Multi-charge ionizable lipid compound and lipid composition thereof

Abstract

The invention discloses a multi-charge ionizable lipid compound and a lipid composition thereof, the multi-charge ionizable lipid compound is a compound as shown in a general formula I, or a pharmaceutically available salt thereof, in the structural general formula I, in the general formula I, in the general formula I, in the general formula I, in the general formula I, in the general formula I, in the general formula I, in the general formula I, in the general formula I; g1, G2, G3 and G4 are respectively and independently selected from C1-C12 straight chain or branched chain alkyl which is substituted or unsubstituted by hydroxyl, C1-C12 straight chain or branched chain alkenyl which is substituted or unsubstituted by hydroxyl, C1-C12 straight chain or branched chain alkylene or alkenylene which is substituted or unsubstituted by hydroxyl, and C1-C12 straight chain or branched chain alkyl or alkylene which contains an ether bond; the multi-charge ionizable lipid compound and the lipid composition thereof provided by the invention can deliver nucleic acid molecules, small molecule compounds, polypeptides or proteins and the like, and a carrier prepared by using the ionizable lipid compound provided by the invention has high encapsulation efficiency on the nucleic acid molecules, and can successfully transfer the nucleic acid molecules into cells and express the nucleic acid molecules.

Description

Technical Field

[0001] The present invention relates to the technical field of multi-charged ionizable lipid compounds, in particular to a multi-charged ionizable lipid compound and a lipid composition thereof. Background Art

[0002] Gene therapy refers to the introduction of exogenous normal genes into target cells to correct or compensate for diseases caused by gene defects and abnormalities to achieve the purpose of treatment. That is, through gene transfer technology, exogenous genes are inserted into appropriate receptor cells of patients so that the products produced by exogenous genes can treat certain diseases. As a new method of disease treatment combining modern medicine and molecular biology, gene therapy has gradually occupied an important position in the medical field. At present, the treatment of "expressing a certain gene" and "expressing a certain protein" is mainly achieved through the introduction of plasmid DNA and mRNA, and the treatment of "inhibiting a certain gene" is mainly achieved through siRNA or microRNA (microRNA, miRNA) (i.e. RNAi technology).

[0003] Genes have great therapeutic potential in regulating protein expression in vivo, but the efficiency of naked genes being internalized by cells is extremely low, and they are easily cleared by the kidneys or rapidly degraded by enzymes in the body during delivery in vivo, which greatly reduces their actual therapeutic effect. Since the first clinical studies on therapeutic genes began in the 2000s, the design of gene molecules and the research on their delivery methods have made significant progress. However, gene therapy still faces several challenges, including low cell permeability and high sensitivity to degradation of certain nucleic acid molecules (including RNA). Therefore, it is necessary to develop more lipid compounds and compositions capable of delivering genes and related preparation methods to promote the delivery of various nucleic acid molecules outside or inside cells for treatment and / or prevention purposes. Summary of the invention

[0004] The purpose of the present invention is to provide a multi-charged ionizable lipid compound and a lipid composition thereof to solve the problems raised by the above-mentioned background technology. The technical solution of the present invention provides a solution that is significantly different from the prior art in order to solve the technical problem that the prior art solution is too single.

[0005] To achieve the above object, the present invention provides the following technical solution: a multi-charged ionizable lipid compound, wherein the multi-charged ionizable lipid compound is a compound described in general formula I, or a pharmaceutically acceptable salt thereof; Formula I In the general structural formula I, G1, G2, G3 and G4 are each independently selected from C1-C 12 Straight chain or branched alkyl, hydroxy substituted or unsubstituted C1-C12 Straight-chain or branched alkenyl, hydroxy-substituted or unsubstituted C1-C 12 Straight or branched chain alkylene or alkenylene and C1-C 12 A straight or branched alkyl or alkylene group; Said m and n are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Said A1 and A2 are absent or are each independently selected from -N(T7)-, -NH-, -NH-CO-, -CO-NH-, -NHC(O)O-, -OC(O)N(H)-, -C(O)N(T7)-, -N(T7)C(O)-, -ON=C(T7)-, -C(T7)=NO-, -OC(O)N(T7)-, -N(T7)C(O)N(T7)-, -N(T7)C(O)O- or -C(T7)=NOC(O)-; Said T7 is absent or each occurrence is independently selected from H, alkyl or cycloalkane, nitrogen-containing or non-nitrogen-containing heterocycle; When A1 and A2 are tertiary amine groups, T7 in -N(T7)- is T4 and / or T5, when A1 and A2 do not exist or are each independently selected from -NH-, -NH-CO-, -CO-NH-, -NHC(O)O-, -OC(O)N(H)-, -C(O)N(T7)-, -N(T7)C(O)-, -ON=C(T7)-, -C(T7)=NO-, -OC(O)N(T7)-, -N(T7)C(O)N(T7)-, -N(T7)C(O)O- or -C(T7)=NOC(O)-, T4 and T5 do not exist; when T4 and T5 do not exist, the dotted lines of T4 and T5 do not exist either; Said T1, T2, T3, T4, T5 and T6 do not exist or are independently selected from H or independently selected from compounds conforming to the chemical structure of general formula II: General formula Ⅱ The L1, L2 and L3 do not exist or are independently selected from H or independently selected from compounds conforming to the chemical structure of the general formula III: General formula III.

[0006] Preferably, G5 and G6 in the general structural formula II are absent or independently selected from C1-C 20 Straight chain or branched alkyl, hydroxy substituted or unsubstituted C1-C 20 Straight-chain or branched alkenyl, hydroxy-substituted or unsubstituted C1-C 20 Straight or branched chain alkylene or alkenylene; Said X1 is absent or selected from -O-, -NH-, -N(R1)-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R1)-, -N(R1)C(O)-, -SS-, -OC(O)O-, -ON=C(R1)-, -C(R1)=NO-, -OC(O)N(R1)-, -N(R1)C(O)N(R1)-, -N(R1)C(O)O-, -C(O)S-, -C(S)O- or -C(R1)=NOC(O)-; R1 is absent or each occurrence is independently selected from H, alkyl, nitrogen-containing or non-nitrogen-containing heterocycle; When X1 is a tertiary amine group, R1 in -N (R1)- is L3; When When C(R1)=NO-, -OC(O)N(R1)-, -N(R1)C(O)N(R1)-, -N(R1)C(O)O-, -C(O)S-, -C(S)O- or -C(R1)=NOC(O)-, L3 does not exist; When L3 does not exist, the dotted line of L3 does not exist either; The D is absent or selected from A1 or A2, -O-, -N(R2)-, -NH-, -NH-CO-, -CO-NH-, -NHC(O)O-, -OC(O)N(H)-, -C(O)N(R2)-, -N(R2)C(O)-, -ON=C(R2)-, -C(R2)=NO-, -OC(O)N(R2)-, -N(R2)C(O)N(R2)-, -N(R2)C(O)O- or -C(R2)=NOC(O)-; R2 is absent or each occurrence is independently selected from H, alkyl, nitrogen-containing or non-nitrogen-containing heterocycle; When D is A1 or A2, L1 and L2 are independently T3 and T4; When D is a tertiary amine group, R2 in -N (R2)- is L2; When D is -NH-, -NH-CO-, -CO-NH-, -NHC(O)O-, -OC(O)N(H)-, -C(O)N(R2)-, -N(R2)C(O)-, -ON=C(R2)-, -C(R2)=NO-, -OC(O)N(R2)-, -N(R2)C(O)N(R2)-, -N(R2)C(O)O-, or -C(R2)=NOC(O)-, L2 is absent; When L2 does not exist, the dotted line of L2 does not exist either.

