Fluorinated lipid nanoparticles as well as preparation method and application thereof

Abstract

The invention provides a fluorinated liposome as well as a preparation method and application thereof. The preparation method of the series of highly fluorinated ionizable lipid molecules with novel structures has the advantages of simple synthesis and high yield. The invention further provides the LNP containing the fluoro ionizable lipid or the composite ionizable lipid, the LNP is good in particle size dispersity, high in nucleic acid encapsulation efficiency and low in in-vivo toxicity, nucleic acid drugs can be efficiently delivered to enable the LNP to have better cell entering efficiency and nucleic acid expression efficiency, the LNP has spleen targeting performance, and the LNP can be used for preparing the LNP. The method has a great application prospect in immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to fluorinated lipid nanoparticles and a preparation method and application thereof. Background Art

[0002] Messenger RNA (mRNA) is an important part of the central dogma of molecular biology. Once exogenous mRNA is delivered into the cytoplasm of a cell, the mRNA can express the encoded protein as a therapeutic drug or vaccine. People have long been interested in developing mRNA-based drugs. However, the physicochemical properties and biological characteristics of mRNA pose great challenges to its delivery. Due to its negative charge, hydrophilicity and large molecular weight, mRNA cannot pass through the cell membrane. In addition, naked mRNA will be rapidly degraded by enzymes in the body. Therefore, mRNA requires a delivery vehicle to transport it into cells and improve its stability. Among the different categories of delivery vehicles, liposome nanoparticles (LNPs) are currently the only mRNA carriers approved by regulatory authorities.

[0003] LNPs are composed of ionizable lipids, helper lipids, cholesterol, and polyethylene glycol (PEG) lipids. Among these components, the chemical structure of the ionizable lipid has a great influence on the delivery efficiency of mRNA. The ionizable lipid is composed of a hydrophobic alkyl tail and one or more amino heads. The ionizable lipid is positively charged in acidic buffer, interacts with negatively charged mRNA to form LNP, then becomes uncharged at physiological pH to avoid rapid clearance in the systemic circulation, and finally becomes positively charged in the endosomes of the cell to promote the release of mRNA. A variety of ionizable lipids have been developed and show good efficiency in delivering mRNA. Despite these advances, there is still a great need for new ionizable lipids to improve the delivery efficiency to reduce the toxicity and immunogenicity associated with mRNA and LNP. Summary of the invention

[0004] The present invention provides a fluorinated modified ionizable lipid as shown in Formula I:

[0005]

[0006] Among them, N represents the nitrogen element;

[0007] M, P, and Q are the same or different, and are independently selected from biodegradable functional groups; preferably, including but not limited to ester groups, amide groups, acetal groups, ether bonds, and thioether bonds;

[0008] L1, L2, L3 are the same or different, and are independently selected from C1-20 alkyl; the C1-20 alkyl is optionally substituted by one or more halogens, hydroxyls, and mercapto groups; the carbon atoms in the C1-20 alkyl are optionally substituted by oxygen atoms or sulfur atoms;

[0009] R1 and R2 are the same or different and are independently selected from C1-6 alkyl; the C1-6 alkyl is optionally substituted by one or more halogens, hydroxyls, and mercapto groups; the carbon atoms in the C1-6 alkyl are optionally substituted by oxygen atoms or sulfur atoms;

[0010] R3 and R4 are the same or different and are independently selected from C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl; the C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl are optionally substituted by one or more halogen, hydroxyl, thiol; the carbon atoms in the C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl are optionally substituted by oxygen atoms or sulfur atoms;

[0011] Among the M, P, Q, L1, L2, L3, R1, R2, R3, and R4 groups, at least one group is substituted by one or more fluorine atoms.

[0012] According to an embodiment of the present invention, M, P, and Q are the same or different and are independently selected from an ester group, an ether bond, and a thioether bond;

[0013] Preferably, M, P, Q are selected from ester groups, such as -O-CO- or -CO-O-.

[0014] According to an embodiment of the present invention, L1 is selected from C1-10 alkyl; the C1-10 alkyl is optionally substituted by one or more halogens, hydroxyls, and mercapto groups; the carbon atoms in the C1-10 alkyl are optionally substituted by oxygen atoms or sulfur atoms;

[0015] Preferably, L1 is selected from C1-6 alkyl; the C1-6 alkyl is optionally substituted by one or more halogen, hydroxyl, and mercapto; the carbon atoms in the C1-6 alkyl are optionally substituted by oxygen atoms and sulfur atoms;

[0016] Further preferably, L1 is selected from C1-3 alkyl; the C1-3 alkyl is optionally substituted by one or more halogen, hydroxyl, and mercapto; the carbon atoms in the C1-3 alkyl are optionally substituted by oxygen atoms and sulfur atoms;

[0017] For example, L1 is selected from C3 alkyl (n-propyl, isopropyl).

