), and PEG derivatives (such as DMG-PEG lipids, or DMG-PEG substances modified by other groups / or PEG and other derivatives), can form stable nanoparticles. The nanoparticles encapsulating mRNA have uniform particle size, high encapsulation efficiency, and good stability, which can improve the transfection efficiency of mRNA in targeted tissues or cells, and have low toxicity, thereby making the preventive and therapeutic effects of mRNA vaccines / drugs more prominent.

[0251] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0252] Example 1: Preparation of Ionizable Lipids

[0253] 1.1 Synthetic steps and characterization of compound AL-6:

[0254]

[0255] 1.1.1 Synthesis of 6-bromohexyl-2-hexyldecanoate

[0256]

[0257] In a 1.0 L three-necked flask, 2-hexylundecanoic acid (55 g, 214.483 mmol, 1 equiv), dichloromethane (550 mL), 6-bromo-1-hexanol (46.60 g, 257.380 mmol, 1.2 equiv), N,N-dimethylaminopyridine (2.62 g, 21.448 mmol, 0.1 equiv), and dicyclohexylcarbodiimide (53.11 g, 257.380 mmol, 1.2 equiv) were added. The reaction mixture was stirred at 20 °C for five hours. The reaction mixture was filtered, and the filter cake was washed with dichloromethane (100 mL x 3). The organic phase was concentrated under reduced pressure. The solution was purified by column chromatography, eluted with n-heptane:ethyl acetate (10:1), and concentrated to give 6-bromohexyl-2-hexyldecanoate (66 g, 73.36%) as a pale yellow oil.

[0258] LCMS-PH-AXTER-SDPC-2022-08B-6-3:460[M+CH3CN+1] +

[0259] 1H NMR(400MHz,Chloroform-d)δ4.096(t,J=6.4Hz,2H),3.437(t,J=6.8Hz,2H),2.398-2.277(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).

[0260] 1.1.2 Synthesis of (1R,4R)-2,5-diaza-bicyclo[2.2.1]heptane hydrochloride

[0261]

[0262] In a 50 mL single-necked flask, 1.0 g (5.044 mmol, 1 equiv) of tert-butyl(1R,4R)-2,5-diaza-bicyclo[2.2.1]heptane and 10 mL (4 M) of dioxane hydrochloric acid gas were added, and the reaction was stirred at room temperature for 2–4 hours. The product was concentrated to dryness to give 1.1 g of (1R,4R)-2,5-diaza-bicyclo[2.2.1]heptane hydrochloride, which is a white, solid crude product.

[0263] LCMS-PH-AXTER-SDPC-2022-08B-6-5:99[M+1] +

[0264] 1 H NMR (400MHz, DMSO-d6) δ4.422 (s, 2H), 3.550 (d, J = 13.6Hz, 2H), 3.329-3.318 (m, 2H), 2.064 (s, 2H).

[0265] 1.1.3 Synthesis of the final product

[0266]

[0267] In a 20 mL single-necked flask, (1R,4R)-2,5-diaza-bicyclo[2.2.1]heptane hydrochloride (0.6 g, 4.457 mmol, 1 equiv), acetonitrile (6 mL), N,N-dimethylethylamine (3.46 g, 26.742 mmol, 6.0 equiv), and 6-bromohexyl-2-hexyldecanoate (4.11 g, 9.805 mmol, 2.2 equiv) were added. The reaction mixture was stirred at 70 °C for 12 hours. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (120 mL), washed three times with 30 mL of water each time, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. Purification was performed using a silica gel column chromatography method. The product was obtained when the methanol / ethyl acetate ratio was (5 / 95). The product was concentrated to dryness to give 1.2 g (HPLC: 94.7%) of product. 1.2 g (HPLC: 94.7%) of the product was diluted with n-heptane (120 mL), then washed once each with methanol / water (3 / 1, 36 mL), acetonitrile / water (3 / 1, 36 mL), and water (36 mL). The product was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 980 mg (HPLC: 95.4%) of the product. The 980 mg (HPLC: 95.4%) product was purified using reverse-phase chromatography: Prep-HPLC (IntelFlash-1, Column: C18 silica gel; mobile phase A: water (0.5% TFA), phase B: acetonitrile; 40% to 95% gradient in 25 min, 95% in 5 min; Detector, ELSD). The collected solution was adjusted to pH 8 with saturated sodium bicarbonate aqueous solution and extracted twice with 60 mL of n-heptane. The combined organic phases were washed once with 30 mL of water and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure to obtain the final product (HPLC: 99.3%) 6-[(1R,4R)-5-{6-[(2-hexyldecyl)oxy]hexyl}-2,5-diazabicyclo[2.2.1]heptane-2-yl]hexyl-2-hexyldecanoate, 0.5344 g of which was delivered.

