Ionizable lipid compound and application thereof

Abstract

The invention provides an ionizable lipid compound and application thereof, the ionizable lipid compound has a structure as shown in a formula (I), and a liposome prepared from the lipid compound provided by the invention is uniform in particle size distribution, carries negative charges on the surface and is low in cytotoxicity. The lipid nanoparticles provided by the invention can deliver the active substances to cells, tissues or organs in a specific targeting manner, so that the targeting delivery of the active substances is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of nucleic acid carriers and relates to an ionizable lipid compound and application thereof. Background Art

[0002] mRNA molecules are large, hydrophilic, negatively charged, and easily degraded. Naked mRNA is almost incapable of crossing lipophilic cell membranes (which also carry a slight negative charge). Therefore, safely and efficiently delivering mRNA to ribosomes in the cytoplasm of target cells is a major challenge in the clinical translation of mRNA technology. While mRNA can pass through porous, lipid-raft-rich membrane domains, as endocytosis proceeds, mRNA ultimately accumulates and is degraded in lysosomes, with only a small portion being released into the cytoplasm. Furthermore, mRNA is also highly susceptible to degradation by extracellular ribonucleases (RNases) before internalization. Recently, with the advancement of nanoscience, a large number of nanocarriers have been constructed to facilitate cytoplasmic delivery of mRNA.

[0003] Because LNPs have good biocompatibility and can protect mRNA from degradation, they are widely used in mRNA delivery. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide an ionizable lipid compound and its application.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In one aspect, the present invention provides an ionizable lipid compound having a structure as shown in formula (I):

[0007]

[0008] Wherein M, Q, and K may be the same or different and are selected from -(CH2) m1 -、-(CH2) n1 C(O)(CH2) p 、-(CH2) n1 C(O)O(CH2) p -, m1 is an integer of 1-6 (e.g., 1, 2, 3, 4, 5 or 6), n1 is an integer of 1-3 (e.g., 1, 2 or 3), and p is an integer of 1-3 (e.g., 1, 2 or 3).

[0009] R' and R" may be the same or different and are selected from -(CH) q -、-(CH) q CH=CH(CH) s -、-(CH2) q NH(CH2)s 、-(CH) q C(O)(CH) s -, wherein q and s are independently an integer from 1 to 3 (eg, 1, 2 or 3).

[0010] (1) R1, R2, R3, R4, R5 and R6 may be the same or different and are selected from C 1-6 Alkyl, C 3-7 Cycloalkyl, 4-7 membered heterocyclic group, C 6-10 Aryl, 5-10 membered heteroaryl, benzoheterocyclyl or heterocyclyl, the above cycloalkyl, heterocyclyl, aryl, heteroaryl, benzoheterocyclyl or heterocyclyl groups are optionally substituted by a group selected from the following: halogen atoms, -(CH2) 1-6 CX3, -OH, -(CH2) 1-3 OH, -(CH2) 1-3 NRaRb、C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkyl hydroxyl, C 1-6 Alkylamino, where R a and R b Each independently selected from H, C 1-20 Alkyl, -(CH2) 1-3 NH2 or -(CH2) 1-20 NH(CH2) 1-20 NH2; X is selected from halogen.

[0011] (2) R1, R2, R3, R4, R5, and R6 may be the same or different and may be selected from substituted or unsubstituted linear or branched C 3-30 Alkyl, substituted or unsubstituted linear or branched C 3-30 Alkenyl, substituted or unsubstituted straight chain or branched C 3-30 Alkynyl, where R c and R d Can be the same or different, selected from C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 wherein n and m are selected from integers of 1 to 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15).

[0012] Preferably, R1, R2, R3, R4, R5 and R6 in (1) are selected from

[0013] in Indicates the attachment site of a group.

[0014] Preferably, R1, R2, R3, R4, R5 and R6 in (2) are selected from in Indicates the attachment site of a group.

[0015] In the structure, at least one of R1, R2, R3, R4, R5 and R6 is selected from (1), and at least one is selected from (2).

[0016] In the present invention, the ionized ester compound is selected from any one of the structures shown in the following formula II-III:

[0017]

[0018] wherein Ra is within the same range as described in (1); R1, R2, R3, R4, R5, and R6 are within the same range as described in (2); and R', R", M, Q, and K are within the same range as described in formula (I).

[0019] Preferably, the ionizable lipid compound has any one of the structures shown in the following formulas A1-A16:

[0020]

[0021]

[0022]

[0023] Preferably, the ionizable lipid compound has any one of the structures shown in the following formulas IV-V:

[0024]

[0025] Wherein Ra is the same as the range described in (1); R b and R c The same ranges as those described in (2); R', R", M, Q, and K are the same ranges as those described in formula (1).

[0026] Preferably, the ionizable lipid compound has any one of the structures shown in the following formulas B1-B16:

[0027]

[0028]

[0029]

[0030] Preferably, the ionizable lipid compound is selected from any one of the compounds in Table 1 below:

[0031] Table 1

[0032]

[0033]

[0034]

[0035]

[0036] In a second aspect, the present invention provides a liposome, wherein the liposome comprises the ionizable lipid compound as described in the first aspect.

[0037] Preferably, the liposome further comprises a co-lipid.

[0038] Preferably, the co-lipid comprises a phospholipid.

[0039] Preferably, the phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (1,2-dioleo ... PC), 1-oleoyl-2-cholesteryl hemisuccinyl sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl Any one or a combination of at least two of phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine or lysophosphatidylethanolamine (LPE).

[0040] Preferably, the co-lipid further comprises structural lipids.

[0041] Preferably, the structured lipid comprises at least one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid or α-tocopherol, or a combination of at least two thereof.

[0042] Preferably, the co-lipid further comprises PEGylated lipids.

[0043] Preferably, the PEGylated lipid comprises any one or a combination of at least two of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide or PEG-modified dialkylamine.

[0044] Preferably, the liposomes include ionizable lipid compounds, phospholipids, structural lipids and PEGylated lipids.

[0045] Preferably, the liposomes comprise, by mole percentage, 20%-80% (for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, etc.) of ionizable lipid compounds, 5%-30% (for example, 5%, 7%, 9%, 11%, 13%, 15%, 17%, 19%, 21%, 23%, 25%, 27%, 29%, 30%, etc.) of phospholipids, 10%-50% (for example, 10%, 20%, 30%, 40%, 50%, etc.) of structural lipids, and 1%-10% (for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc.) of pegylated lipids.

[0046] In a third aspect, the present invention provides a lipid complex comprising the liposome according to the second aspect and an active substance;

[0047] Preferably, the active substance includes any one of small molecule compounds, nucleic acid molecules or protein molecules, or a combination of at least two of them.

[0048] Preferably, the nucleic acid molecule comprises any at least one or a combination of at least two of DNA, mRNA, siRNA, aiRNA, miRNA, dsRNA, aRNA or lncRNA.

[0049] Preferably, the protein molecule comprises any one or a combination of at least two of an enzyme, an antibody, a short peptide, a polypeptide or a recombinant protein.

[0050] Preferably, the method for preparing the lipid complex comprises the following steps: mixing liposomes and active substances by a mixer to obtain the lipid complex.

[0051] In a fourth aspect, the present invention provides a lipid nanoparticle, wherein the lipid nanoparticle comprises the liposome described in the second aspect or the lipid complex described in the third aspect.

[0052] Preferably, the particle size of the lipid nanoparticles is 40-300 nm (for example, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 220 nm, 240 nm, 260 nm, 280 nm, 300 nm, etc.).

[0053] In a fifth aspect, the present invention provides a reagent, a kit, a preparation or a pharmaceutical composition, each of which independently comprises any one of the ionizable lipid compound described in the first aspect, the liposome described in the second aspect, the lipid complex described in the third aspect or the lipid nanoparticles described in the fourth aspect, or a combination of at least two thereof.

[0054] In a sixth aspect, the present invention provides a use of the ionizable lipid compound according to the first aspect, the liposome according to the second aspect, the lipid complex according to the third aspect, the lipid nanoparticle according to the fourth aspect, or the reagent, kit, preparation or pharmaceutical composition according to the fifth aspect in the preparation of a product having any one of the following functions (1) to (4):

[0055] (1) Encapsulation of active substances;

[0056] (2) delivering the encapsulated active substance to cells, tissues, or organs;

[0057] (3) enabling the delivered active substance to exert its active effect in cells, tissues or organs;

[0058] (4) Prevent, diagnose or treat diseases.