[0018] According to an embodiment of the present invention, L2 and L3 are the same or different and are independently selected from C1-10 alkyl; the C1-10 alkyl is optionally substituted by one or more halogens, hydroxyls, and mercapto groups; the carbon atoms in the C1-10 alkyl are optionally substituted by oxygen atoms or sulfur atoms;

[0019] Preferably, L2 and L3 are the same or different and are independently selected from C3-8 alkyl; the C3-8 alkyl is optionally substituted by one or more halogens, hydroxyls, and mercapto groups; the carbon atoms in the C3-8 alkyl are optionally substituted by oxygen atoms or sulfur atoms;

[0020] Further preferably, L2 and L3 are the same or different and are independently selected from C5-8 alkyl; the C5-8 alkyl is optionally substituted by one or more halogens, hydroxyls, and mercapto groups; the carbon atoms in the C5-8 alkyl are optionally substituted by oxygen atoms or sulfur atoms;

[0021] For example, L2 and L3 are selected from C7 alkyl (heptyl).

[0022] According to an embodiment of the present invention, R1 and R2 are the same or different and are independently selected from C1-3 alkyl; the C1-3 alkyl is optionally substituted by one or more halogens, hydroxyls, and mercapto groups; the carbon atoms in the C1-3 alkyl are optionally substituted by oxygen atoms or sulfur atoms;

[0023] Preferably, R1 and R2 are the same or different and are independently selected from methyl; the methyl group is optionally substituted by one or more halogen, hydroxyl, and mercapto groups.

[0024] According to an embodiment of the present invention, R3 and R4 are the same or different and are independently selected from C5-15 alkyl, C5-15 alkenyl, C5-15 alkynyl; the C5-15 alkyl, C5-15 alkenyl, C5-15 alkynyl are optionally substituted with one or more halogen, hydroxyl, thiol; the carbon atoms in the C5-15 alkyl, C5-15 alkenyl, C5-15 alkynyl are optionally substituted with oxygen atoms or sulfur atoms;

[0025] Preferably, R3 and R4 are the same or different, and are independently selected from C6-12 alkyl, C6-12 alkenyl, C6-12 alkynyl; the C6-12 alkyl, C6-12 alkenyl, C6-12 alkynyl are optionally substituted with one or more halogen, hydroxyl, thiol; the carbon atoms in the C6-12 alkyl, C6-12 alkenyl, C6-12 alkynyl are optionally substituted with oxygen atoms or sulfur atoms;

[0026] Further preferably, R3 and R4 are the same or different, and are independently selected from C8-10 alkyl, C8-10 alkenyl, C8-10 alkynyl; the C8-10 alkyl, C8-10 alkenyl, C8-10 alkynyl are optionally substituted by one or more halogen, hydroxyl, thiol; the carbon atoms in the C8-10 alkyl, C8-10 alkenyl, C8-10 alkynyl are optionally substituted by oxygen atoms or sulfur atoms;

[0027] For example, R3 and R4 are the same or different, and are each independently selected from C9 alkyl, C9 alkenyl, and C9 alkynyl.

[0028] According to an embodiment of the present invention, at least one of the L1, L2, L3, R1, R2, R3, and R4 groups is substituted by one or more fluorine atoms;

[0029] Preferably, at least one of the R3 and R4 groups is substituted by one or more fluorine atoms;

[0030] Further preferably, the R3 and R4 groups are substituted by 1-30 fluorine atoms; for example, 5-25, 10-20, 15-18, 16, etc.

[0031] According to an embodiment of the present invention, the fluorinated modified ionizable lipid is selected from the following compounds:

[0032]

[0033] The present invention also provides lipid nanoparticles comprising the fluorinated ionizable lipid represented by the above formula I.

[0034] According to an embodiment of the present invention, the lipid nanoparticles include a carrier and an encapsulated nucleic acid, wherein the carrier includes a fluorinated ionizable lipid as shown in the above formula I, an auxiliary phospholipid, a cholesterol-like substance and a pegylated lipid.

[0035] According to an embodiment of the present invention, the nucleic acid is selected from one or more of RNA and DNA.