[0268] LCMS-PH-AXTER-SDPC-2022-08B-6-0:775.7[M+1] +

[0269] 1H NMR(300MHz,Chloroform-d)δ4.059(t,J=6.6Hz,4H),3.282(s,2H)2.682(s,4H),2.632-2.513(m,2H),2.501-2.39 2(m,2H),2.380-2.261(m,2H),1.720(s,2H),1.714-1.541(m,8H),1.513-1.196(m,56H),0.876(t,J=7.2Hz,12H).

[0270] The proton spectrum of AL-6 is shown below. Figure 1 The purity characterization diagram is shown below. Figure 2 .

[0271] 1.2 Synthetic steps and characterization of compound AL-17:

[0272]

[0273] 1.2.1 Synthesis of (1R,4R)-2,5-diazabicyclo[2.2.1]heptanyl hydrochloride

[0274]

[0275] At room temperature, tert-butyl(1R,4R)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid ester (9 g, 45.394 mmol, 1 equiv) was dissolved in dioxane hydrochloride solution (90 mL) in a 250 mL three-necked flask, and the mixture was stirred for 2 hours at room temperature. The final reaction solution was directly evaporated under reduced pressure to dryness to give (1R,4R)-2,5-diazabicyclo[2.2.1]heptane dihydrochloride (7.4 g, 95.29%) as a white solid.

[0276] LCMS-PH-AXTER-SDPC-2022-07B-17-1:99[M+H] +

[0277] 1 H NMR (300MHz, DMSO-d6, ppm) δ10.038 (s, 4H), 4.477-4.363 (m, 2H), 3.542 (d, J = 12.391Hz, 2H), 3.269 (dd, J = 12.394, 2.741Hz, 2H), 2.058 (s, 2H).

[0278] 1.2.2 Synthesis of tert-butyl N-{2-[(1R,4R)-5-{2-[(tert-butylcarbonyl)aminoethyl}-2,5-diazabicyclo[2.2.1]heptane-2-yl]ethyl}carbamate

[0279]

[0280] At room temperature, in a 100 mL three-necked flask, (1R,4R)-2,5-diazabicyclo[2.2.1]heptane dihydrochloride (3 g, 17.537 mmol, 1 equivalent), methanol (30 mL), N-Boc-2-diaminoacetaldehyde (8.37 g, 52.611 mmol, 3 equivalent), NaBH(OAc)3 (18.58 g, 87.685 mmol, 5 equivalent), and glacial acetic acid (3.16 g, 52.611 mmol, 3 equivalent) were added sequentially. The reaction mixture was stirred at room temperature for another 10 hours. The reaction was confirmed to be complete by LCMS. The reaction mixture was quenched in saturated sodium carbonate solution (90 mL), extracted with DCM (3 x 100 mL), the organic phases were combined, dried over MgSO4, filtered, evaporated to dryness, and the crude product was purified by silica gel column chromatography (PE / EA (1:1)). tert-butyl N-{2-[(1R,4R)-5-{2-[(tert-butylcarbonyl)aminoethyl}-2,5-diazabicyclo[2.2.1]heptane-2-yl]ethyl}carbamate (3.9 g, 52.11%) was obtained.

[0281] LCMS-PH-AXTER-SDPC-2022-07B-17-2:385[M+H] +

[0282] 1 H NMR (300MHz, Chloroform-d, ppm) δ5.100 (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).