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] The present invention provides a series of novel ionizable lipid compounds. The liposomes prepared using these lipid compounds exhibit uniform particle size distribution, negative surface charge, and low cytotoxicity. The lipid nanoparticles provided by the present invention can specifically deliver active substances (such as small molecules, siRNA, mRNA, and proteins) to cells, tissues, or organs, achieving targeted delivery of active substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 The particle size and PDI test results of the lipid nanoparticles prepared in the present invention are shown in FIG.

[0062] Figure 2 This is a graph showing the results of measuring the encapsulation efficiency of lipid nanoparticles prepared in the present invention;

[0063] Figure 3 This is a graph showing the relative luminescence intensity measurement results of the lipid nanoparticles prepared in the present invention in 293T cells;

[0064] Figure 4 This is a graph showing the cell viability results of lipid nanoparticles of different concentrations prepared in the present invention measured in 293T cells. DETAILED DESCRIPTION

[0065] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0066] Example 1 Synthesis of ionizable lipid I-1

[0067] (1) Synthesis of intermediate a-4

[0068]

[0069] 1) Compound a-1 (2.4 g, 15 mmol) was dissolved in DCM (150 ml, 0.10 M), followed by the addition of compound a-2 (3.6 g, 37.5 mmol) and stirring for 10 min. Finally, NaBH(OAc)3 (9.537 g, 45 mmol) was added and stirred at room temperature for 3 h. The reaction was monitored by TLC. The mixture was quenched by the addition of saturated NaHCO3 solution (80 ml), extracted with DCM (60 × 3 ml), and the organic phases were combined and dried over anhydrous Na2SO4. After filtration and concentration under reduced pressure, the residue was purified by column chromatography (DCM:MeOH = 10:1) to afford compound a-3 as a light yellow viscous liquid (2.078 g, 6.495 mmol, 43.3%).

[0070] MS (ESI) m / z: [M+H] + 321.45. 1 H NMR (500MHz, Methanol-d4) δ7.60 (s, 4H), 3.61 (s, 4H), 3.22 (t, J = 6.7Hz, 2H), 2.51 (t, J = 6.8Hz, 2H), 1.45 (s, 9H).

[0071] 2) Synthesis of Intermediate a-4: Compound a-3 (2.078 g, 6.495 mmol) was dissolved in EA (15.0 ml, 0.20 M), and a 4 M solution of hydrogen chloride in ethyl acetate (13.0 ml, 51.96 mmol) was added. The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC. The mixture was quenched by the addition of saturated NaHCO₃ solution (60 ml), and extracted with EA (60 ml x 3). The organic phases were combined and used without purification to obtain compound a-4 (1.66 g, 6.4 mmol, 100%).

[0072] MS (ESI) m / z: [M+H] + 221.21. 1 H NMR (500MHz, Methanol-d4) δ 8.21 (s, 4H), 4.41 (s, 4H), 3.17 (s, 2H), 2.98 (d, J = 7.5Hz, 2H).

[0073] (2) Synthesis of Compound Ⅰ-1

[0074]

[0075] 1) Synthesis of compound b-3: Referring to the method of step (1) 1) of Example 1, compound b-3 was obtained as a white viscous liquid (2.052 g, 3.91 mmol, 78.2%) using b-1 and b-2 as starting materials.

[0076] MS (ESI) m / z: [M+H] + 526.34. 1 H NMR (500MHz, CDCl3) δ4.40 (t, J = 5.7Hz, 1H), 3.34 (s,

[0077] 6H),2.41–2.34(m,6H),1.60(dd,J=9.2,5.6Hz,2H),1.50–1.47(m,2H),1.42–1.38(m,4H),1.28–1.21(m,44H),0.90(t,J=6.8Hz,6H).

[0078] 2) Synthesis of Compound b-4: Compound b-3 (2.052 g, 3.91 mmol) was dissolved in H₂O (8 ml, 0.50 M), followed by the addition of HCl (0.59 ml, 19.55 mmol) and stirring at room temperature for 3 h. The reaction was monitored by TLC and quenched by the addition of saturated NaHCO₃ solution (60 ml). The mixture was extracted with DCM (30 ml x 3). The organic phases were combined and dried over anhydrous Na₂SO₄. After filtration and concentration under reduced pressure, the residue was purified by column chromatography (PE:EA = 10:1) to afford compound b-4 as a yellow viscous liquid (1.676 g, 3.5 mmol, 89.7%).

[0079] MS (ESI) m / z: [M+H] + 780.51. 1 H NMR (500MHz, CDCl3) δ9.78 (d, J = 1.7Hz, 1H), 2.49–

[0080] 2.35(m,8H),1.78–1.71(m,2H),1.40–1.34(m,4H),1.28–1.21(m,44H),0.90(t,J=6.8Hz,6H).

[0081] 3) Synthesis of Compound I-1: Referring to the method of step (1) 1) of Example 1, using b-4 and a-4 as starting materials, compound I-1 was obtained as a light yellow viscous liquid (15 mg, 0.0043 mmol, 14.5%).

[0082] MS (ESI) m / z: [M+H] + 1148.32. 1 H NMR (500MHz, CDCl3) δ7.51 (s, 4H), 3.57 (s, 4H), 2.48–2.42 (m, 16H), 2.36 (t, J = 7.5Hz, 4H), 1.53–1.45 (m, 8H), 1.44–1.35 (m, 8H), 1.28–1.22 (m, 88H), 0.89 (d, J = 7.1Hz, 12H).

[0083] Example 2 Synthesis of ionizable lipid I-2

[0084]

[0085] (1) Synthesis of compound c-2: Referring to the method of step (1) 1) of Example 1, compound c-2 was obtained as a white viscous liquid (3.45 g, 8.34 mmol, 69.7%) using b-1 and c-1 as starting materials.

[0086] MS (ESI) m / z: [M+H] + 414.73. 1 H NMR(500MHz,Chloroform-d)δ4.40(t,J=5.7Hz,1H),

[0087] 3.34(s,6H),2.44–2.35(m,6H),1.60(dd,J=9.2,5.6Hz,2H),1.52–1.47(m,2H),1.45–1.39(m,4H),1.28–1.22(m,28H),0.90(t,J=6.9Hz,6H).

[0088] (2) Synthesis of compound c-3: Referring to the method of step (2) 2) of Example 1, compound c-3 was obtained as a yellow viscous liquid (1.82 g, 4.95 mmol, 82.9%) using c-2 as the starting material.

[0089] MS (ESI) m / z: [M+H] + 368.66. 1 H NMR(500MHz,Chloroform-d)δ9.77(t,J=1.9Hz,1H),

[0090] 2.49–2.33(m,8H),1.83–1.70(m,2H),1.48–1.37(m,4H),1.28–1.22(m,28H),0.90(t,J=6.8Hz,6H).

[0091] (3) Synthesis of Compound I-2: Referring to the method of step (1) 1) of Example 1, using c-3 and a-4 as starting materials, compound I-2 was obtained as a light yellow viscous liquid (133.4 mg, 0.145 mmol, 28.8%).

[0092] MS (ESI) m / z: [M+H] + 923.92. 1 H NMR(500MHz,Chloroform-d)δ7.51(s,4H),3.57(s,4H),2.48–2.42(m,16H),2.37(t,J=7.5H z, 4H), 1.51–1.45 (m, 8H), 1.40 (d, J = 7.9Hz, 8H), 1.28–1.22 (m, 56H), 0.90 (t, J = 6.9Hz, 12H).

[0093] Example 3 Synthesis of ionizable lipid I-3

[0094]

[0095] (1) Synthesis of compound d-2: Referring to the method of step (1) 1) of Example 1, compound d-2 was obtained as a white viscous liquid (2.55 g, 5.43 mmol, 65.2%) using b-1 and d-1 as starting materials.

[0096] MS (ESI) m / z: [M+H] + 470.55. 1 H NMR(500MHz,Chloroform-d)δ4.40(t,J=5.7Hz,1H),

[0097] 3.34(s,6H),2.47–2.35(m,6H),1.60(dd,J=9.1,5.7Hz,2H),1.54–1.47(m,2H),1.44–1.37(m,4H),1.28(s,36H),0.90(t,J=6.9Hz,6H).