[0007] Preferably, the G7 is absent or independently selected from hydroxy-substituted or unsubstituted C6-C 18 Straight chain alkyl, hydroxy substituted or unsubstituted C6-C 26 Branched alkyl, hydroxy substituted or unsubstituted C4-C 22 Straight-chain or branched alkenyl, hydroxy-substituted or unsubstituted C2-C 22 Straight or branched chain alkylene or alkenylene; Said X2 is absent or independently selected from -O-, -NH-, -N(R4)-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N((R4)-, -N(R4)C(O)-, -SS-, -OC(O)O-, -ON=C((R4)-, -C(R4)=NO-, -OC(O)N((R4)-, -N(R4)C(O)N((R4)-, -N(R4)C(O)O-, -C(O)S-, -C(S)O- or -C(R4)=NOC(O)-; R4 is absent or each occurrence is independently selected from H, alkyl, nitrogen-containing or non-nitrogen-containing heterocycle; When X2 does not exist, then R3 does not exist either; The R3 is independently selected from C6-C 14 Straight-chain or branched alkyl or alkenyl.

[0008] Preferably, A1 and A2 are independently selected from -N(T7)-, -NH-, -NH-CO-, -CO-NH-, -NHC(O)O-, -OC(O)N(H)-, G2 and G3 are independently selected from hydroxyl-substituted or unsubstituted C1-C4 straight chain or branched alkyl or alkylene, and G1 and G4 are independently selected from hydroxyl-substituted or unsubstituted C1-C6 straight chain or branched alkyl or alkylene or C1-C4 straight chain or branched alkyl or alkylene containing an ether bond.

[0009] Preferably, the general formula I, general formula II and general formula III contain multiple protonatable tertiary amine group structures, including but not limited to , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

[0010] Preferably, when X 2和 When R3 does not exist, G7 includes but is not limited to , , , , , , , , , , , , , , , , .

[0011] Preferably, when said R3 exists, R3 includes but is not limited to , , , , , , , , , , , , , , , , .

[0012] Preferably, when X2 and / or R3 exist, G7 includes but is not limited to , , , , , , , , , , , , .

[0013] Preferably, the ionizable lipid compound includes but is not limited to one or more of the following compounds: , , , , , , .

[0014] Preferably, the multi-charged ionizable lipid compound has the following general structural formula: Formula IV In the general structural formula IV, p and q are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8; said t is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; W is a head group containing multiple protonatable tertiary amine groups; The X3 does not exist or is part of the chemical bond formed by connecting to the W head group; The R 10 Independently selected from C5-C 15 Straight or branched alkyl, C5-C 15 Straight chain or branched alkenyl or C5-C 15 straight chain or branched chain alkynyl; X3, p, q and R in the different side chains connected to the W head group in the general structure IV 10 Same or different.

[0015] Preferably, the head group is , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

[0016] Preferably, p is 3 to 6.

[0017] Preferably, q is 0 to 4.

[0018] Preferably, the multi-charged ionizable lipid compound has the following general structural formula: Formula V In the general structural formula V, p is 0, 1, 2, 3, 4, 5, 6, 7 or 8; said t is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; W is a head group containing multiple protonatable tertiary amine groups; Said X3 is absent or is part of a chemical bond formed with the W head group; The R 100 Independently selected from C5-C 15 Straight or branched alkyl, C5-C15 Straight chain or branched alkenyl or C5-C 15 straight chain or branched chain alkynyl; In the general structural formula V, X3, p and R in different side chains connected to the W head group 100 Same or different.

[0019] Preferably, the head group is , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

[0020] Preferably, p is 0 to 6.

[0021] Preferably, the multi-charged ionizable lipid compound has the following general structural formula: Formula VI In the general structural formula VI, p is 0, 1, 2, 3, 4, 5, 6, 7 or 8; Said t or z is each independently 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; W is a head group containing multiple protonatable tertiary amine groups; Said X3 is absent or is part of a chemical bond formed with the W head group; The R 100 Independently selected from C5-C 15 Straight or branched alkyl, C5-C 15 Straight chain or branched alkenyl or C5-C 15 straight chain or branched chain alkynyl; The R 200 Independently selected from C 12 -C 24 Straight or branched alkyl, C 12 -C 24 Straight or branched alkenyl or C 12 -C 24 straight chain or branched chain alkynyl; In the general structure VI shown, X3, p, R 100 and R 200 Same or different.

[0022] Preferably, the head group is , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

[0023] Preferably, p is 0 to 6.

[0024] A lipid composition, comprising an active agent and a carrier for delivering the active agent; the carrier comprises an ionizable lipid compound; the ionizable lipid compound comprises one or more of the ionizable lipid compounds represented by structural formula I, structural formula II, structural formula III, structural formula IV, structural formula V and structural formula VI or their pharmaceutically acceptable salts.

[0025] Preferably, the lipid composition comprises a neutral lipid, a PEG lipid that reduces aggregation, a sterol and an ionizable lipid compound, wherein the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC) or 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), and the sterol includes one or more of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and corticosteroids.

[0026] Preferably, the molar ratio of the ionizable lipid is 20% to 60%, the molar ratio of the neutral lipid is 5% to 25%, the molar ratio of the sterol is 25% to 55%, and the molar ratio of the PEG lipid is 0.5% to 15%.

[0027] Preferably, the active agent includes one or more of a nucleic acid molecule, a small molecule compound, a polypeptide and a protein, wherein the nucleic acid molecule includes but is not limited to single-stranded DNA, double-stranded DNA, short isomers, agomir, antagomir, antisense molecules, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), locked nucleic acid (LNA), peptide nucleic acid (PNA) or morpholino oligonucleotide.

[0028] Preferably, the mass ratio of the carrier to the active agent is 5:1-50:1.

[0029] Preferably, the lipid composition is used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

[0030] Compared with the prior art, the present invention has the following beneficial effects: The multi-charged ionizable lipid compound and lipid composition provided by the present invention, compared with the single-charged lipid, when the mass ratio of the ionizable lipid to the nucleic acid drug is the same, the multi-charged lipid generally has a higher N / P ratio than the single-charged lipid, is more conducive to the encapsulation, cellular uptake and lysosomal escape of the nucleic acid, and can improve the efficiency of nucleic acid delivery; The carrier prepared by using the ionizable lipid compound of the present invention has high encapsulation efficiency for nucleic acid molecules, can successfully transport nucleic acid molecules into cells and express them, has strong stability, is conducive to the release of active agents into cells, and increases the expression amount; The multi-charged ionizable lipids of the present invention can achieve long-term expression of nucleic acid drugs in vivo, significantly reduce the frequency of drug use while ensuring the effect, and the synthesis method is diverse and simple, which broadens the types and ranges of ionizable lipid compounds to promote the extracellular or intracellular delivery of various nucleic acid molecules for treatment and / or prevention, which is of great significance to the development and application of nucleic acid therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a transmission electron microscopy image of nanolipid particles; Figure 2 Schematic diagram of the stability test of nanolipid particles within 20 hours; Figure 3 Schematic diagram of the toxic feedback of nanolipid particles on ATP7B knockout hepatocytes; Figure 4 Schematic diagram of the feedback effect of nanolipid particles on the proliferation of ATP7B knockout hepatocytes. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] See also Figure 1-Figure 4 , the present invention provides a technical solution: a multi-charged ionizable lipid compound and a lipid composition thereof, the multi-charged ionizable lipid compound is a compound of general formula I, or a pharmaceutically acceptable salt thereof; Formula I In the general structural formula I, G1, G2, G3 and G4 are each independently selected from hydroxy-substituted or unsubstituted C1-C 12 Straight chain or branched alkyl, hydroxy substituted or unsubstituted C1-C 12 Straight-chain or branched alkenyl, hydroxy-substituted or unsubstituted C1-C 12 Straight or branched chain alkylene or alkenylene and C1-C 12 A straight or branched alkyl or alkylene group; m and n are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; A1 and A2 are absent or are each independently selected from -N(T7)-, -NH-, -NH-CO-, -CO-NH-, -NHC(O)O-, -OC(O)N(H)-, -C(O)N(T7)-, -N(T7)C(O)-, -ON=C(T7)-, -C(T7)=NO-, -OC(O)N(T7)-, -N(T7)C(O)N(T7)-, -N(T7)C(O)O- or -C(T7)=NOC(O)-; T7 is absent or each occurrence is independently selected from H, alkyl or cycloalkane, nitrogen-containing or non-nitrogen-containing heterocycle; When A1 and A2 are tertiary amine groups, T7 in -N(T7)- is T4 and / or T5, when A1 and A2 do not exist or are each independently selected from -NH-, -NH-CO-, -CO-NH-, -NHC(O)O-, -OC(O)N(H)-, -C(O)N(T7)-, -N(T7)C(O)-, -ON=C(T7)-, -C(T7)=NO-, -OC(O)N(T7)-, -N(T7)C(O)N(T7)-, -N(T7)C(O)O- or -C(T7)=NOC(O)-, T4 and T5 do not exist; when T4 and T5 do not exist, the dotted lines of T4 and T5 do not exist either; T1, T2, T3, T4, T5 and T6 are absent or are independently selected from H or independently selected from compounds conforming to the chemical structure of general formula II: General formula Ⅱ L1, L2 and L3 do not exist or are independently selected from H or independently selected from compounds conforming to the chemical structure of general formula III: General formula III.