[0283] 1.2.3 Synthesis of 2-[(1R,4R)-5-(2-aminoethyl)-2,5-diazabicyclo[2.2.1]heptane-2-yl]acetamide

[0284]

[0285] At room temperature, tert-butyl N-{2-[(1R,4R)-5-{2-[(tert-butylcarbonyl)aminoethyl}-2,5-diazabicyclo[2.2.1]heptane-2-yl]ethyl}carbamate (3.5 g, 9.102 mmol, 1 equiv) and dioxane hydrochloride solution (35 mL) were added sequentially to a 100 mL single-necked flask. The reaction was allowed to proceed at room temperature for 2 hours, and the reaction was confirmed to be complete by LCMS. The crude product was evaporated to dryness and then purified by ion exchange resin (PL-HCO3 MP SPE 500 mg / 6 mL, 50 / pk, eluted with ACN / H2O (1:4)) to give 2-[(1R,4R)-5-(2-aminoethyl)-2,5-diazabicyclo[2.2.1]heptane-2-yl]acetamide (2 g, crude).

[0286] LCMS-PH-AXTER-SDPC-2022-07B-17-3:185[M+H] +

[0287] 1H NMR (300MHz, DMSO-d6, ppm) δ7.607-7.097 (m, 4H), 3.272 (s, 2H), 2.728 (h, J = 7.149, 6.463Hz, 6H), 2.620 (s, 6H), 1.585 (s, 2H).

[0288] 1.2.4 Synthesis of the final product

[0289]

[0290] At room temperature, 2-[(1R,4R)-5-(2-aminoethyl)-2,5-diazabicyclo[2.2.1]heptane-2-yl]acetamide (800 mg, 4.341 mmol, 1 equiv), i-PrOH (8 mL), and 2-(octyldithio)ethyl acrylate (6000.42 mg, 21.705 mmol, 5 equiv) were added sequentially to a 40 mL reaction flask, and the reaction was carried out at 70 °C for 60 hours. The final reaction system was concentrated under reduced pressure, and the crude product was purified by Prep-HPLC (column, XB-Phenyl gel; mobile phase, i-PrOH in Water (0.1% TFA), 50% to 90% gradient in 20 min; detector, UV 200 nm; flow: 90 mL / min). The received product was rotary evaporated to remove the organic solution. The pH of the residual aqueous phase was adjusted to 9 with sodium carbonate, and the alkaline aqueous phase was extracted with EtOAc (2 x 20 mL). The organic phases were combined, washed once with saturated brine (1 x 20 mL), dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The product was dissolved in n-heptane (20 mL), decolorized with activated carbon (200 mg), filtered, and evaporated to dryness to obtain final product 7 (1.0039 g, 17.32%), which was a yellow oil.

[0291] LCMS-PH-AXTER-SDPC-2022-07B-17-0:1289.7[M+H] +

[0292] 1H NMR(300MHz,Chloroform-d,ppm)δ4.342(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).

[0293] For the proton spectrum of AL-17, please see [link / reference]. Figure 3 For detailed HPLC purity characterization chromatograms, please refer to [link / reference]. Figure 4 .

[0294] 1.3 Synthetic steps and characterization of compound AL-18:

[0295]

[0296] 1.3.1 Synthesis of 3-(acetylsulfonyl)-2-[(acetylsulfonyl)methyl]propionic acid

[0297]

[0298] In a 250 mL round-bottom flask, 20 g of 3-bromo-2-(bromomethyl)propionic acid (81.335 mmol, 1 equiv), 23.22 g of 1-(sulfonyl potassium) acetone (203.337 mmol, 2.5 equiv), and 1 equiv of NaOH (1 M) were added and dissolved in 100 mL of water at room temperature. The resulting mixture was stirred at room temperature for 16 hours. Acidification with 1 M HCl was performed until a white emulsion formed. The mixture was extracted three times with ethyl acetate, and the combined organic layers were washed with brine (pH ≈ 1) and dried over anhydrous Na₂SO₄. The filtrate was filtered and concentrated under reduced pressure to give 3-(acetylsulfonyl)-2-[(acetylsulfonyl)methyl]propionic acid as a yellow oil, which could be used directly in the next reaction without further purification.

[0299] LCMS-PH-AXTER-SDPC-2022-07B-18-3:235[M-1]-

[0300] 1.3.2 Synthesis of 3-sulfonyl-2-(sulfonylmethyl)propionic acid

[0301]

[0302] In a three-necked round-bottom flask, 3-(acetylsulfonyl)-2-[(acetylsulfonyl)methyl]propionic acid (13 g, 55.085 mmol, 1 equiv) and NaOH (1 M, 10 V) were added in batches, maintaining the temperature at 0 °C. The resulting mixture was stirred at room temperature for another 16 hours, then cooled to 0 °C and acidified with HCl (6 M) until a white emulsion was formed. The mixture was extracted three times with ethyl acetate, and the combined organic phases were dried over anhydrous Na₂SO₄. The filtrate was filtered and concentrated under reduced pressure to give 3-sulfonyl-2-(sulfonylmethyl)propionic acid as a yellow oil.