[0098] (2) Synthesis of compound d-3: Referring to the method of step (2) 2) of Example 1, compound d-2 was used as the starting material to obtain compound d-3 as a yellow viscous liquid (1.23 g, 2.9 mmol, 85.8%).

[0099] MS (ESI) m / z: [M+H] + 424.59. 1 H NMR(500MHz,Chloroform-d)δ9.77(d,J=1.9Hz,1H),

[0100] 2.47–2.36(m,8H),1.78(t,J=7.0Hz,2H),1.41(t,J=7.3Hz,4H),1.28–1.23(m,36H),0.90(t,J=7.1Hz,6H).

[0101] (3) Synthesis of compound I-3: Referring to the method of step (1) 1) of Example 1, compound I-3 was obtained as a light yellow viscous liquid (109.1 mg, 0.1053 mmol, 25.3%) using d-3 and a-4 as starting materials.

[0102] MS (ESI) m / z: [M+H] + 1036.14. 1H NMR(500MHz,Chloroform-d)δ7.51(s,4H),3.57(s,4H),2.49–2.43(m,16H),2.37(t,J=7.4 Hz, 4H), 1.46–1.41 (m, 8H), 1.43–1.38 (m, 8H), 1.28–1.22 (m, 72H), 0.90 (t, J = 6.8Hz, 12H).

[0103] Example 4 Synthesis of ionizable lipid II-5

[0104]

[0105] (1) Synthesis of compound a4: First, compound b-2 (1.282 g, 8.0 mmol) was dissolved in DCM (80 ml, 0.10 M), and then compound a-5 (3.395 g, 16.0 mmol) was added and stirred for 10 min. Finally, NaBH(OAc)3 (5.086 g, 24 mmol) was added and stirred at room temperature for 3 h. After completion of the reaction, the reaction was monitored by TLC. Saturated NaHCO3 solution (80 ml) was added to quench the reaction, and the mixture was extracted with DCM (60 ml × 3). The organic phases were combined and dried over anhydrous Na2SO4. After filtration and concentration under reduced pressure, the residue was purified by column chromatography (PE:EA = 10:1) to obtain compound e-1 as a light yellow viscous liquid (2.519 g, 4.56 mmol, 57%).

[0106] MS (ESI) m / z: [M+H] + 553.56. 1 H NMR (500MHz, CDCl3) δ3.14(s,2H),2.49(s,2H),2.38(t,J=7.5Hz,4H),1.44(s,9H),1.40(s,4H),1.26–1.22(m,44H),0.88(t,J=6.8Hz,6H).

[0107] (2) Synthesis of Compound e-2: Compound e-1 (2.208 g, 4.0 mmol) was first dissolved in EA (20.0 ml, 0.20 M), and then a 4 M hydrogen chloride solution in ethyl acetate (8.0 ml, 32.0 mmol) was added. The mixture was stirred at room temperature for 4 h. After completion of the reaction, the mixture was quenched by adding a saturated NaHCO₃ solution (40 ml), and extracted with EA (30 ml × 4). The organic phases were combined and dried over anhydrous Na₂SO₄. After filtration and concentration under reduced pressure, the residue was purified by column chromatography (DCM:MeOH = 10:1) to obtain compound e-2 as a light yellow viscous liquid (1.719 g, 3.80 mmol, 95%).

[0108] MS (ESI) m / z: [M+H] + 453.67. 1 H NMR (500MHz, CDCl3) δ2.84 (t, J = 6.1Hz, 2H), 2.59 (t,

[0109] J=6.1Hz,2H),2.45(t,J=7.6Hz,4H),1.43(s,4H),1.27–1.22(m,44H),0.89(t,J=6.9Hz,6H).

[0110] (3) Synthesis of compound e-3: Compound e-2 (1.50 g, 3.32 mmol) was first dissolved in a mixed solution of MeCN (33.2 ml, 0.1 M) and THF (10 ml). Then, a-6 (1.865 g, 6.64 mmol), K2CO3 (1.376 g, 9.96 mmol), and KI (0.11 g, 0.664 mmol) were added. The temperature was raised to reflux and stirred for 6 h. The reaction was monitored by TLC. After completion, saturated NH4Cl (40 ml) was added to quench the reaction. The mixture was extracted with EA (30 ml × 4). The organic phases were combined and dried over anhydrous Na2SO4. After filtration and concentration under reduced pressure, the residue was purified by column chromatography (PE:EA = 3:1) to obtain compound e-3 as a light yellow viscous liquid (2.014 g, 2.357 mmol, 71%).

[0111] MS (ESI) m / z: [M+H] + 855.66. 1 H NMR (500MHz, CDCl3) δ7.84(s,4H),7.70(s,4H),3.70(t,J=7.4Hz,4H),2.47(d,J=16.7Hz,8H),2.39 (s,4H),1.69(t,J=7.7Hz,4H),1.48(s,4H),1.41(s,4H),1.27–1.22(m,44H),0.88(t,J=7.4Hz,6H).

[0112] (4) Synthesis of compound e-4: Compound e-3 (2.0 g, 2.23 mmol) was dissolved in EtOH (22.3 ml, 0.10 M), and hydrazine hydrate (0.446 g, 8.92 mmol) was added. The mixture was heated to 40°C and stirred for 8 h. The reaction was monitored by TLC. Silica gel was added, the mixture was spin-dried, and the mixture was purified by column chromatography using a dry method [DCM: ammonia methanol (7 M in MeOH) = 4:1] to obtain compound e-4 as a light yellow viscous liquid (1.053 g, 1.619 mmol, 72.6%).

[0113] MS (ESI) m / z: [M+H] + 595.65. 1 H NMR(500MHz, CDCl3)δ3.29–2.99(m,8H),2.96(t,J=

[0114] 8.2Hz,2H),2.80(s,2H),1.94–1.86(m,4H),1.73–1.60(m,4H),1.38–1.21–1.18(m,52H),0.89(t,J=6.8Hz,6H).

[0115] (5) Synthesis of Compound II-5: Compound e-4 (178 mg, 0.3 mmol) was first dissolved in DCM (6 ml, 0.05 M), followed by the addition of compound a-2 (144.0 mg, 1.5 mmol) and stirring for 10 min. Finally, NaBH(OAc)3 (455.1 mg, 2.1 mmol) was added and stirred at room temperature for 12 h. The reaction was monitored by TLC and quenched by the addition of saturated NaHCO3 solution (15 ml). The mixture was extracted with DCM (6 ml × 3), and the organic phases were combined and dried over anhydrous Na2SO4. After filtration and concentration under reduced pressure, the residue was purified by column chromatography (DCM:MeOH = 5:1) to obtain compound II-5 as a colorless viscous liquid (148.0 mg, 0.162 mmol, 54%).

[0116] MS (ESI) m / z: [M+H] + 915.81. 1 H NMR (500MHz, CDCl3) δ7.55(s,8H),3.73(s,8H),2.67(s,12H),2.44(s,4H),1.53–1.48(m,12H),1.26–1.22(m,44H),0.88(t,J=7.2Hz,6H).

[0117] Example 5 Synthesis of ionizable lipid II-6

[0118]

[0119] (1) Synthesis of compound II-6: Referring to the method of step (5) of Example 4, compound II-6 was synthesized using e-4 and a-6 as starting materials. It was a colorless viscous liquid (152.1 mg, 0.162 mmol, 55%).

[0120] MS (ESI) m / z: [M+H] + 979.73. 1H NMR (500MHz, CDCl3) δ2.74–2.67(m,8H),2.51(s,4H),2.43–2.38(m,12H),2.2 4–2.18(m,8H),1.46–1.42(m,12H),1.28–1.22(m,44H),0.90(t,J=6.8Hz,6H).

[0121] Example 6 Synthesis of ionizable lipid II-9

[0122]

[0123] (1) Synthesis of compound II-6: Referring to the method of step (5) of Example 4, e-4 and a-7 were used as starting materials to obtain a colorless viscous liquid (143.7 mg, 0.147 mmol, 49%).