[0036] According to an embodiment of the present invention, the nucleic acid includes but is not limited to one or more of mRNA, circular RNA, siRNA, microRNA, antisense nucleic acid and plasmid.

[0037] According to an embodiment of the present invention, the nucleic acid selected in the present invention includes, for example, at least one of CpG-FAM and mFLuc (mRNA encoding firefly luciferase).

[0038] According to an embodiment of the present invention, the fluorinated ionizable lipid accounts for 20 mol% to 70 mol% of the total lipids in the LNP, such as 30 mol%, 40 mol%, 50 mol%, 60 mol%.

[0039] According to an embodiment of the present invention, the auxiliary phospholipid accounts for 2 mol% to 20 mol% of the total lipids in the LNP, for example 4 mol%, 8 mol%, 10 mol%, 12 mol%, 4 mol%, 16 mol%, 18 mol%.

[0040] According to an embodiment of the present invention, the auxiliary phospholipid includes but is not limited to 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylcholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanolamine (DOPE), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylethanolamine (POPE), 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine (DSPE), 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylethanolamine (DPPE), preferably DSPC.

[0041] According to an embodiment of the present invention, the cholesterol-like substance accounts for 10 mol% to 60 mol% of the total lipids in the LNP, for example, 20 mol%, 30 mol%, 40 mol%, 50 mol%.

[0042] According to an embodiment of the present invention, the cholesterol-like substances include but are not limited to one or more of cholesterol, β-sitosterol, cholestanol, cholestanone, cholestenone, 7β-hydroxycholesterol, 7α-hydroxycholesterol, preferably cholesterol.

[0043] According to an embodiment of the present invention, the PEGylated lipid accounts for 0.3 mol% to 30 mol% of the total lipid in the LNP, preferably 0.5 mol% to 2.5 mol%, such as 1.0 mol%, 1.5 mol%, 2.0 mol%.

[0044] According to an embodiment of the present invention, the PEGylated lipids include but are not limited to one or more of 1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-rac-glyceryl-3-methoxypolyethylene glycol (DSG-PEG), 1,2-dipalmitoyl-rac-glyceryl-3-methoxypolyethylene glycol (DPG-PEG), and 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine-methoxypolyethylene glycol (DSPE-PEG), preferably DMG-PEG.

[0045] In the present invention, the above-mentioned "total lipids" refers to the sum of fluorinated ionizable lipids, auxiliary phospholipids, cholesterol substances and PEGylated lipids.

[0046] According to an embodiment of the present invention, the encapsulation rate of the nucleic acid is 20-99%, preferably 70%-90%, such as 70%, 75%, 80%, 85%, 90%.

[0047] According to an embodiment of the present invention, the molar ratio of the nitrogen element contained in the fluorinated ionizable lipid to the phosphorus element contained in the nucleic acid is (1-50):1; preferably (5-10):1, and exemplarily 5.67:1.

[0048] According to an embodiment of the present invention, the hydrated particle size of the fluorinated lipid nanoparticles is less than 500 nm, preferably less than 300 nm, further preferably less than 250 nm, such as 80-180 nm.

[0049] According to an embodiment of the present invention, the polydispersity coefficient of the fluorinated lipid nanoparticles is less than 0.4, preferably less than 0.3, further preferably less than 0.2, for example, 0.119, 0.102, or 0.093.

[0050] In some embodiments, the molar ratio of the fluorinated ionizable lipid: auxiliary phospholipid: cholesterol substance: polyethylene glycol lipid is 20-80:5-20:20-60:0.1-5, preferably 30-70:8-15:30-50:0.5-3, for example 50:10:38.5:1.5.

[0051] According to an embodiment of the present invention, the fluorinated lipid nanoparticles are capable of:

[0052] 1) Improve nucleic acid expression efficiency, and / or

[0053] 2) Improve the ability of nucleic acids to enter cells, and / or

[0054] 3) Improve the spleen targeting of nucleic acids.