[0303] LCMS-PH-AXTER-SDPC-2022-07B-18-3:151[M-1]-

[0304] 1.3.3 Synthesis of 3-(octetrazyl)-2-[(octetrazyl)methyl]propionic acid

[0305]

[0306] In a round-bottom flask, 2-(octyldithioyl)pyridine (37.2 g, 140.053 mmol, 3 equiv), AcOH (584 mg, 9.737 mmol, 0.2 equiv), and MeOH (37 mL, 5 V) were added dropwise. 3-sulfonyl-2-(sulfonylmethyl)propionic acid (7.4 g, 48.684 mmol, 1 equiv) (diluted with 5 V MeOH) was added dropwise at room temperature. The resulting mixture was stirred at room temperature for 16 hours and then rotary evaporated under reduced pressure. Purification was performed by silica gel column chromatography, eluting with n-heptane / EA (20:1) to give 3-(octyldithioyl)-2-[(octyldithioyl)methyl]propionic acid as a yellow oil.

[0307] LCMS-PH-AXTER-SDPC-2022-07B-18-4:439[M-1]-

[0308] 1H NMR(400MHz,Chloroform-d)δ3.354-3.287(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,4H),1.561-1.270(m,20H),0.922-0.877(m,6H).

[0309] 2.3.4 Synthesis of 4-bromobutyl-3-(octanodithioyl)-2-[(octanodithioyl)methyl]propionate

[0310]

[0311] At room temperature, 3-(octanodithioyl)-2-[(octanodithioyl)methyl]propionic acid (4.7 g, 10.656 mmol, 1 equiv), 4-bromobutane-1-ol (4.9 g, 31.973 mmol, 3 equiv), DMAP (390 mg, 3.197 mmol, 0.3 equiv), and DCM (25 mL, 5V) were added dropwise to the mixture. EDCI (3.1 g, 15.986 mmol, 1.5 equiv) (dissolved in 25 mL of DCM) was added dropwise to the mixture. The resulting mixture was stirred at room temperature for 16 hours. The mixture was filtered, the filter cake was washed with DCM, and the filtrate was concentrated under reduced pressure. Purification was performed by silica gel column chromatography, eluting with n-heptane, to give 4-bromobutyl-3-(octanodithioyl)-2-[(octanodithioyl)methyl]propionic acid ester as a yellow oil.

[0312] 1H NMR(400MHz,Chloroform-d)δ4.198(t,J=6.4Hz,2H),3.464(t,J=6.4Hz,2H),3.278-3.243(m,1H),3.067-2.792(m,4H),2.730 (t,J=7.2Hz,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).

[0313] 1.3.5 Synthesis of 2-(octyldithio)pyridine

[0314]

[0315] 2,2′-Bipyridine disulfide (60 g, 273.972 mmol, 2 equiv) was dissolved in ethanol (100 mL, 5V). HOAc (1.6 g, 27.397 mmol, 0.2 equiv) was added at room temperature, followed by dropwise addition of 1-octylthiol (20 g, 136.986 mmol, 1 equiv) (diluted with 100 mL EtOH) under nitrogen protection. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure and purified by column chromatography, eluting with PE / EA (10:1) to give 2-(octyldithioyl)pyridine as a yellow oil.

[0316] LCMS: 256[M+1]+

[0317] 1H NMR(400MHz,Chloroform-d)δ1H NMR (400MHz, DMSO-d6) δ8.560-8.371(m,1H),7.832(td,J=7.7,1.9Hz,1H),7.764(d,J=8.0Hz,1H),7.277-7.191(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).