[0124] MS (ESI) m / z: [M+H] + 979.81. 1 H NMR(500MHz, CDCl3)δ7.22–7.15(m,4H),6.97–6.91(m,8H),3.83(d,J=3.9Hz,8H),2.53–2.4 6(m,8H),2.47–2.35(m,8H),1.52–1.42(m,12H),1.28–1.22(m,44H),0.90(t,J=7.1Hz,6H).

[0125] Example 7 Synthesis of ionizable lipid II-3

[0126]

[0127] (1) Synthesis of compound f-1: Referring to the method of step (1) of Example 4, compound f-1 was obtained as a light yellow viscous liquid (2.460 g, 4.96 mmol, 62%) using d-1 and a-5 as starting materials.

[0128] MS (ESI) m / z: [M+H] + 497.51. 1 H NMR (500MHz, CDCl3) δ3.14(s,2H),2.49(s,2H),2.38(t,J=7.5Hz,4H),1.44(s,9H),1.40(s,4H),1.26–1.21(m,36H),0.88(t,J=6.8Hz,6H).

[0129] (2) Synthesis of compound f-2: Referring to the method of step (2) of Example 4, compound f-2 was obtained as a light yellow viscous liquid (1.52 g, 3.88 mmol, 97%) using f-1 as the starting material.

[0130] MS (ESI) m / z: [M+H] + 397.44. 1 H NMR (500MHz, CDCl3) δ2.84 (t, J = 6.1Hz, 2H), 2.59 (t,

[0131] J=6.1Hz,2H),2.45(t,J=7.6Hz,4H),1.43(s,4H),1.27–1.21(m,36H),0.89(t,J=6.9Hz,6H).

[0132] (3) Synthesis of compound f-3: Referring to the method of step (3) of Example 4, f-1 and a-6 were used as starting materials to obtain compound f-3 as a light yellow viscous liquid (1.79 g, 2.25 mmol, 75%).

[0133] MS (ESI) m / z: [M+H] + 799.62. 1 H NMR (500MHz, CDCl3) δ7.84(s,4H),7.70(s,4H),3.70(t,J=7.4Hz,4H),2.47(d,J=16.7Hz,8H),2.39 (s,4H),1.69(t,J=7.7Hz,4H),1.48(s,4H),1.41(s,4H),1.27–1.22(m,36H),0.88(t,J=7.4Hz,6H).

[0134] (5) Synthesis of compound f-4: Referring to the method of step (4) of Example 4, compound f-4 was obtained as a light yellow viscous liquid (0.936 g, 1.74 mmol, 77.3%) using f-3 as the starting material.

[0135] MS (ESI) m / z: [M+H] + 539.59. 1 H NMR(500MHz, CDCl3)δ3.29–2.99(m,8H),2.96(t,J=

[0136] 8.2Hz,2H),2.80(s,2H),1.94–1.86(m,4H),1.73–1.60(m,4H),1.38–1.21–1.15(m,44H),0.89(t,J=6.8Hz,6H).

[0137] (6) Synthesis of compound II-3: Referring to the method of step (5) of Example 4, compound II-3 was obtained as a colorless viscous liquid (117.3 mg, 0.136 mmol, 45.6%) using f-4 and a-2 as starting materials.

[0138] MS (ESI) m / z: [M+H] + 859.74. 1 H NMR (500MHz, CDCl3) δ7.55 (s, 8H), 3.73 (s, 8H), 2.67 (s, 12H), 2.44 (s, 4H), 1.53 (s, 12H), 1.26–1.22 (m, 36H), 0.88 (t, J = 7.2Hz, 6H).

[0139] Example 8 Synthesis of ionizable lipid II-4

[0140]

[0141] (1) Synthesis of compound II-4: Referring to the method of step (5) of Example 4, compound II-4 was obtained as a colorless viscous liquid (148.0 mg, 0.162 mmol, 54%) using f-4 and a-7 as starting materials.

[0142] MS (ESI) m / z: [M+H] + 923.67. 1 H NMR (500MHz, CDCl3) δ2.74–2.67(m,8H),2.51(s,4H),2.43–2.38(m,12H),2.2 4–2.18(m,8H),1.46–1.42(m,12H),1.28–1.22(m,36H),0.90(t,J=6.8Hz,6H).

[0143] Example 9 Synthesis of ionizable lipid II-10

[0144]

[0145] (1) Synthesis of compound II-10: Referring to the method of step (5) of Example 4, compound II-10 was obtained as a colorless viscous liquid (121.7 mg, 0.132 mmol, 44%) using f-4 and a-8 as starting materials.

[0146] MS (ESI) m / z: [M+H] + 923.45. 1 H NMR(500MHz, CDCl3)δ7.22–7.16(m,4H),6.97–

[0147] 6.91(m,8H),3.83(d,J=3.9Hz,8H),2.53–2.47(m,8H),2.47–2.35(m,8H),1.52–1.42(m,12H),1.28–1.22(m,36H),0.90(t,J=7.1Hz,6H).

[0148] Example 10 Synthesis of ionizable lipid II-1

[0149]

[0150] (1) Synthesis of compound g-2: Compound g-1 (4.84 g, 20.0 mmol) was dissolved in DCM (100 ml, 0.20 M), and then DMP (5.086 g, 24.0 mmol) was added and stirred at room temperature for 20 min. The reaction was monitored by TLC. Saturated Na2S2O3 solution and saturated NaHCO3 solution were added to quench the reaction. The mixture was stirred vigorously for 20 min until the solution became clear. The mixture was extracted with DCM (80 ml × 3). The organic phases were combined and dried over anhydrous Na2SO4. After filtration and concentration under reduced pressure, the residue was purified by column chromatography (PE:EA=80:1) to obtain compound g-2 as a light yellow oily liquid (3.31 g, 16.75 mmol, 67%).

[0151] MS (ESI) m / z: [M+H] + 241.34. 1 H NMR (500MHz, CDCl3, ppm) δ9.75 (s, 1H), 2.41 (t, J = 7.3Hz, 2H) 1.63–1.61 (m, 2H), 1.34–1.28 (m, 24H), 0.88 (t, J = 6.7Hz, 3H).

[0152] (2) Synthesis of compound g-3: Referring to the method of step (1) of Example 4, compound g-3 was obtained as a light yellow viscous liquid (3.06 g, 5.04 mmol, 63%) using g-2 and a-5 as starting materials.

[0153] MS (ESI) m / z: [M+H] + 609.62. 1 H NMR (500MHz, CDCl3) δ3.14(s,2H),2.49(s,2H),2.38(t,J=7.5Hz,4H),1.44(s,9H),1.40(s,4H),1.26–1.21(m,52H),0.88(t,J=6.8Hz,6H).

[0154] (3) Synthesis of compound g-4: Referring to the method of step (2) of Example 4, compound g-4 was obtained as a light yellow viscous liquid (1.91 g, 3.76 mmol, 94%) using g-3 as the starting material.

[0155] MS (ESI) m / z: [M+H] + 509.52. 1 H NMR (500MHz, CDCl3) δ2.84(t,J=6.1Hz,2H),2.59(t,J=6.1Hz,2H),2.45(t,J=7.6Hz,4H),1.43(s,4H),1.27–1.22(m,52H),0.89(t,J=6.9Hz,6H).

[0156] (4) Synthesis of compound g-5: Referring to the method of step (3) of Example 4, using g-4 and a-6 as starting materials, compound g-5 was obtained as a light yellow viscous liquid (2.056 g, 2.26 mmol, 60.1%).

[0157] MS (ESI) m / z: [M+H] + 911.73. 1 H NMR (500MHz, CDCl3) δ7.84(s,4H),7.70(s,4H),3.70(t,J=7.4Hz,4H),2.47(d,J=16.7Hz,8H),2.39 (s,4H),1.69(t,J=7.7Hz,4H),1.48(s,4H),1.41(s,4H),1.27–1.22(m,52H),0.88(t,J=7.4Hz,6H).

[0158] (5) Synthesis of compound g-6: Referring to the method of step (4) of Example 4, compound g-6 was obtained as a light yellow viscous liquid (1.072 g, 1.650 mmol, 76.3%) using g-5 as the starting material.