[0034] In the general structural formula II, G5 and G6 are absent or independently selected from C1-C 20 Straight chain or branched alkyl, hydroxy substituted or unsubstituted C1-C 20 Straight-chain or branched alkenyl, hydroxy-substituted or unsubstituted C1-C 20 Straight or branched chain alkylene or alkenylene; X1 is absent or selected from -O-, -NH-, -N(R1)-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R1)-, -N(R1)C(O)-, -SS-, -OC(O)O-, -ON =C(R1)-, -C(R1)=NO-, -OC(O)N(R1)-, -N(R1)C(O)N(R1)-, -N(R1)C(O)O-, -C(O)S-, -C(S)O- or -C(R1)=NOC(O)-; R1 is absent or each occurrence is independently selected from H, alkyl, nitrogen-containing or non-nitrogen-containing heterocycle; When X1 is a tertiary amine group, R1 in -N (R1)- is L3; When When C(R1)=NO-, -OC(O)N(R1)-, -N(R1)C(O)N(R1)-, -N(R1)C(O)O-, -C(O)S-, -C(S)O- or -C(R1)=NOC(O)-, L3 does not exist; When L3 does not exist, the dotted line of L3 does not exist either; D is absent or selected from A1 or A2, -O-, -N(R2)-, -NH-, -NH-CO-, -CO-NH-, -NHC(O)O-, -OC(O)N(H)-, -C(O)N(R2)-, -N(R2)C(O)-, -ON=C(R2)-, -C(R2)=NO-, -OC(O)N(R2)-, -N(R2)C(O)N(R2)-, -N(R2)C(O)O- or -C(R2)=NOC(O)-; R2 is absent or each occurrence is independently selected from H, alkyl, nitrogen-containing or non-nitrogen-containing heterocycle; When D is A1 or A2, L1 and L2 are independently T3 and T4; When D is a tertiary amine group, R2 in -N (R2)- is L2; When D is -NH-, -NH-CO-, -CO-NH-, -NHC(O)O-, -OC(O)N(H)-, -C(O)N(R2)-, -N(R2)C(O)-, -ON=C(R2)-, -C(R2)=NO-, -OC(O)N(R2)-, -N(R2)C(O)N(R2)-, -N(R2)C(O)O-, or -C(R2)=NOC(O)-, L2 is absent; When L2 does not exist, the dotted line of L2 does not exist either.

[0035] G7 is absent or independently selected from hydroxy-substituted or unsubstituted C6-C 18 Straight chain alkyl, hydroxy substituted or unsubstituted C6-C 26 Branched alkyl, hydroxy substituted or unsubstituted C4-C 22 Straight-chain or branched alkenyl, hydroxy-substituted or unsubstituted C2-C 22 Straight or branched chain alkylene or alkenylene; X2 is absent or independently selected from -O-, -NH-, -N(R4)-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N((R4)-, -N(R4)C(O)-, -SS-, -OC(O)O-, -ON=C((R4)-, -C(R4)=NO-, -OC(O)N((R4)-, -N(R4)C(O)N((R4)-, -N(R4)C(O)O-, -C(O)S-, -C(S)O- or -C(R4)=NOC(O)-; R4 is absent or each occurrence is independently selected from H, alkyl, nitrogen-containing or non-nitrogen-containing heterocycle; When X2 does not exist, then R3 does not exist either; R3 is independently selected from C6-C 14Straight-chain or branched alkyl or alkenyl.

[0036] A1 and A2 are independently selected from -N(T7)-, -NH-, -NH-CO-, -CO-NH-, -NHC(O)O-, -OC(O)N(H)-, G2 and G3 are independently selected from hydroxy-substituted or unsubstituted C1-C4 straight chain or branched alkyl or alkylene, G1 and G4 are independently selected from hydroxy-substituted or unsubstituted C1-C6 straight chain or branched alkyl or alkylene or C1-C4 straight chain or branched alkyl or alkylene containing ether bonds.

[0037] The general structures I, II and III contain multiple protonatable tertiary amine group structures, including but not limited to , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

[0038] When X 2和 When R3 does not exist, G7 includes but is not limited to , , , , , , , , , , , , , , , , .

[0039] When R3 exists, R3 includes but is not limited to , , , , , , , , , , , , , , , , .

[0040] When X2 and / or R3 are present, G7 includes but is not limited to , , , , , , , , , , , , .

[0041] Ionizable lipid compounds include, but are not limited to, one or more of the following compounds: , , , , , , .

[0042] The multi-charged ionizable lipid compound has the following general structural formula: Formula IV In the general structural formula IV, p and q are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8; t is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; W is a head group containing multiple protonatable tertiary amine groups; X3 is absent or is part of the chemical bond formed with the W head group; R 10 Independently selected from C5-C 15 Straight or branched alkyl, C5-C 15 Straight chain or branched alkenyl or C5-C 15 straight chain or branched chain alkynyl; X3, p, q and R in the different side chains connected to the W head group in the general structure IV 10 Same or different.

[0043] The head base is , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

[0044] p is 3 to 6.

[0045] q is 0 to 4.

[0046] The multi-charged ionizable lipid compound has the following general structural formula: Formula V In the general structural formula V, p is 0, 1, 2, 3, 4, 5, 6, 7 or 8; t is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; W is a head group containing multiple protonatable tertiary amine groups; X3 is absent or is part of a chemical bond formed with the W head group; R 100 Independently selected from C5-C 15 Straight or branched alkyl, C5-C 15 Straight chain or branched alkenyl or C5-C 15 straight chain or branched chain alkynyl; X3, p and R in different side chains connected to the W head group in the general structure V 100 Same or different.

[0047] The head base is , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

[0048] p is 0 to 6.