[0318] 1.3.6. Synthesis of the final product

[0319]

[0320] In a 50 mL round-bottom flask, 4-bromobutyl-3-(octyldithioyl)-2-[(octyldithioyl)methyl]propionic acid (3.5 g, 6.076 mmol, 3 equiv), (1R,4R)-2,5-diazabicyclo[2.2.1]heptane (269 mg, 2.025 mmol, 1 equiv), and K₂CO₃ (838 mg, 6.076 mmol, 3 equiv) were added, followed by the addition of MeCN (30 mL, 10 V). The mixture was stirred at room temperature for 16 hours. The mixture was extracted three times with ethyl acetate, and the combined organic layers were dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography (HPLC) under the following conditions: column: xb-phenyl gel; mobile phase: i-PrOH / MeCN (1 / 1) and water (with 0.1% TFA added), gradient from 50% to 90% for 20 min; detector: UV 200 nm; flow rate: 90 mL / min). The resulting fraction was concentrated under vacuum to remove the organic solvent and extracted with heptane (2 × 40 mL). The combined organic layers were washed with brine (1 × 40 mL) and dried over anhydrous Na₂SO₄. The filtrate was concentrated under reduced pressure to give the crude product (600 mg, purity 82.8%), which was then prepared again by high-performance preparative chromatography (HPLC) under the following conditions: column: xb-phenyl gel; mobile phase: i-PrOH / MeCN (1 / 1) and water (without additives), gradient from 50% to 90% for 25 min; detector: UV 200 nm; flow rate: 90 mL / min). The resulting solution was concentrated under vacuum to remove the organic solvent. Extracted twice with n-heptane (2 x 40 mL). The combined organic layers were washed with brine (1 x 40 mL) and dried over anhydrous Na₂SO₄. The filtrate was filtered and concentrated under reduced pressure to give the final product 4-[(1R,4R)-5-(4-{[3-(octyldithioyl)-2-[(octyldithioyl)methyl]propyl]oxy}butyl)-2,5-diazabicyclo[2.2.1]heptane-2-yl]butyl3-(octyldithioyl)-2-[(octyldithioyl)methyl]propionate (130 mg, purity 63.4%) as a yellow oil.

[0321] LCMS-PH-AXTER-SDPC-2022-07B-18-3:1087[M+1] +

[0322] 1H NMR(400MHz,Chloroform-d)δ4.197-4.121(m,4H),3.454-3.339(m,2H),3.245(p,J=6.8Hz,2H),3.304-2.944(m,8H) ),2.796-2.481(m,14H),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).

[0323] For the proton spectrum of AL-18, please see [link / reference]. Figure 5 For detailed HPLC purity characterization chromatograms, please refer to [link / reference]. Figure 6 .

[0324] Example 2: mRNA synthesis process, quality control methods, and results

[0325] Preparation of mRNA for delivery:

[0326] Plasmids containing transcription templates were cultured in a shaker at 30°C for 24 hours. The resulting plasmids were then extracted and purified using a plasmid large-scale extraction kit. The purified plasmids were digested with Bsa I and purified using Beads to obtain linearized transcription templates. Concentration was determined using Nanodrop One, and template purity was analyzed using an Agilent Fragment Analyzer. The purity of the transcription template was determined to be 98-100% using the dsDNA915Reagent kit.

[0327] mRNA was prepared using a two-step method. The IVT system was incubated at 37°C for 3 hours. The DNA template was digested with DNase I, purified using RNA Clean Beads, and the concentration was determined using Nanodrop One and adjusted to 1 mg / mL. The RNA for capping was treated at 65°C for 10 minutes, then immediately placed on ice to open the secondary structure. The capping system was incubated at 37°C for 1 hour, purified using RNA Clean Beads, and the concentration was determined using Nanodrop One and adjusted to 1 mg / mL. After aliquoting, the RNA was stored at -20°C.

[0328] The purity of the transcription template was analyzed using the Agilent Fragment Analyzer, and the purity was determined to be 95%-100% using the RNA kit (15nt). The A260 / A280 ratio was approximately 1.9 using Nanodrop One. dsRNA residue was detected at 1.8-2.5 ng / μg using ELISA. DNA residue was detected at 3-30 pg / μg using quantitative real-time PCR.

[0329] The electrophoresis diagram of the transcription template, the peak diagram of the transcription template integrity, the electrophoresis diagram after in vitro transcription reaction (IVT), the peak diagram of IVT RNA integrity, and the peak diagram of mRNA integrity are shown below. Figure 7 , 8 9, 10, and 11.