[0159] MS (ESI) m / z: [M+H] + 651.72. 1 H NMR (500MHz, CDCl3) δ3.29–2.99 (m, 8H), 2.96 (t, J = 8.2Hz, 2H), 2.80 (s, 2H), 1. 94–1.86(m,4H),1.73–1.60(m,4H),1.38–1.21(m,60H),0.89(t,J=6.8Hz,6H).

[0160] (6) Synthesis of Compound II-1: Referring to the method of step (5) of Example 4, using g-6 and a-2 as starting materials, compound II-1 was obtained as a colorless viscous liquid (154.2 mg, 0.159 mmol, 53.3%).

[0161] MS (ESI) m / z: [M+H] + 971.87. 1 H NMR (500MHz, CDCl3) δ7.55 (s, 8H), 3.73 (s, 8H), 2.67 (s, 12H), 2.44 (s, 4H), 1.53 (s, 12H), 1.26–1.20 (m, 52H), 0.88 (t, J = 7.2Hz, 6H).

[0162] Example 11 Synthesis of ionizable lipid II-2

[0163]

[0164] (1) Synthesis of Compound II-2: Referring to the method of step (5) of Example 4, using g-6 and a-7 as starting materials, compound II-2 was obtained as a colorless viscous liquid (150.8 mg, 0.146 mmol, 48.6%).

[0165] MS (ESI) m / z: [M+H] + 1035.42. 1 H NMR (500MHz, CDCl3) δ2.74–2.67(m,8H),2.51(s,4H),2.43–2.36(m,12H),2 .24(m,8H),1.46–1.42(m,12H),1.28–1.21(m,52H),0.90(t,J=6.8Hz,6H).

[0166] Example 12 Synthesis of ionizable lipid II-8

[0167]

[0168] (1) Synthesis of compound II-8: Referring to the method of step (5) of Example 4, using g-6 and a-8 as starting materials, compound II-8 was obtained as a colorless viscous liquid (180.2 mg, 0.174 mmol, 58.1%).

[0169] MS (ESI) m / z: [M+H] +1035.38. H NMR (500MHz, CDCl3) δ7.22–7.15(m,4H),6.97–6.91(m,8H),3.83(d,J=3.9Hz,8H),2.53–2.4 7(m,8H),2.47–2.35(m,8H),1.52–1.42(m,12H),1.28–1.22(m,52H),0.90(t,J=7.1Hz,6H).

[0170] Example 13 Synthesis of ionizable lipid II-13

[0171]

[0172] (1) Synthesis of compound h-2: Referring to the method of step (1) of Example 10, compound h-2 was obtained as a light yellow oily liquid (4.210 g, 16.00 mmol, 63.8%) using h-1 as the starting material.

[0173] MS (ESI) m / z: [M+H] + 265.45. 1 H NMR (500MHz, CDCl3, ppm) δ9.75 (s, 1H), 5.42–5.28 (m, 4H), 2.76 (t, J=6.4Hz, 2H), 2.41 (t, J= 7.3Hz, 2H), 2.04–1.92 (m, 4H), 1.63–1.61 (m, 2H), 1.34–1.28 (m, 14H), 0.88 (t, J = 6.7Hz, 3H).

[0174] (2) Synthesis of compound h-3: Referring to the method of step (1) of Example 4, h-2 and a-5 were used as starting materials to obtain compound h-3 as a light yellow viscous liquid (3.30 g, 5.032 mmol, 62.9%).

[0175] MS (ESI) m / z: [M+H] + 657.62. 1 H NMR (500MHz, CDCl3, ppm) δ5.41–5.29 (m, 8H), 3.15 (s, 2H), 2.77 (t, J = 6.2Hz, 4H), 2.49 (s, 2H), 2.40 –2.37(m,4H),2.04(dd,J=13.1,6.4Hz,8H),1.44(s,9H),1.33–1.28(m,36H),0.88(t,J=6.2Hz,6H).

[0176] (3) Synthesis of compound h-4: Referring to the method of step (2) of Example 4, compound h-4 was obtained as a light yellow viscous liquid (2.180 g, 3.92 mmol, 98%) using h-3 as the starting material.

[0177] MS (ESI) m / z: [M+H] + 557.32. 1 H NMR (400MHz, CDCl3, ppm) δ5.41–5.29(m,8H),2.78–2.71(m,6H),2.47(t,J=5.9Hz,2H),2. 41–2.38(m,4H),2.04(dd,J=13.4,6.6Hz,8H),1.41–1.27(m,36H),0.88(t,J=6.6Hz,6H).

[0178] (4) Synthesis of compound h-5: Referring to the method of step (3) of Example 4, h-4 and a-6 were used as starting materials to obtain compound h-5 as a light yellow viscous liquid (2.590 g, 2.708 mmol, 69.1%).

[0179] MS (ESI) m / z: [M+H] + 959.46. 1 H NMR (500MHz, CDCl3) δ7.84(s,4H),7.70(s,4H),5.41–5.29(m,8H),3.70(t,J=7.4Hz,4H),2.47(d,J=16.7Hz,8H),2.44(t,J=7.4Hz ,4H),2.07–1.92(m,8H),2.39(s,4H),1.69(t,J=7.7Hz,4H),1.48(s,4H),1.41(s,4H),1.27–1.22(m,32H),0.88(t,J=7.4Hz,6H).

[0180] (5) Synthesis of compound h-6: Referring to the method of step (4) of Example 4, compound h-6 was obtained as a light yellow viscous liquid (1.430 g, 2.049 mmol, 75.7%) using h-5 as the starting material.

[0181] MS (ESI) m / z: [M+H] + 699.48. 1H NMR (500MHz, CDCl3) δ5.41–5.29(m,8H),3.29–2.99(m,8H),2.96(t,J=8.2Hz,2H),2.80(s,2H),2.44(t,J=7. 4Hz,4H),2.07–1.93(m,8H),1.94–1.86(m,4H),1.73–1.60(m,4H),1.38–1.21(m,40H),0.89(t,J=6.8Hz,6H).

[0182] (6) Synthesis of compound II-13: Referring to the method of step (5) of Example 4, h-6 and a-2 were used as starting materials to obtain compound II-13 as a colorless viscous liquid (153 mg, 0.141 mmol, 47.3%).

[0183] MS (ESI) m / z: [M+H] + 1019.98. 1 H NMR(500MHz, Methanol-d4)δ7.58(s,8H),5.34–5.29(m,8H),3.59(s,8H),2.78(t,J=6.5Hz,4H),2.69(s,4H),2.56–2.47(m,8H) ,2.44(t,J=7.4Hz,4H),2.07–1.93(m,8H),1.59(t,J=7.6Hz,4H),1.50–1.46(m,8H),1.35–1.31(m,32H),0.92(t,J=6.7Hz,6H).

[0184] Example 14 Synthesis of ionizable lipid II-12

[0185]

[0186] (1) Synthesis of Compound II-12: Referring to the method of step (5) of Example 4, using h-6 and a-7 as starting materials, compound II-12 was obtained as a colorless viscous liquid (160.0 mg, 0.148 mmol, 49.3%).

[0187] MS (ESI) m / z: [M+H] + 1083.46. 1H NMR (500MHz, CDCl3) δ5.34–5.28(m,8H),2.74–2.67(m,8H),2.51(s,4H),2.43–2.38(m,12H),2.40(t,J=7.4H z,4H),2.24–2.20(m,8H),1.46–1.42(m,12H),2.07–1.92(m,8H),1.28–1.22(m,32H),0.90(t,J=6.8Hz,6H).

[0188] Example 15 Synthesis of ionizable lipid II-14

[0189]

[0190] (1) Synthesis of Compound II-14: Referring to the method of step (5) of Example 4, using h-6 and a-9 as starting materials, compound II-14 was obtained as a colorless viscous liquid (148.0 mg, 0.162 mmol, 54%).

[0191] MS (ESI) m / z: [M+H] + 1083.59. 1 H NMR (500MHz, CDCl3) δ7.22–7.17(m,4H),6.97–6.91(m,8H),5.34–5.28(m,8H),3.83(d,J=3.9Hz,8H),2.53–2.4 8(m,8H),2.47–2.35(m,12H),2.07–1.94(m,8H)1.52–1.42(m,12H),1.28–1.23(m,32H),0.90(t,J=7.1Hz,6H).