[0049] The multi-charged ionizable lipid compound has the following general structural formula: Formula VI In the general structural formula VI, p is 0, 1, 2, 3, 4, 5, 6, 7 or 8; t or z are each independently 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; W is a head group containing multiple protonatable tertiary amine groups; X3 is absent or is part of a chemical bond formed with the W head group; R 100 Independently selected from C5-C 15 Straight or branched alkyl, C5-C 15 Straight chain or branched alkenyl or C5-C 15 straight chain or branched chain alkynyl; R 200 Independently selected from C 12 -C 24 Straight or branched alkyl, C 12 -C 24 Straight or branched alkenyl or C 12 -C 24 straight chain or branched chain alkynyl; In the general structure VI shown, X3, p, R 100 and R 200 Same or different.

[0050] The head base is , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

[0051] p is 0 to 6.

[0052] The lipid composition includes an active agent and a carrier for delivering the active agent; the carrier includes an ionizable lipid compound; the ionizable lipid compound includes one or more of the ionizable lipid compounds shown in structural formula I, structural formula II, structural formula III, structural formula IV, structural formula V and structural formula VI or their pharmaceutically acceptable salts.

[0053] The lipid composition comprises a neutral lipid, an aggregation-reducing PEG lipid, a sterol, and an ionizable lipid compound, wherein the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC) or 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), and sterols include one or more of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and corticosteroids.

[0054] The molar ratio of ionizable lipids is 20% to 60%, the molar ratio of neutral lipids is 5% to 25%, the molar ratio of sterols is 25% to 55%, and the molar ratio of PEG lipids is 0.5% to 15%.

[0055] The active agent includes one or more of a nucleic acid molecule, a small molecule compound, a polypeptide and a protein. The nucleic acid molecule includes but is not limited to single-stranded DNA, double-stranded DNA, short isomers, agomir, antagomir, antisense molecules, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), locked nucleic acid (LNA), peptide nucleic acid (PNA) or morpholino oligonucleotide.

[0056] The mass ratio of the carrier to the active agent is 5:1-50:1.

[0057] The lipid composition is used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs. The small molecule compounds include but are not limited to effective ingredients of therapeutic and / or preventive agents. The therapeutic and / or preventive agents are existing known drugs, such as anti-tumor drugs, anti-infective drugs, local anesthetics, antidepressants, anticonvulsants, antibiotics / antibacterial agents, antifungal drugs, antiparasitic drugs, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-glaucoma agents, anesthetics or imaging agents.

[0058] The ionizable lipid compound also includes one or more other ionizable lipid compounds, and the other ionizable lipid compounds refer to other charged lipid compounds disclosed or undisclosed; Other ionizable lipid compounds include, but are not limited to: 3-(Docosylamino)-N1,N1,4-triadecyl-1-piperazineethylamine (KL10), N1-[2-(Docosylamino)ethyl]-N1,N4,N4-triadecyl-1,4-piperazinedienamide (KL22), 14,25-tricosyl-15,18,21,24-tetraazaoctaconane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl- 4-Dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioloxy-N,N-dimethylaminopropane (DODMA); 2-({8-[(3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl- CLinDMA(2R)), (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethylethyl-3-[(9Z-12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA(2S)), (12Z,15Z)-N,Ndimethyl-2-nonyldocayl-12,15-den-1-amine, N,N-dimethyl-1-{(1S,2R)-2-octylcyclopropyl}heptadecan-8-amine; N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC ); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:1); N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[bis(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); dioctadecylamino-glycyl spermine (DOGS); 3b-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol); dioctadecylamino-glycyl spermine (DOGS); 3b-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol); Dimethylammonium bromide (DDAB); SAINT-2, N-methyl-4-(dioleyl) methylpyridine; 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE); 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORIE); 1,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride (DORI); di-alkylated amino acids (DILA2); dioleyl dimethylammonium chloride (DODAC); 1-palmitoyl-2-oleoyl -sn-glycero-3-ethylphosphocholine (POEPC); 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC); (R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride (DODAPen-Cl); (R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride (DOPen-G); and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-ammonium chloride (DOTAPen); Cationic lipids having a head group charged at physiological pH, such as a primary amine (e.g., DODAGN', N'-dioctadecyl-N-4,8-diaza-10-aminodecanoyl glycine amide) and a guanidinium head group (e.g., bis-guanidinium-spermidine-cholesterol (BGSC), bis-guanidinium trisaminoethylamine-cholesterol (BGTC), PONA, and (R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride (DOPen-G)). Another suitable cationic lipid is (R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride (DODAPen-Cl). The cationic lipid is a specific enantiomer or racemic form, and includes various salt forms (e.g., chlorides or sulfates) of the above cationic lipids; N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP-Cl), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium sulfate (DOTAP-sulfate); Dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptathriacontane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), morpholino cholesterol (Mo-CHOL).

[0059] Ionizable lipid compounds can be represented as a hydrophobic tail attached to a head group containing an ionizable tertiary amine group; It can be thought of as a combination of a head group and five hydrophobic tails as follows: , another typical ionizable lipid compound: It can be thought of as a combination of the following head group and eight hydrophobic tails: .

[0060] Suitable ionizable lipids include compounds consisting of any combination of the head groups and hydrophobic tails listed below: Suitable head groups are shown in Table 1: Table 1

[0061] Suitable hydrophobic tails include those shown in Table 2: Table 2 , , , , ; These compounds are prepared by at least one of the techniques described herein or known organic synthesis techniques. The following are several typical synthetic routes for the synthesis of the compounds: Synthesis route of compound 1: ; Here are the steps: 1. Add THF (30 mL) to compound 1-1 (4 mmol, 1 eq, 945.28 mg) and dissolve at RT, 300 rpm; 2. Add compound 1-2 (2.5 eq, 10 mmol, 2.69 g), introduce high-purity nitrogen, 400 rpm, RT and react for 30 min; 3. Add STAB (2.5 eq, 10 mmol, 2.119 g) and THF (20 mL) and react under RT and 500 rpm high-purity nitrogen protection for 24 h; 4. Add saturated NaHCO3 aqueous solution (150 mL) to the solution, RT, 400 rpm, 45 min to quench the unreacted STAB; 5. Transfer the solution into a separatory funnel, add DCM (150 mL), shake vigorously for 5 min, let stand for 10 min, collect the lower organic phase, and repeat 3 times; 6. Drying with anhydrous Na2SO4; 7. Vacuum filtration was performed to remove insoluble salts, and the filtrate was rotary evaporated to obtain a crude product, which was purified by silica gel column, the solvent was DCM:ultra (85:15), ultra was a mixture (mixing ratio DCM / MeOH / NH4OH(aq)=75 / 22 / 3), and a colorless transparent oily liquid compound 1 was obtained.