[0330] Example 3: Assembly of LNP-mRNA using ionizable lipid-encapsulated mRNA.

[0331] Ionizable lipids (SM-102, AL-6, AL-17), DSPC (distearylphosphatidylcholine, 1,2-distearyl-sn-glycerol-3-phosphocholine), cholesterol, and DMG-PEG2000 were mixed thoroughly in an ethanol phase at different ratios to form the organic phase. mRNA was dissolved in sodium citrate solution (pH=4.0) and thoroughly mixed to form the aqueous phase. The aqueous and organic phases were transferred separately to suitable BD syringes, ensuring air bubbles were removed as much as possible. Using a PNI nanoparticle preparation instrument, the mixture was stirred at a volume ratio of 3:1 (aqueous to organic phase) and a flow rate of 12 mL / min. After the samples were allowed to stabilize, they were purified, concentrated, and filtered for sterilization. The resulting product was lipid nanoparticles encapsulated with mRNA (LNP-mRNA). The final product, LNP-mRNA, underwent physicochemical quality control.

[0332] Physicochemical quality control methods and results of LNP-mRNA:

[0333] 1) Particle size and PDI (distribution): The particle size and distribution of LNP were determined using a nanoparticle size analyzer. The results are detailed in [link to relevant documentation]. Figure 12 .

[0334] 2) Encapsulation efficiency (EE%): After staining total mRNA and free mRNA with RiboGreen, the encapsulation efficiency was measured using a microplate reader. The results are detailed in [link to results]. Figure 12 .

[0335] 3) pH: The final product pH was measured to be 7.2-7.4 using a pH meter.

[0336] 4) Osmotic pressure: The osmotic pressure of LNP-mRNA was measured using a freezing point osmoremeter. The osmotic pressure at a concentration of 100 μg / mL was 280-310 mOsmol / kg.

[0337] 5) mRNA integrity: The integrity (i.e., mRNA purity) of the encapsulated mRNA was detected using Agilent Fragment Analyzers, and the purity was greater than 90% when determined using an RNA kit (15nt).

[0338] Example 4: In vitro expression detection of LNP-mRNA

[0339] according to Figure 13 The procedure shown is used to perform cell expression screening experiments on the LNP-mRNA prepared in Example 2. The specific steps are as follows:

[0340] 1) Cell plating: After digestion of 293T cells, the cell density was adjusted to 2×10⁻⁶. 4 / well is seeded into 96-well plates, 100 μL / well, and incubated overnight in a cell culture incubator.

[0341] 2) Experimental grouping: LNP(AL-6)-Luc and LNP(AL-17)-Luc series products were used as sample groups, LNP(SM-102)-Luciferase was used as a positive control group, and only 100 μL of culture medium and cell mixture was added as a negative control group.

[0342] 3) Cell transfection: LNP-Luc does not require transfection. Perform three-fold serial dilutions with 100 ng mRNA per well as the highest concentration, for a total of six dilutions. Set up three replicates for each sample. Add 10 μL of each dilution of LNP-Luciferase evenly to the 96-well cell plate, mix well, and incubate in an incubator.

[0343] 4) Kit detection: 48 hours after transfection, follow the ONE-Glo test results. TM Follow the instructions in the EX Luciferase Assay System kit manual, use a BioTek SYNERGY microplate reader to detect the luminescence intensity of Luciferase, and take the average OD value for comparison.

[0344] The results are as follows Figure 14 As shown, LNP(AL-6)-Luc series products can be normally expressed in cells, and the expression level increases with increasing mRNA concentration. At an mRNA concentration of 1 μg / mL, the expression level in the AL-6 group was significantly higher than that in the positive control group (LNP(SM-102)-Luc). This demonstrates that LNP-mRNA prepared from AL-6 is efficiently expressed in vitro.

[0345] Figure 15 The expression of LNP(AL-6)-Luc series products in 293T cells was shown.

[0346] Example 5: In vitro toxicity detection of LNP-mRNA

[0347] according to Figure 13 The procedure shown is as follows for performing cytotoxicity experiments.

[0348] 1) Cell plating: After digestion of 293T cells, the cell density was adjusted to 2×10⁻⁶.4 / well is seeded into 96-well plates, 100 μL / well, and incubated overnight in a cell culture incubator.