[0192] Example 16 Synthesis of ionizable lipid II-15

[0193]

[0194] (1) Synthesis of Compound II-14: Referring to the method of step (5) of Example 4, h-6 and a-10 were used as starting materials to obtain Compound II-14 as a colorless viscous liquid (148.0 mg, 0.162 mmol, 54%).

[0195] MS (ESI) m / z: [M+H] + 1289.72. 1H NMR(500MHz,Chloroform-d)δ7.57(s,4H),7.49(d,J=7.7Hz,4H),7.45(d,J=7.8Hz,4H),7.37(t,J=7.7Hz,4H),5.33–5.30(m,8H),3.56(s,8H),2.75(t ,J=6.8Hz,4H),2.45(s,4H),2.40–2.36(m,8H),2.31(t,J=7.4Hz,4H),2.03 –1.92(m,8H),1.46–1.44(m,4H),1.33–1.23(m,40H),0.86(t,J=7.1Hz,6H).

[0196] Example 17 Synthesis of Ionizable Lipid II-11

[0197]

[0198] (1) Synthesis of compound i-2: Referring to the method of step (1) of Example 4, i-1 and a-5 were used as starting materials to obtain compound i-2 as a light yellow viscous liquid (2.983 g, 6.78 mmol, 67.8%).

[0199] MS (ESI) m / z: [M+H] + 441.73. 1 H NMR (500MHz, Chloroform-d) δ3.14(s,2H),2.49(s,2H),2.38(t,J=7.5Hz,4H),1.44(s,9H),1.40(s,4H),1.26–1.21(m,28H),0.88(t,J=6.8Hz,6H).

[0200] (2) Synthesis of compound i-3: Referring to the method of step (2) of Example 4, compound i-3 was obtained as a light yellow viscous liquid (2.261 g, 6.651 mmol, 98.1%) using i-2 as the starting material.

[0201] MS (ESI) m / z: [M+H] + 341.63. 1 H NMR(500MHz,Chloroform-d)δ2.84(t,J=6.1Hz,2H),

[0202] 2.59(t,J=6.1Hz,2H),2.45(t,J=7.6Hz,4H),1.43(s,4H),1.27–1.21(m,28H),0.89(t,J=6.9Hz,6H).

[0203] (3) Synthesis of compound i-4: Referring to the method of step (3) of Example 4, i-3 and a-6 were used as starting materials to obtain compound i-4 as a light yellow viscous liquid (3.689 g, 4.968 mmol, 74.7%).

[0204] MS (ESI) m / z: [M+H] + 743.34. 1 H NMR(500MHz,Chloroform-d)δ7.84(s,4H),7.70(s,4H),3.70(t,J=7.4Hz,4H),2.47(d,J=16.7Hz,8H),2 .39(s,4H),1.69(t,J=7.7Hz,4H),1.48(s,4H),1.41(s,4H),1.27–1.23(m,28H),0.88(t,J=7.4Hz,6H).

[0205] (4) Synthesis of compound i-5: Referring to the method of step (4) of Example 4, compound i-5 was obtained as a light yellow viscous liquid (1.635 g, 3.393 mmol, 68.3%) using i-4 as the starting material.

[0206] MS (ESI) m / z: [M+H] + 483.45. 1 H NMR(500MHz,Chloroform-d)δ3.29–2.99(m,8H),2.96(t,J=8.2Hz,2H),2.80(s,2H ),1.94–1.86(m,4H),1.73–1.60(m,4H),1.38–1.21(m,36H),0.89(t,J=6.8Hz,6H).

[0207] (5) Synthesis of compound II-11: Referring to the method of step (5) of Example 4, using i-5 and a-2 as starting materials, compound II-11 was obtained as a colorless viscous liquid (102.7 mg, 0.128 mmol, 42.7%).

[0208] MS (ESI) m / z: [M+H] + 803.68. 1 H NMR (500MHz, Chloroform-d) δ7.55(s,8H),3.73(s,8H),2.67(s,12H),2.44(s,4H),1.53(s,12H),1.26–1.21(m,28H),0.88(t,J=7.2Hz,6H).

[0209] Example 18 Synthesis of ionizable lipid II-7

[0210]

[0211] (1) Synthesis of compound II-7: Referring to the method of step (5) of Example 4, i-5 and a-7 were used as starting materials to obtain compound II-7 as a colorless viscous liquid (127.0 mg, 0.147 mmol, 48.9%).

[0212] MS (ESI) m / z: [M+H] + 867.58. 1 H NMR (500MHz, CDCl3) δ5.34–5.28(m,8H),2.74–2.67(m,8H),2.51(s,4H),2.43–2.38(m,8 H), 2.40 (t, J = 7.4Hz, 4H), 1.46–1.42 (m, 12H), 1.28–1.23 (m, 28H), 0.90 (t, J = 6.8Hz, 6H).

[0213] Example 19 Preparation of lipid nanoparticles by encapsulating mRNA with ionizable lipids

[0214] The materials used for assembling lipid nanoparticles include: (1) ionizable lipid compounds: such as the ionizable lipids designed and synthesized in the present invention; (2) structural lipids: such as Cholesterol (purchased from Sigma-Aldrich); (3) phospholipids: such as DSPC (Distearoylphosphatidylcholine, purchased from AVT); (4) PEGylated lipid compounds: such as DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, purchased from AVT); (5) nucleic acid fragment active ingredients: such as Luciferase mRNA (self-made).

[0215] Preparation method of lipid nanoparticles: ionizable lipid: phospholipid: cholesterol: PEGylated lipid are mixed in anhydrous ethanol in a ratio of 48.5:10:40:1.5 (molar percentage) to form an alcohol phase; then Luciferase mRNA is dissolved in 50mM sodium acetate solution (pH=5) to form an aqueous phase, wherein the mass ratio of mRNA to ionizable lipid is 1:10, and the volume ratio of the alcohol phase to the aqueous phase is 1:3.

[0216] Then, an automated high-throughput microfluidic system was used to prepare LNPs, with the lipid molecule DLin-MC3-DMA (purchased from AVT) as a positive control. The LNP mixing speed was set to 16 mL / min, and the mixing flow rate ratio of the alcohol phase and the aqueous phase was 1:3. The prepared LNPs (N / P ratio of 6) were diluted with phosphate buffered saline (PBS) and then placed in an ultrafiltration tube with a molecular weight cutoff of 30 kDa. The tubes were centrifuged at 4°C and 3000 rpm for 30 minutes, and the elution was repeated twice to remove ethanol and sodium acetate as much as possible. Finally, the concentrated LNPs were obtained and stored in a 4°C refrigerator for future use.

[0217] Methods for preparing lipid nanoparticles include, but are not limited to, microfluidic mixing systems and also include T-type mixers and ethanol injection methods.

[0218] Example 20 Particle size and polydispersity index (PDI) of lipid nanoparticles

[0219] After LNP preparation is completed, its particle size and polydispersity index (PDI) need to be evaluated first. The numerical value is closely related to in vitro cell transfection and effective delivery in vivo.

[0220] The freshly prepared LNP sample was equilibrated at room temperature, diluted with PBS at a ratio of 1:100, and then placed in a sample cell. The particle size and PDI size were measured using a dynamic light scattering instrument (Malvern Zetasizer Nano-ZS).

[0221] Depend on Figure 1 As can be seen, the lipid nanoparticles prepared by the present invention have a particle size range of 60-200 nm; with the exception of II-4 and II-7, the polydispersity index of the remaining particles is less than 0.3. As shown in I-1, I-2, and I-3, when the ionizable lipid is composed of a bipyrazole head and four hydrophobic tail chains, the LNP particle size is relatively small, approximately between 60 and 100 nm. However, when the ionizable lipid head is connected to four pyrazole rings and the number of tail chains is two, the particle size increases significantly to approximately 140 nm.

[0222] From the PDI of II-4 and II-7, it can be seen that the ionizable lipid with 4 3Fs attached to the head should have a hydrophobic tail chain length of not less than 12 carbons. When the carbon chain length is between 14 and 18, lipid nanoparticles with a particle size of less than 200 nm and uniform dispersion (PDI less than 0.3) can be obtained.