[0062] Synthesis route of compound 2: ; Here are the steps: Step 1: Synthesis of compound 2-2 1. Add THF (190 mL) to compound 2-1 (15 mmol, 1.0 eq) and cool to 0 ° C. Add LAH THF solution (1 mol / L, 22.5 mmol, 1.5 eq, 22.5 mL) dropwise under stirring at 500 rpm. After the addition of LAH solution, return the reaction solution to room temperature RT and stir overnight; 2. Cool the reaction solution to 0°C, and slowly add ultrapure water (0.85 mL) - NaOH solution (1N, 1 mol / L, 0.85 mL) - ultrapure water (2.6 mL) drop by drop in the following order; 3. Filter through diatomaceous earth, collect the filtrate, and dry the filtrate by rotary evaporation to remove the solvent to obtain a colorless oily fatty alcohol crude product compound 2-2; Step 2: Synthesis of compound 2-3 1. Dissolve compound 2-2 (1 eq, 15 mmol) in DCM (160 mL) at RT, 300 rpm; 2. Add NaHCO3 (8.821 g, 105 mmol, 7 eq), followed by DMP (7.634 g, 18 mmol, 1.2 eq), and react at RT, 500 rpm for 1 h; 3. Remove insoluble matter by suction filtration, and then dilute the filtrate into petroleum ether (160 mL); 4. Wash the petroleum ether organic phase with saturated NaHCO3 solution (300 mL / time) 3 times, collect the organic phase, then wash the organic phase with saturated NaCl solution (300 mL / time) 3 times, collect the organic phase; 5. Add anhydrous Na2SO4 to the organic phase, shake and stand for 30 minutes to absorb residual water; remove Na2SO4 from the dried organic phase by suction filtration, collect the organic phase filtrate, and then transfer the organic phase to a rotary evaporator to remove the solvent to obtain a crude product compound 2-3; Step 3: Synthesis of compound 2 1. Add THF (30 mL) to compound 2-4 (4 mmol, 1 eq) and dissolve at RT, 300 rpm; 2. Add compound 2-3 (2.5 eq, 10 mmol), introduce high-purity nitrogen, 400 rpm, and react at RT for 30 min; 3. Add STAB (2.5eq, 10mmol, 2.119g), add THF (20mL), RT, 500rpm high-purity nitrogen protection reaction for 24h, then add saturated NaHCO3 aqueous solution (150mL) to the solution, RT, 400rpm, 45min to quench the unreacted STAB; 4. Transfer the solution into a separatory funnel, add DCM (150 mL), shake vigorously for 5 min, let stand for 10 min, collect the lower organic phase, repeat 3 times, and then dry over anhydrous Na2SO4; 5. Vacuum filtration was performed to remove insoluble salts, and the filtrate was rotary evaporated to obtain a crude product, which was purified by silica gel column, the solvent was DCM:ultra (85:15), and ultra was a mixture (mixing ratio DCM / MeOH / NH4OH(aq)=75 / 22 / 3) to obtain a colorless, transparent, oily liquid compound 2.

[0063] Synthesis route of compound 3: ; Here are the steps: 3-2 (135 mmol, 9.0 eq) was added to compound 3-1 (15 mmol, 1.0 eq), and then heated to 70°C under the protection of high-purity nitrogen, and reacted for 96 hours with stirring at 500 rpm to obtain a crude compound 3.

[0064] Synthesis route of compound 4: ; Here are the steps: 1. Dissolve compound 4-1 (3 mmol, 1 eq) and DCC (1.1 eq, 3.3 mmol) in anhydrous DCM (15 mL) at RT, 300 rpm; 1. DMAP (10 mg, 0.082 mmol) was added to the reaction solution, and then compound 4-2 (1.5 eq, 4.5 mmol) was added to the reaction system. The reaction solution was stirred at 500 rpm for 2 h at 0 ° C, and then the reaction solution was heated to RT and stirred at 500 rpm for 24 h; 3. Add ultrapure water to the reaction product to separate and collect the organic phase, repeatedly wash the organic phase with ultrapure water, and then dry the organic phase with anhydrous Na2SO4; 4. Filter and rotary evaporate to remove the solvent, and purify on a silica gel column (hexane: ethyl acetate = 95:5) to obtain compound 4.

[0065] Synthesis route of compound 5: ; Here are the steps: Step 1: Synthesis of compound 5-2 1. Add compound 5-1 (17.7 mmol) to a KOH aqueous solution (1.25%, 400 mL) at 50°C and 300 rpm in an oil bath and stir until a transparent clear liquid is formed; 2. Add another portion of KOH aqueous solution (1.25%, 400 mL) to the clear oleic acid solution, add KMnO4 (5.0 g, 31.64 mmol) to the solution, and stir for 15 min; 3. Add sodium thiosulfate (10% aqueous solution) and sodium sulfite Na2SO3 (20% aqueous solution) to the reaction solution to quench the reaction; 4. Add hydrochloric acid solution (37%) to acidify for 2-3h until a white loose precipitate is formed, and filter to obtain compound 5-2; 5. Compound 5-2 was transferred into a vacuum drying oven and dried under vacuum at a constant temperature of 45°C with a yield of 90%. Compound 5-2 was then purified by recrystallization. After crystallization was completed, compound 5-2 was filtered and dried; Step 2: Synthesis of compound 5-3 1. Add DCM (45 mL) to compound 5-2 (1 eq, 3.792 mmol), then add NaIO4 aqueous solution (3 eq, 11.376 mmol, 2.433 g, 54.072 mg / mL, 45 mL) to the DCM solution, stir at RT, 300 rpm; 2. Add tetrabutylammonium hydrogen sulfate TBA-HS aqueous solution (0.5eq, 1.896mmol, 643.75mg, 5mL) to the two-phase solution and stir at RT, 500rpm for 10h; 3. The reaction solution was separated and the organic phase was collected. The organic phase was dried over anhydrous Na2SO4, then filtered and rotary evaporated to remove the solvent, and purified on a silica gel column (hexane: ethyl acetate = 1:1) to obtain compound 5-3; Step 3: Synthesis of compound 5-5 1. Dissolve compound 5-3 (3 mmol, 1 eq) and EDCI (632.61 mg, 1.1 eq, 3.3 mmol) in anhydrous DCM (15 mL) at RT, 300 rpm; 2. DMAP (10 mg, 0.082 mmol) was added to the reaction solution, and then compound 5-4 (1.5 eq, 4.5 mmol) was added to the reaction system. The reaction solution was stirred at 0°C and 500 rpm for 2 h; 3. The reaction solution was heated to RT and stirred at 500 rpm for 24 h, and then ultrapure water was added to the reaction product to separate and collect the organic phase, and the organic phase was repeatedly washed with ultrapure water and dried with anhydrous Na2SO4; 4. Filter and rotary evaporate to remove the solvent, and purify on a silica gel column (hexane: ethyl acetate = 95:5) to obtain compound 5-5; Step 4: Synthesis of compound 5 1. Add THF (30 mL) to compound 5-6 (4 mmol, 1 eq) and dissolve at RT, 300 rpm; 2. Add compound 5-5 (2.5 eq, 10 mmol), introduce high-purity nitrogen, 400 rpm, RT and react for 30 min; 3. Add STAB (2.5eq, 10mmol, 2.119g), add THF (20mL), RT, 500rpm high-purity nitrogen protection reaction for 24h, then add saturated NaHCO3 aqueous solution (150mL) to the solution, RT, 400rpm, 45min to quench the unreacted STAB; 4. Transfer the solution into a separatory funnel, add DCM (150 mL), shake vigorously for 5 min, let stand for 10 min, collect the lower organic phase, repeat 3 times, and dry over anhydrous Na2SO4; 5. Vacuum filtration was performed to remove insoluble salts, and the filtrate was rotary evaporated to obtain a crude product, which was purified by silica gel column, the solvent was DCM:ultra (85:15), and ultra was a mixture (mixing ratio DCM / MeOH / NH4OH(aq)=75 / 22 / 3) to obtain a colorless, transparent, oily liquid compound 5.