[0349] 2) Experimental grouping: LNP(AL-6)-Luc and LNP(SM-102)-Luc were used as sample groups. Only 100 μL of culture medium was added as a blank control group. Only 100 μL of a mixture of culture medium and cells was added as a negative control group. Staurosporine (STS) apoptosis inducer was added to the mixture of 100 μL of culture medium and cells as a positive control group.

[0350] 3) Cell transfection: LNP-Luc does not require transfection. Perform three-fold serial dilutions (buffer dilutions) at a maximum concentration of 100 ng mRNA per well, for a total of four dilutions. Add 10 μL of each dilution of LNP-Luc evenly to a 96-well cell plate, with three replicates per sample. Mix well and incubate. Add 10 μL of 100 μM STS (diluted with PBS) to the 96-well cell plate, with six replicates. Mix well and incubate.

[0351] 4) Kit detection: Incubate for 48 hours, then... Following the instructions for the Luminescent Cell V(I)bilityAssay kit, the luminescence intensity was measured using a BioTek SYNERGY microplate reader, and the average OD values ​​were compared. The cell inhibition rate was calculated using the formula "(negative control group - sample group / positive control group) ÷ (negative control group - blank control group) × 100%", and a dose-response-inhibition curve was plotted.

[0352] The results are as follows Figure 16 As shown, the series of LNP-mRNA products formed by AL-6 had no significant effect on cell proliferation at concentrations less than 1 μg / mL, indicating that there was no obvious toxicity within the detection concentration range.

[0353] Example 6: In vivo expression detection of LNP-mRNA

[0354] To elucidate the in vivo expression capacity and toxicity of LNP-mRNA assembled using AL-6, an hEPO ELISA expression detection assay and a toxicity assay were designed. The expression level and toxicity were measured in mice with intact innate immunity, as follows:

[0355] Experimental procedure (refer to) Figure 17 :

[0356] 1) Encapsulation of LNP-mRNA: hEPO mRNA was dissolved in an aqueous buffer and mixed thoroughly to form the aqueous phase. Ionizable lipids (SM-102 or AL-6), DSPC, cholesterol, and DMG-PEG2000 were dissolved in anhydrous ethanol and mixed thoroughly in a certain ratio to form the organic phase. The aqueous and organic phases were transferred to syringes separately, and hEPO-LNP was prepared using a PNI microfluidic nanoparticle preparation instrument with parameters set (aqueous to organic phase volume ratio of 3:1, flow rate of 12 mL / min). LNP-hEPO was concentrated, purified, sterilized, filtered, and quality-controlled before being injected into mice.

[0357] 2) Mouse tail vein injection: Mice were immobilized, and the tail vein was selected for injection. The injection dose of LNP-hEPO was 5 μg. LNP(AL-6)-hEPO was set as the sample group, LNP(SM-102)-hEPO was set as the positive control group, and those injected with only the solvent were set as the negative control group. Two mice were injected with each LNP-hEPO sample.

[0358] 3) Submandibular blood collection in mice: Blood was collected from the submandibular region at 6 h, 24 h, and 48 h after injection. The blood was collected in EDTA anticoagulant tubes, gently mixed, and labeled for later use.

[0359] 4) Serum extraction: After obtaining whole blood, centrifuge at 2000g for 10 minutes, and the supernatant is plasma. Aliquot and store at -80℃.

[0360] 5) Detect hEPO expression using a human Erythropoietin ELISA kit: Follow the instructions in the manual, setting up two replicates for each serum sample. Measure the OD value using a microplate reader, and calculate the expression level based on the standard curve and dilution ratio.

[0361] The results are as follows Figure 18 As shown, the LNP-hEPO series products prepared from AL-6 can be successfully expressed in mice, and the overall expression level (AUC) is higher than that of the positive control (SM-102). This experiment shows that LNPs formed by AL-6 can efficiently mediate the expression of mRNA in vivo.

[0362] Example 7: In vivo toxicity detection of LNP-mRNA

[0363] In vivo toxicity testing procedure (refer to) Figure 17 The specific steps are as follows:

[0364] 1) Encapsulation of LNP-mRNA: Luciferase mRNA was dissolved in an aqueous buffer and mixed thoroughly to form the aqueous phase; ionizable lipids (SM-102 or AL-6), DSPC, cholesterol, and DMG-PEG2000 were dissolved in anhydrous ethanol and mixed thoroughly in a specific ratio to form the organic phase. The aqueous and organic phases were transferred separately to syringes, and LNP-Luciferase was prepared using a PNI microfluidic nanomaterial preparation system. LNP-Luciferase was concentrated, purified, sterilized, filtered, and quality-controlled before being injected into mice.