[0223] The size of lipid nanoparticles has a great influence on their uptake by cells. In most cases, the particle size is controlled between 50-200nm, which is conducive to the effective uptake and transfection of lipid nanoparticles. Figure 1The test results of lipid nanoparticle size and PDI indicate that the present invention prepared 16 lipid nanoparticles with uniform particle size distribution and a size suitable for entering cells.

[0224] Example 21 Determination of lipid nanoparticle encapsulation efficiency

[0225] The lipid nanoparticle encapsulation efficiency was determined using Quant-iT RiboGreen RNA Assay (purchased from Invitrogen). This coefficient reflects the degree of binding between lipid nanoparticles and mRNA fragments. The lipid nanoparticle sample was diluted in TE buffer (10mM Tris HCl, 1mM EDTA, pH = 7.5), and a portion of the sample solution was taken out and added with 0.5% Triton X100 and allowed to stand at 37°C for 30 minutes. After adding the reaction solution for 3 minutes, the fluorescence value was read on a multifunctional microplate reader (purchased from Thermofisher, model: Thermo Varioskan LUX) at an absorption band of 480nm and an emission band of 520nm to obtain the encapsulation efficiency value. Encapsulation efficiency EE (%) = (total mRNA - free mRNA) / total mRNA × 100%

[0226] The results are as follows Figure 2 As shown in the figure, lipid nanoparticles I-1, I-2, and I-3 had high mRNA encapsulation efficiencies, ranging from approximately 90% to 100%. However, II-2, II-13, and II-15 had poor mRNA encapsulation capabilities, with encapsulation efficiencies of less than 50%. The remaining lipid nanoparticles all had encapsulation efficiencies above 60%.

[0227] Combined with the chemical structure of ionizable lipids, we can know that the head containing a tertiary amine as the core, such as I-1, I-2 and I-3, has an encapsulation efficiency of more than 90%; however, if the head is increased to two tertiary amine structures (such as II-1, II-3 and II-5), the encapsulation efficiency will drop significantly (about 60%). Therefore, it can be concluded that it is best for the ionizable lipid head to have a tertiary amine structure.

[0228] In addition, according to the encapsulation efficiency of I-1, I-2 and I-3 (all maintained above 90%), it can be seen that increasing the number of tail chains does not lead to a decrease in the encapsulation efficiency.

[0229] Example 22 In vitro screening of lipid nanoparticles

[0230] This experiment used 293T cells to evaluate the mRNA transfection efficiency based on LNP. The culture medium used was DMEM high glucose medium (supplemented with 10% fetal bovine serum). One day before transfection, 2×10 4Cells were seeded into 96-well plates at a density of 100 μL per well and cultured overnight at 37°C, 5% CO2, and 90% humidity. After 12 hours, LNPs were added dropwise to the cells for transfection. The mRNA concentrations were 2 μg / mL and 1 μg / mL, and 5 replicates were set for each concentration. The commercialized ionizable lipid DLin-MC3-DMA (MC3 for short) was used as a positive transfection control. After 24 hours of incubation, the luciferase substrate ONE-Glo was added. TM Luciferase Assay System was added to a 96-well culture plate at 100 μL / well, and the cell fluorescence intensity was detected using a multifunctional microplate reader (ThermoScientfic, model: Thermo Varioskan LUX).

[0231] like Figure 3 As shown in Figure 2, at a concentration of 1 μg / mL, the transfection efficiency of I-8-LNP and II-5-LNP in 293T cells was as high as 10 7 The results are above 1:1, which is higher than the transfection ability of the positive control Dlin-MC3-DMA. This indicates that LNPs prepared from ionizable lipids I-8 and II-5 have good cell transfection efficiency after encapsulating mRNA.

[0232] The chemical structures of the ionizable lipids I-1, I-2, and I-3, each with a bipyrazole ring as its core, reveal that the transfection efficiency of lipid nanoparticles is positively correlated with the length of the ionizable lipid tail chain. Among the three, a lipid tail chain length of 14 carbon atoms exhibits the highest transfection efficiency. When the ionizable lipid is a bipyrazole tertiary amine head, lipid nanoparticles with two hydrophobic tail chains of 14 carbon atoms exhibit the highest transfection efficiency; other chain lengths result in decreased transfection efficiency. Lipid nanoparticles containing four thiazole head groups exhibit higher transfection efficiency. For ionizable lipids composed of four tertiary amine heads composed of 3F-, longer tail chains are associated with improved transfection efficiency.

[0233] Example 23 Lipid Nanoparticle Cytotoxicity Detection

[0234] The cytotoxicity of lipid nanoparticles was evaluated using 293T cells. 293T cells were plated into 96-well plates at a cell seeding density of 3 × 10 4Cells were incubated at 37°C, 5% CO2, and 90% humidity for 12 hours. Luciferase mRNA-encapsulated lipid nanoparticles were co-incubated with 293T cells at varying mRNA concentrations (4 μg / mL, 2 μg / mL, and 1 μg / mL), with five replicates per well. The cells were incubated at 37°C, 5% CO2, and 90% humidity. A commercially available ionizable lipid, DLin-MC3-DMA (MC3), served as a positive control. After 24 hours of incubation, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C, 5% CO2, and 90% humidity. After 1 hour, cell viability was calculated using a microplate reader with absorbance set at 450 nm.

[0235] The results are as follows Figure 4 It can be seen that the cytotoxicity of the lipid nanoparticles of the ionizable lipids I-1, I-2 and I-3 with a tertiary amine head connected to two pyrazoles decreases with the increase of the chain length of the two hydrophobic tail chains of the ionizable lipids; it can be seen from the ionizable lipids II-1, II-3 and II-5 that when four pyrazoles are introduced into the ionizable lipid head, the cytotoxicity of the lipid nanoparticles decreases with the increase of the tail chain length; it can be seen from the ionizable lipids II-6, II-7 and II-12 that when four 3Fs are introduced into the ionizable lipid head, the cytotoxicity of the lipid nanoparticles is relatively small, and at high doses (4μg / mL), the cell survival rate is above 75%; in addition, when the ionizable lipid II-14 head contains four thiazoles and the two hydrophobic tail chains are 18 carbons long, the cytotoxicity of the lipid nanoparticles is small, and the cell survival rate is above 80%, indicating that the lipid nanoparticles have low cytotoxicity and good biocompatibility.

[0236] Example 24 In vivo biodistribution of representative lipid nanoparticles

[0237] In order to detect the in vivo distribution of lipid nanoparticles, II-1, II-5, II-12 and II-14 were selected for animal experiments.

[0238] All animal studies were conducted in accordance with animal ethics guidelines, and the protocol was approved by the Veterinary Services Department. SPF female BALB / c mice, 7-8 weeks old, weighing 20 to 22 g, were purchased from Guangdong Yaokang Biotechnology Co., Ltd. All animals were acclimated for 7 days prior to the experiment. During the experiment, they had free access to food and water and were placed under a 12 / 12-hour light / dark cycle. The room temperature was 25°C and the humidity was 50%. The mice were randomly divided into groups to evaluate the targeted delivery efficiency of different nanoparticles in mice.

[0239] The administration method is divided into two ways: tail vein injection and intramuscular injection, and the intramuscular injection site is the right muscle of the mouse hind limb.

[0240] Lipid nanoparticles were prepared using the molar ratio of each lipid in the lipid nanoparticles (ionizable lipid:phospholipid:cholesterol:PEGylated lipid = 48.5:10:40:1.5). Commercially available ionizable lipid SM102 was used as a positive control, and 1×PBS was injected as a negative control.

[0241] Lipid nanoparticles were injected into mice at a single dose of 0.5 mg / kg mRNA, resulting in a 10 μg injection in a 100 μL volume. Six hours later, each mouse received an intraperitoneal injection of 3 mg of the luciferase substrate, D-luciferin. The heart, liver, spleen, lung, kidney, and muscle were then harvested. In vivo bioluminescence was measured using an IVIS imaging system.

[0242] The specific detection steps are as follows: First, the luciferase substrate D-luciferin was diluted with physiological saline to a concentration of 15 mg / mL. Each mouse was then intraperitoneally injected with 200 μL of this 15 mg / mL D-luciferin solution. The mice were then anesthetized in an anesthesia chamber. After 10 minutes, the anesthetized mice were removed and placed in an IVIS instrument for in vivo imaging. The organ specificity of the lipid nanoparticles after in situ muscle delivery is shown in Table 2 (n = 3).