[0066] Synthesis route of compound 6: ; Here are the steps: Step 1: Synthesis of compound 6-3 1. Add diisopropylethylamine (133.45 mg, 1.0 eq) and DMAP (100.9 mg, 0.835 mmol, 0.2 eq) to a solution of compound 6-2 (4.13 mmol, 1.0 eq) and 6-1 (5 mmol, 1.2 eq) in DCM (15 mL), stir at room temperature for 5 min, then add EDCI (1.425 g, 7.435 mmol, 1.8 eq), and stir the reaction mixture at room temperature for 12 h; 2. The reaction mixture was diluted with DCM (150 mL), washed with saturated NaHCO3, ultrapure water and saturated NaCl solution; 3. The combined organic layers were dried over Na2SO4, and the solvent was removed by rotary evaporation to obtain a crude product, which was purified by column chromatography (the eluent was a n-hexane solution containing 0.1% EA (volume percentage)), and the pure product fraction was evaporated to obtain a colorless oily compound 6-3; Step 2: Synthesis of compound 6-5 1. Add THF (190 mL) to compound 6-4 (15 mmol, 1.0 eq) and cool to 0 ° C. Add LAH THF solution (1 mol / L, 22.5 mmol, 1.5 eq, 22.5 mL) dropwise under stirring at 500 rpm. After the addition of LAH solution, return the reaction solution to room temperature and stir overnight; 2. Cool the reaction solution to 0°C, and slowly add ultrapure water (0.85 mL) - NaOH solution (1N, 1 mol / L, 0.85 mL) - ultrapure water (2.6 mL) drop by drop in the following order; 3. Filter with diatomaceous earth, collect the filtrate, and dry the filtrate by rotary evaporation to remove the solvent to obtain a colorless oily fatty alcohol crude product compound 6-5; Step 3: Synthesis of compound 6-6 1. Compound 6-5 (1 eq, 15 mmol) was dissolved in DCM (160 mL) at RT and 300 rpm, and then NaHCO3 (8.821 g, 105 mmol, 7 eq) was added, followed by DMP (7.634 g, 18 mmol, 1.2 eq), and the reaction solution was reacted at RT and 500 rpm for 1 h; 2. Remove the insoluble matter by suction filtration, dilute the filtrate into petroleum ether (160 mL), then wash the petroleum ether organic phase with saturated NaHCO3 solution (300 mL / time) 3 times, and collect the organic phase; 3. Wash the organic phase with saturated NaCl solution (300 mL / time) for 3 times, collect the organic phase, add anhydrous Na2SO4 to the organic phase, shake and stand for 30 minutes to absorb residual water; 4. The dried organic phase was filtered to remove Na2SO4, the organic phase filtrate was collected, and the organic phase was transferred to a rotary evaporator to remove the solvent to obtain a crude product compound 6-6; Step 4: Synthesis of Compounds 6-8 1. Add THF (30 mL) to compound 6-7 (4 mmol, 1 eq) and dissolve at RT, 300 rpm; 2. Add compound 6-6 (1 eq, 4 mmol), introduce high-purity nitrogen, 400 rpm, and react at RT for 30 min; 3. Add STAB (1.25 eq, 5 mmol, 1.06 g) and THF (20 mL) and react under RT and 500 rpm high-purity nitrogen protection for 24 h; 4. Add saturated NaHCO3 aqueous solution (150 mL) to the solution, RT, 400 rpm, 45 min to quench the unreacted STAB, then transfer the solution to a separatory funnel, add DCM (150 mL), shake vigorously for 5 min, let stand for 10 min, collect the lower organic phase, repeat 3 times, and dry over anhydrous Na2SO4; 5. Vacuum filtration was performed to remove insoluble salts, and the filtrate was rotary evaporated to obtain a crude product, which was purified by a silica gel column, the solvent was DCM:ultra (85:15), and ultra was a mixture (mixing ratio DCM / MeOH / NH4OH (aq) = 75 / 22 / 3) to obtain compound 6-8; Step 5: Synthesis of compound 6 1. A solution of compound 6-8 (0.74 mmol, 1.0 eq), compound 6-3 (1.47 mmol, 2.0 eq), K2CO3 (306.3 mg, 2.22 mmol, 3.0 eq), Cs2CO3 (71.7 mg, 0.22 mmol, 0.3 eq) and NaI (32.9 mg, 0.22 mmol, 0.3 eq) in acetonitrile (15 mL) was stirred at 90°C overnight; 2. The reaction mixture was diluted with EA, washed with water and concentrated, and the residue was purified by chromatography to give Compound 6.

[0067] Synthesis route of compound 7: ; Here are the steps: 1. Add DMF (15 mL) to compound 7-1 (1.0 mmol, 1.0 eq), and add 7-2 (6 mmol, 6.0 eq), EDC (6 mmol, 6.0 eq) and NHS (6 mmol, 6.0 eq), and stir at room temperature for 24 h; 2. Dilute with DCM (150 mL), saturated NaHCO3、 The mixture was washed with ultrapure water and saturated NaCl solution, the combined organic layers were dried over Na2SO4, and the solvent was removed by rotary evaporation to obtain a crude product; 3. The crude product was purified by column chromatography (the eluent was a n-hexane solution containing 0.1% EA (volume percentage)), and the pure product fractions were evaporated to obtain compound 7.

[0068] At least one of the above organic synthesis techniques is used to prepare an ionizable lipid compound. Some typical ionizable lipid compounds and compound structures are shown in Table 3; Table 3 ; Experimental testing Preparation of lipid nanoparticles (LNP formulations): The cationic lipid compound was dissolved in ethanol with DSPC, cholesterol and DMG-PEG2000 at a molar ratio of 50:10:38.5:1.5 to prepare an ethanol lipid solution, and MusATP7B mRNA was dissolved in 10 to 50 mM citrate buffer (pH = 4) and diluted to obtain an mRNA aqueous solution; Liposomes were prepared by mixing an ethanol lipid solution and an aqueous mRNA solution in a volume ratio of 1:3 using a microfluidic device to obtain a total lipid to mRNA weight ratio of about 15-30:1; The ethanol was removed by static dialysis for 12-24 h and replaced with DPBS; The nanolipid particles were then filtered through a 0.2 μm sterile filter to obtain a LNP formulation encapsulating MusATP7B mRNA using ionizable lipid / DSPC / cholesterol / DMG-PEG2000 (50 / 10 / 38.5 / 1.5 mol%).

[0069] Characterization of Nanolipid Particles (LNP Formulations): The microstructure of the nanolipid particles was observed by transmission electron microscopy. The nanoparticles prepared by the microfluidic mixer were dropped onto a 200-mesh carbon film, and 2% (w / v) phosphotungstic acid was added to the copper mesh. The mesh was stained for 2 minutes and dried at room temperature. The mesh was then placed under a transmission electron microscope for observation and photography. The microscopic morphology of the nanolipid particles is shown in FIG. Figure 1 shown.

[0070] The size and polydispersity index (PDI) of the nanolipid particles were determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) in 173° backscattering detection mode. The test results are shown in Table 4; The encapsulation efficiency of the nanolipid particles was determined using the Quant-it Ribogreen RNA quantification kit (Thermo Fisher Scientific, UK). The test results are shown in Table 4; Table 4 ; The above results prove that the particle size of the nanolipid particles prepared by the ionizable lipids in this patent is about 90-120 nm, and has a very high encapsulation effect on mRNA.

[0071] Stability test of nanolipid particles: The changes in the particle size of the nanolipid particles were observed every 2 h at 4 °C in PBS supplemented with 10% FBS. Figure 2 As shown; The results showed that the nanolipid particles prepared by the ionizable lipid compound of the present invention could still maintain a stable particle size in a 10% FBS PBS solution within 20 hours, indicating that the nanolipid particles are relatively stable, providing a basis for their wider application; In vitro cytotoxicity test of nanolipid particles: The CCK-8 method was used to test the effects of different nanolipid particles on the toxicity of ATP7B knockout hepatocytes at different concentrations. ATP7B knockout hepatocytes (1x104 cells / well) were evenly seeded in a 96-well plate. The corresponding concentration of nanolipid particles was added to each well and cultured for 48 hours. Then, CCK-8 solution (10 μL / well) was added and cultured for 4 h. The absorbance (OD) at 450 nm was measured by an enzyme reader. The experiment was repeated 5 times, and the cell survival rate of each group was calculated according to the following formula: Cell survival rate (100%) = 100% × (ODtreated - ODblank) / (ODcontrol - ODblank); Among them, ODtreated is the OD value of cells treated with nanolipid particles, ODcontrol is the OD value of normal cells, and ODblank is the OD value of CCK-8 solution. The results are shown in Figure 3 shown.