[0365] 2) Mouse tail vein injection: Mice were immobilized, and the tail vein was selected for injection. The injection doses of LNP-Luciferase were 5, 15, and 30 μg. LNP(AL-6)-Luc was designated as the sample group, LNP(SM-102)-Luc as the positive control group (30 μg), and mice injected with only the solvent were designated as the negative control group. Two mice were injected with each LNP-Luciferase sample.

[0366] 3) Mouse weighing: The mice were weighed daily and the weight data was recorded for 7 consecutive days. The weight gain or loss was calculated as a percentage.

[0367] 4) Submandibular blood collection in mice: Blood was collected from the submandibular region before injection (0h) and at 24h and 96h after injection. The blood was collected in EDTA anticoagulant tubes, gently mixed, and labeled for later use.

[0368] 5) Serum extraction: After obtaining whole blood, centrifuge at 2000g for 10 minutes, and the supernatant is plasma. Aliquot and store at -80℃.

[0369] 6) Biochemical tests: The levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) were measured using the IUBIO iChem340 blood biochemistry analyzer. The concentration values ​​were statistically analyzed, the average values ​​were calculated, and the differences between groups were compared and curves were plotted.

[0370] The results are as follows Figure 19 As shown in the results of the mouse toxicity experiment, the mice remained relatively healthy after injection of different doses of LNP(AL-6)-Luc, with no deaths observed. The mice experienced a significant decrease in body weight after injection, with the degree of decrease showing a clear correlation with the injection dose. The weight gradually returned to normal after two days, indicating that the effect of AL-6 on mouse body weight is reversible.

[0371] One day after injection, ALT and AST levels in mice increased, and the degree of increase was significantly correlated with the injection dose. Four days later, liver enzymes returned to pre-injection levels, indicating that the effect of AL-6 on liver function is reversible.

[0372] In this experiment, a high dose of LNP(SM-102)-Luc was simultaneously injected, and its effects on mouse body weight and ALT / AST levels were comparable to those of an equivalent dose of AL-6. Within the experimental system of this invention, the effects of the high-dose LNP(AL-6)-Luc group and LNP(SM-102)-Luc on mice were not significantly different, indirectly indicating that AL-6 and SM-102 have comparable toxicity.

[0373] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An ionizable lipid or a pharmaceutically acceptable salt thereof, characterized in that, The ionizable lipid has the following structure:

2. The ionizable lipid or a pharmaceutically acceptable salt thereof as described in claim 1, characterized in that, The ionizable lipid has the following structure: or 。 3. A lipid nanoparticle, characterized in that, The lipid nanoparticles comprise the ionizable lipids as described in claim 1 or 2, or a pharmaceutically acceptable salt thereof.

4. The lipid nanoparticles as described in claim 3, characterized in that, The lipid nanoparticles comprise the ionizable lipids, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of ionizable lipids:DSPC:cholesterol:DMG-PEG2000 is (30-65):(5-30):(30-55):(1-5).

5. A lipid nanoparticle drug formulation, characterized in that, The lipid nanoparticle pharmaceutical formulation comprises the lipid nanoparticles as described in claim 3 or 4, a bioactive substance encapsulated in the lipid nanoparticles, and a pharmaceutically acceptable carrier.

6. A method for preparing a lipid nanoparticle drug formulation as described in claim 5, characterized in that, The method includes: (a) The ionizable lipid as described in claim 1 or 2 or a pharmaceutically acceptable salt thereof and optionally an auxiliary lipid are mixed with an organic solvent to obtain a lipid organic phase; (b) The bioactive substance is mixed with an aqueous solvent to obtain an aqueous phase containing the bioactive substance; (c) The lipid organic phase from step (a) is mixed with the aqueous phase from step (b) to obtain the lipid nanoparticle drug formulation.

7. Use of an ionizable lipid as described in claim 1 or 2, or a pharmaceutically acceptable salt thereof, for the preparation of a drug delivery system.

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