[0243] The fluorescence distribution ratio of lipid nanoparticles in mouse organs was calculated as follows: fluorescence value of (specific organ) / total fluorescence value of (heart, liver, spleen, lung, kidney, and muscle) × 100%. The experimental results are presented as the average ratio of mice within the same test group. The results are shown in Tables 2 and 3.

[0244] Table 2 Fluorescence distribution of mouse organs after tail vein injection of lipid nanoparticles

[0245]

[0246] Table 3 Fluorescence distribution of mouse organs after intramuscular injection of lipid nanoparticles

[0247]

[0248] As shown in Table 2, after intravenous injection, ionizable lipids II-1 and II-5 were mainly delivered to the lungs, while II-12 and II-14 were mainly delivered to the liver and spleen, respectively.

[0249] From the chemical structures of II-1 and II-5, it can be seen that both have a tertiary amine head connected to four pyrazoles and two hydrophobic tail chains. Therefore, it is inferred that the reason for the difference in in vivo targeting of lipid nanoparticles may be the head structure of the ionizable lipid; and it was found that the longer the two hydrophobic tail chains of the ionizable lipid, the stronger the targeting of the lipid nanoparticles to the lungs.

[0250] It can be seen from II-12 that when the ionizable lipid head is connected to four 3F tertiary amines, its lipid nanoparticles mainly reach the liver after intravenous injection; it can be seen from II-15 that when the ionizable lipid head is a tertiary amine connected to four thiazoles, its lipid nanoparticles can reach the spleen and 45% can reach the liver after intravenous injection.

[0251] According to the distribution ratio of lipid nanoparticles in mouse organs after intramuscular injection in Table 3, it can be seen that after intramuscular injection, II-1 and II-5 have low selectivity in the body (heart, liver, spleen, lung, kidney and muscle), and the fluorescence distribution ratio is less than 45%; although II-12 and II-14 have the same tertiary amine structure in the head, the groups attached to the head are different. However, the experimental results show that after intramuscular injection, the above lipid nanoparticles are all targeted to the muscle, and the targeted distribution ratio is as high as more than 90%.

[0252] The results show that the lipid nanoparticles prepared based on ionizable lipids of the present invention have good in vivo delivery efficiency and show a targeting trend in the liver, spleen, and muscles.

[0253] The applicant states that while the present invention illustrates the ionizable lipid compounds and their applications through the aforementioned embodiments, the present invention is not limited to the aforementioned process steps, nor does it imply that the present invention must rely on the aforementioned process steps for implementation. Those skilled in the art will appreciate that any improvements to the present invention, equivalent substitutions for the raw materials used, addition of auxiliary components, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims

1. An ionizable lipid compound, characterized in that The ionizable lipid compound has a structure as shown in formula (I): Wherein M, Q, and K may be the same or different and are selected from -(CH2) m1 -、-(CH2) n1 C(O)(CH2) p 、-(CH2) n1 C(O)O(CH2) p -, m1 is an integer from 1 to 6, n1 is an integer from 1 to 3, and p is an integer from 1 to 3; R' and R" may be the same or different and are selected from -(CH) q -、-(CH) q CH=CH(CH) s -、-(CH2) q NH(CH2) s 、-(CH) q C(O)(CH) s -, wherein q and s are independently integers from 1 to 3; (1) R1, R2, R3, R4, R5 and R6 may be the same or different and are selected from C1-6 alkyl, C 3-7 Cycloalkyl, 4-7 membered heterocyclic group, C 6-10 Aryl, 5-10 membered heteroaryl, benzoheterocyclyl, heterocyclyl and heterocyclyl, the above alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, benzoheterocyclyl or heterocyclyl and heterocyclyl groups are optionally substituted by a group selected from the following: halogen atoms, (2) R1, R2, R3, R4, R5, and R6 may be the same or different and may be selected from substituted or unsubstituted linear or branched C 3-30 Alkyl, substituted or unsubstituted linear or branched C 3-30 Alkenyl, substituted or unsubstituted straight chain or branched C 3-30 Alkynyl, where R c and R d Can be the same or different, selected from C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 Alkynyl, n and m are selected from integers of 1-15; In the structure, at least one of R1, R2, R3, R4, R5 and R6 is selected from (1), and at least one is selected from (2).

2. Preferably, in (1), R1, R2, R3, R4, R5 and R6 are selected from Preferably, R1, R2, R3, R4, R5 and R6 in (2) are selected from in Indicates the attachment site of a group.

3. The ionizable lipid compound according to claim 1 or 2, characterized in that The ionized ester compound is selected from any one of the structures shown in the following formula II-III: wherein Ra is within the same range as described in (1); R1, R2, R3, R4, R5, and R6 are within the same range as described in (2); and R', R", M, Q, and K are within the same range as described in formula (I).

4. The ionizable lipid compound according to any one of claims 1 to 3, characterized in that The ionizable lipid compound has any one of the structures shown in the following formulas A1-A16: Preferably, the ionizable lipid compound has any one of the structures shown in the following formulas IV-V: Wherein Ra is the same as the range described in (1); R b、 R c The same range as described in (2); R', R", M, Q, K are the same as those described in formula (1); Preferably, the ionizable lipid compound has any one of the structures shown in the following formulas B1-B16:

5. The ionizable lipid compound according to any one of claims 1 to 4, characterized in that The ionizable lipid compound is selected from any one of the following compounds:

6. A liposome, characterized in that The liposome comprises an ionizable lipid compound according to any one of claims 1 to 5; Preferably, the liposome further comprises a co-lipid; Preferably, the co-lipid comprises a phospholipid; Preferably, the phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-diondecanoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2 -Di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphyton Alkyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-bisdocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoyl Any one or a combination of at least two of palmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine or lysophosphatidylethanolamine; Preferably, the co-lipid further comprises structural lipids; Preferably, the structured lipid comprises at least one or a combination of at least two of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid or α-tocopherol; Preferably, the co-lipid further comprises PEGylated lipids; Preferably, the PEGylated lipid comprises any one or a combination of at least two of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide or PEG-modified dialkylamine; Preferably, the liposomes comprise ionizable lipid compounds, phospholipids, structural lipids and PEGylated lipids; Preferably, the liposomes comprise, by molar percentage, 20%-80% of ionizable lipid compounds, 5%-30% of phospholipids, 10%-50% of structural lipids, and 1%-10% of PEGylated lipids.

7. A lipid complex, characterized in that The lipid complex comprises the liposome according to claim 6 and an active substance; Preferably, the active substance comprises any one or a combination of at least two of a small molecule compound, a nucleic acid molecule or a protein molecule; Preferably, the nucleic acid molecule comprises any at least one or a combination of at least two of DNA, mRNA, siRNA, aiRNA, miRNA, dsRNA, aRNA or lncRNA; Preferably, the protein molecule comprises any one or a combination of at least two of an enzyme, an antibody, a short peptide, a polypeptide or a recombinant protein; Preferably, the method for preparing the lipid complex comprises the following steps: mixing liposomes and active substances by a mixer to obtain the lipid complex.

8. A lipid nanoparticle, characterized in that The lipid nanoparticles include the liposomes according to claim 6 or the lipid complexes according to claim 7; Preferably, the particle size of the lipid nanoparticles is 40-300 nm.

9. A reagent, kit, preparation or pharmaceutical composition, characterized in that: The reagent, kit, preparation or pharmaceutical composition each independently comprises any one or a combination of at least two of the ionizable lipid compound according to any one of claims 1 to 5, the liposome according to claim 6, the lipid complex according to claim 7 or the lipid nanoparticle according to claim 8.

10. Use of the ionizable lipid compound according to any one of claims 1 to 5, the liposome according to claim 6, the lipid complex according to claim 7, the lipid nanoparticle according to claim 8, or the reagent, kit, preparation or pharmaceutical composition according to claim 9 in the preparation of a product having any one of the following functions (1) to (4): (1) Encapsulation of active substances; (2) delivering the encapsulated active substance to cells, tissues, or organs; (3) enabling the delivered active substance to exert its active effect in cells, tissues or organs; (4) Prevent, diagnose or treat diseases.

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