[0072] In vitro cell proliferation assay of nanolipid particles: The CCK-8 method was used to test the effects of different nanolipid particles at different concentrations on the proliferation of ATP7B-knockout hepatocytes; ATP7B knockout hepatocytes (1x104 cells / well) in the logarithmic growth phase were evenly inoculated into a 96-well plate, each well contained 200 μL of complete medium, and cultured statically; Then, the corresponding concentration of nanolipid particles was added to each well and cultured for 48 h; then CCK-8 solution (10 μL / well) was added and cultured for 4 h; The absorbance value (OD) at 450 nm was measured by an enzyme-labeled instrument; the experiment was repeated 5 times, and the cell survival rate of each group was calculated according to the following formula: Cell survival rate (100%) = 100% × (ODtreated - ODblank) / (ODcontrol - ODblank); Among them, ODtreated is the OD value of cells treated with nanolipid particles, ODcontrol is the OD value of normal cells, and ODblank is the OD value of CCK-8 solution. The results are shown in Figure 4 shown.

[0073] The results showed that the toxicity of nanolipid particles prepared from different ionizable lipid compounds to cells was similar, and with the increase of the concentration of nanolipid particles, the cells showed slight toxicity.

[0074] In vivo testing of nanolipid particles: Nanolipid particles were systemically administered to 6-8 week old male balb / c mice with ATP7B knockout gene at a dose of 0.5 mg / kg via tail vein injection. The mice were euthanized 6 h after administration, and liver tissues were obtained. After tissue grinding, the tissues were centrifuged at 10000 g for 10 min at 4 °C to separate the tissue supernatant, which was quickly frozen and stored at -80 °C for analysis. ELSA analysis was performed using a commercially available kit, and the ATP7B expression levels (ng / mL) measured in each test group are shown in Table 5 ; Table 5 Nanolipid particles Ionizable lipids used ATP7B expression level (ng / mL) 1 Compound 1 420 2 Compound 2 409 3 Compound 3 412.7 4 Compound 4 330.2 5 Compound 5 339.5 6 Compound 6 448.7 7 Compound 7 391 8 Compound 8 477.5 9 Compound 9 526.8 10 Compound 10 469.7 11 Compound 11 414.9 12 Compound 12 1017.9 13 Compound 13 641.4 14 Compound 14 445.7 15 Compound 15 474.5 16 Compound 16 648.3 17 Compound 17 482.1 18 Compound 18 508 19 Compound 19 377.9 20 Compound 20 622.7 21 Compound 21 426.2 22 Compound 22 537.3 23 Compound 23 404 24 Compound 24 453.9 25 Compound 25 414.1 26 Compound 26 427.4 27 Compound 27 626.8 28 Compound 28 1035.9 29 Compound 29 946.9 30 Compound 30 936.6 31 Compound 31 1088.3 32 Compound 32 781.4 33 Compound 33 725.2 34 Compound 34 651.7 35 Compound 35 454.9 36 Compound 36 547 37 Compound 37 502.7 38 Compound 38 515.5 39 Compound 39 225.8 ; The above results prove that the nanolipid particles prepared by the ionizable lipids of this patent can carry MusATP7B mRNA into the liver tissue of mice and express it.

[0075] Experiments have shown that the ionizable lipid compound of the present invention can deliver nucleic acid molecules, small molecule compounds, polypeptides or proteins, etc. The carrier prepared using the ionizable lipid compound of the present invention has high encapsulation efficiency for nucleic acid molecules and can successfully transport nucleic acid molecules into cells for expression.

[0076] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A multi-charged ionizable lipid compound, characterized in that: The multi-charged ionizable lipid compound is a compound of formula I, or a pharmaceutically acceptable salt thereof; Formula I In the general structural formula I, G1, G2, G3 and G4 are each independently selected from C1-C 12 Straight chain or branched alkyl, hydroxy substituted or unsubstituted C1-C 12 Straight-chain or branched alkenyl, hydroxy-substituted or unsubstituted C1-C 12 Straight or branched chain alkylene or alkenylene and C1-C 12 A straight or branched alkyl or alkylene group; Said m and n are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Said A1 and A2 are absent or are each independently selected from -N(T7)-, -NH-, -NH-CO-, -CO-NH-, -NHC(O)O-, -OC(O)N(H)-, -C(O)N(T7)-, -N(T7)C(O)-, -ON=C(T7)-, -C(T7)=NO-, -OC(O)N(T7)-, -N(T7)C(O)N(T7)-, -N(T7)C(O)O- or -C(T7)=NOC(O)-; Said T7 is absent or each occurrence is independently selected from H, alkyl or cycloalkane, nitrogen-containing or non-nitrogen-containing heterocycle; When A1 and A2 are tertiary amine groups, T7 in -N(T7)- is T4 and / or T5, when A1 and A2 do not exist or are each independently selected from -NH-, -NH-CO-, -CO-NH-, -NHC(O)O-, -OC(O)N(H)-, -C(O)N(T7)-, -N(T7)C(O)-, -ON=C(T7)-, -C(T7)=NO-, -OC(O)N(T7)-, -N(T7)C(O)N(T7)-, -N(T7)C(O)O- or -C(T7)=NOC(O)-, T4 and T5 do not exist; when T4 and T5 do not exist, the dotted lines of T4 and T5 do not exist either; Said T1, T2, T3, T4, T5 and T6 do not exist or are independently selected from H or independently selected from compounds conforming to the chemical structure of general formula II: Formula Ⅱ The L1, L2 and L3 do not exist or are independently selected from H or independently selected from compounds conforming to the chemical structure of the general formula III: General formula III.

2. A multi-charged ionizable lipid compound according to claim 1, characterized in that: In the general structural formula II, G5 and G6 are absent or independently selected from C1-C 20 Straight chain or branched alkyl, hydroxy substituted or unsubstituted C1-C 20 Straight-chain or branched alkenyl, hydroxy-substituted or unsubstituted C1-C 20 Straight or branched chain alkylene or alkenylene; Said X1 is absent or selected from -O-, -NH-, -N(R1)-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R1)-, -N(R1)C(O)-, -SS-, -OC(O)O-, -ON=C(R1)-, -C(R1)=NO-, -OC(O)N(R1)-, -N(R1)C(O)N(R1)-, -N(R1)C(O)O-, -C(O)S-, -C(S)O- or -C(R1)=NOC(O)-; R1 is absent or each occurrence is independently selected from H, alkyl, nitrogen-containing or non-nitrogen-containing heterocycle; When X1 is a tertiary amine group, R1 in -N (R1)- is L3; When When C(R1)=NO-, -OC(O)N(R1)-, -N(R1)C(O)N(R1)-, -N(R1)C(O)O-, -C(O)S-, -C(S)O- or -C(R1)=NOC(O)-, L3 does not exist; When L3 does not exist, the dotted line of L3 does not exist either; The D is absent or selected from A1 or A2, -O-, -N(R2)-, -NH-, -NH-CO-, -CO-NH-, -NHC(O)O-, -OC(O)N(H)-, -C(O)N(R2)-, -N(R2)C(O)-, -ON=C(R2)-, -C(R2)=NO-, -OC(O)N(R2)-, -N(R2)C(O)N(R2)-, -N(R2)C(O)O- or -C(R2)=NOC(O)-; R2 is absent or each occurrence is independently selected from H, alkyl, nitrogen-containing or non-nitrogen-containing heterocycle; When D is A1 or A2, L1 and L2 are independently T3 and T4; When D is a tertiary amine group, R2 in -N (R2)- is L2; When D is -NH-, -NH-CO-, -CO-NH-, -NHC(O)O-, -OC(O)N(H)-, -C(O)N(R2)-, -N(R2)C(O)-, -ON=C(R2)-, -C(R2)=NO-, -OC(O)N(R2)-, -N(R2)C(O)N(R2)-, -N(R2)C(O)O-, or -C(R2)=NOC(O)-, L2 is absent; When L2 does not exist, the dotted line of L2 does not exist either.

3. A multi-charged ionizable lipid compound according to claim 2, characterized in that: In the general structural formula III, the G7 is absent or independently selected from a hydroxy-substituted or unsubstituted C6-C 18 Straight chain alkyl, hydroxy substituted or unsubstituted C6-C 26 Branched alkyl, hydroxy substituted or unsubstituted C4-C 22 Straight-chain or branched alkenyl, hydroxy-substituted or unsubstituted C2-C 22 Straight or branched chain alkylene or alkenylene; Said X2 is absent or independently selected from -O-, -NH-, -N(R4)-, -S-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N((R4)-, -N(R4)C(O)-, -SS-, -OC(O)O-, -ON=C((R4)-, -C(R4)=NO-, -OC(O)N((R4)-, -N(R4)C(O)N((R4)-, -N(R4)C(O)O-, -C(O)S-, -C(S)O- or -C(R4)=NOC(O)-; R4 is absent or each occurrence is independently selected from H, alkyl, nitrogen-containing or non-nitrogen-containing heterocycle; When X2 does not exist, then R3 does not exist either; The R3 is independently selected from C6-C 14 Straight or branched chain alkyl or alkenyl.

4. A multi-charged ionizable lipid compound according to claim 3, characterized in that: A1 and A2 are independently selected from -N(T7)-, -NH-, -NH-CO-, -CO-NH-, -NHC(O)O-, -OC(O)N(H)-, G2 and G3 are independently selected from hydroxyl-substituted or unsubstituted C1-C4 straight chain or branched alkyl or alkylene, and G1 and G4 are independently selected from hydroxyl-substituted or unsubstituted C1-C6 straight chain or branched alkyl or alkylene or C1-C4 straight chain or branched alkyl or alkylene containing ether bonds.

5. A multi-charged ionizable lipid compound and lipid composition thereof according to claim 4, characterized in that: The general structural formulas I, II and III contain multiple protonatable tertiary amine group structures, including but not limited to: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

6. A multi-charged ionizable lipid compound according to claim 5, characterized in that: When the X 2和 When R3 does not exist, G7 includes but is not limited to 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 7. A multi-charged ionizable lipid compound according to claim 6, characterized in that: When said R3 exists, R3 includes but is not limited to 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 8. A multi-charged ionizable lipid compound according to claim 7, characterized in that: When X2 and / or R3 exist, G7 includes but is not limited to 、 、 、 、 、 、 、 、 、 、 、 、 。 9. A multi-charged ionizable lipid compound according to claim 8, characterized in that: The ionizable lipid compound includes but is not limited to one or more of the following compounds: 、 、 、 、 、 、 。 10. A multi-charged ionizable lipid compound according to claim 9, characterized in that: The multi-charged ionizable lipid compound has the following general structural formula: Formula IV In the general structural formula IV, p and q are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8; said t is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; W is a head group containing multiple protonatable tertiary amine groups; The X3 does not exist or is part of the chemical bond formed by connecting to the W head group; The R 10 Independently selected from C5-C 15 Straight or branched alkyl, C5-C 15 Straight chain or branched alkenyl or C5-C 15 straight chain or branched chain alkynyl; X3, p, q and R in the different side chains connected to the W head group in the general structure IV 10 Same or different.

11. A multi-charged ionizable lipid compound according to claim 10, characterized in that: The head group is , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

12. A multi-charged ionizable lipid compound according to claim 11, characterized in that: The p is 3 to 6.

13. A multi-charged ionizable lipid compound according to claim 12, characterized in that: The q is 0 to 4.

14. A multi-charged ionizable lipid compound according to claim 13, characterized in that: The multi-charged ionizable lipid compound has the following general structural formula: Formula V In the general structural formula V, p is 0, 1, 2, 3, 4, 5, 6, 7 or 8; said t is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; W is a head group containing multiple protonatable tertiary amine groups; Said X3 is absent or is part of a chemical bond formed with the W head group; The R 100 Independently selected from C5-C 15 Straight or branched alkyl, C5-C 15 Straight chain or branched alkenyl or C5-C 15 straight chain or branched chain alkynyl; In the general structural formula V, X3, p and R in different side chains connected to the W head group 100 Same or different.

15. A multi-charged ionizable lipid compound according to claim 14, characterized in that: The head group is , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

16. A multi-charged ionizable lipid compound according to claim 15, characterized in that: The p is 0 to 6.

17. A multi-charged ionizable lipid compound according to claim 16, characterized in that: The multi-charged ionizable lipid compound has the following general structural formula: Formula VI In the general structural formula VI, p is 0, 1, 2, 3, 4, 5, 6, 7 or 8; Said t or z is each independently 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; W is a head group containing multiple protonatable tertiary amine groups; Said X3 is absent or is part of a chemical bond formed with the W head group; The R 100 Independently selected from C5-C 15 Straight or branched alkyl, C5-C 15 Straight chain or branched alkenyl or C5-C 15 straight chain or branched chain alkynyl; The R 200 Independently selected from C 12 -C 24 Straight or branched alkyl, C 12 -C 24 Straight or branched alkenyl or C 12 -C 24 straight chain or branched chain alkynyl; In the general structure VI shown, X3, p, R 100 and R 200 Same or different.

18. A multi-charged ionizable lipid compound according to claim 17, characterized in that: The head group is , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

19. A multi-charged ionizable lipid compound according to claim 18, characterized in that: The p is 0 to 6.

20. A lipid composition, using a multi-charged ionizable lipid compound as claimed in claim 19, characterized in that: The lipid composition comprises an active agent and a carrier for delivering the active agent; the carrier comprises an ionizable lipid compound; the ionizable lipid compound comprises one or more of the ionizable lipid compounds represented by structural formula I, structural formula II, structural formula III, structural formula IV, structural formula V and structural formula VI or their pharmaceutically acceptable salts.

21. A lipid composition according to claim 20, characterized in that: The lipid composition comprises a neutral lipid, an aggregation-reducing PEG lipid, a sterol and an ionizable lipid compound, wherein the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1 '-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC) or 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), and the sterol includes one or more of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and corticosteroids.

22. The method according to claim 21, characterized in that: The molar ratio of the ionizable lipid is 20% to 60%, the molar ratio of the neutral lipid is 5% to 25%, the molar ratio of the sterol is 25% to 55%, and the molar ratio of the PEG lipid is 0.5% to 15%.

23. A lipid composition according to claim 22, characterized in that: The active agent includes one or more of a nucleic acid molecule, a small molecule compound, a polypeptide and a protein, wherein the nucleic acid molecule includes but is not limited to single-stranded DNA, double-stranded DNA, short isomers, agomir, antagomir, antisense molecules, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), locked nucleic acid (LNA), peptide nucleic acid (PNA) or morpholino oligonucleotide.

24. A lipid composition according to claim 23, characterized in that: The mass ratio of the carrier to the active agent is 5:1-50:

1.

25. A lipid composition according to claim 24, characterized in that: The lipid composition is used for preparing nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

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