[0055] The present invention also provides a method for preparing F-L319 fluorinated ionizable lipid, the preparation method comprising the following steps:

[0056] 1) Compound 1 (9-bromo-1-nonene) reacts with magnesium (Mg) and ethyl formate in solvent-1 to obtain compound 2 (19-1,18-diene-10-ol);

[0057] 2) Compound 2 (nonadecanediol) reacts with 4-bromobutyryl chloride under alkaline conditions to obtain compound 3 (nonadecanediol-1,18-diene-10-ol);

[0058] 3) Compound 3 (4-bromobutyric acid-1,18-diene-10-nonadecanedioic acid) reacts with an oxidant under metal catalyst conditions to obtain compound 4 (9-((4-bromobutyryl)oxy)heptadecanedioic acid);

[0059] 4) Compound 4 (9-((4-bromobutyryl)oxy)heptadecanedioic acid) reacts with dimethylamine under alkaline conditions to obtain compound 5 (9-((4-(dimethylamino)butyryl)oxy)heptadecanedioic acid);

[0060] 5) Compound 5 (9-((4-(dimethylamino)butyryl)oxy)heptadecanedioic acid) reacts with 1H,1H,9H-hexadecanefluorononanol to obtain compound F-L319 (9-((4-(dimethylamino)butyryl)oxy)heptadecanedioic acid di(2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexadecanefluorononyl) ester);

[0061] The reaction formula is as follows:

[0062]

[0063] According to an embodiment of the present invention, in step 1), solvent-1 is selected from tetrahydrofuran.

[0064] According to an embodiment of the present invention, in step 2), the base is selected from triethylamine.

[0065] According to an embodiment of the present invention, in step 3), the metal catalyst is selected from ruthenium trichloride; preferably, the oxidant is selected from sodium periodate.

[0066] According to an embodiment of the present invention, in step 4), the base is selected from potassium carbonate.

[0067] According to an embodiment of the present invention, in step 5), the reaction is carried out under the conditions of EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and DMAP (4-(N,N-dimethylamino)pyridine).

[0068] The present invention also provides a composite ionizable lipid, wherein the composite ionizable lipid comprises a first ionizable lipid and a second ionizable lipid;

[0069] The second ionizable lipid is selected from the fluorinated modified ionizable lipid shown in the above formula I or the fluorinated modified ionizable lipid shown in the following formula II:

[0070]

[0071] Among them, N represents the nitrogen element;

[0072] W and X are the same or different, and are independently selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl; the C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl are optionally substituted by one or more halogen, hydroxyl, thiol; the carbon atoms in the C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl are optionally substituted by oxygen atoms or sulfur atoms;

[0073] A and B are the same or different, and are independently selected from biodegradable functional groups; preferably, including but not limited to ester groups, amide groups, acetal groups, ether bonds, and thioether bonds;

[0074] R 1 , R 2 are the same or different, and are independently selected from C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl; the C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl are optionally substituted by one or more halogen, hydroxyl, thiol; the carbon atoms in the C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl are optionally substituted by oxygen atoms or sulfur atoms;

[0075] Y is selected from C1-10 alkyl, which is optionally substituted with one or more halogens;

[0076] Z is selected from -OH, -NH2, -SH, -NR 3 R 4 ;

[0077] R 3 , R 4 are the same or different, each independently selected from C1-5 alkyl, wherein the C1-5 alkyl is optionally substituted by one or more halogens;

[0078] C is selected from hydrogen, C1-5 alkyl, and the C1-5 alkyl is optionally substituted by one or more halogens;

[0079] C, Y, W, X, A, B, Z, R 1 , R 2 In the group, at least one group is substituted by one or more fluorine atoms.

[0080] According to an embodiment of the present invention, W and X are the same or different and are independently selected from C3-10 alkyl and C3-10 alkenyl; the C3-10 alkyl and C3-10 alkenyl are optionally substituted by one or more halogens, hydroxyls and mercapto groups; the carbon atoms in the C3-10 alkyl and C3-10 alkenyl are optionally substituted by oxygen atoms and sulfur atoms;

[0081] Preferably, W and X are the same or different and are independently selected from C5-7 alkyl and C5-7 alkenyl; the C5-7 alkyl and C5-7 alkenyl are optionally substituted with one or more halogens, hydroxyls and thiol groups; the carbon atoms in the C5-7 alkyl and C5-7 alkenyl are optionally substituted with oxygen atoms and sulfur atoms;

[0082] Further preferably, W is a C7 alkyl or C7 alkenyl, and X is a C5 alkyl or C5 alkenyl; for example, W is a C7 alkyl (such as n-heptyl -(CH2)6CH3), and X is a C5 alkyl (such as n-pentyl -(CH2)4CH3).

[0083] According to an embodiment of the present invention, A and B are selected from ester groups, such as -O-CO- or -CO-O-.

[0084] According to an embodiment of the present invention, R 1 is selected from C10-20 alkyl, C10-20 alkenyl, C10-20 alkynyl; the C10-20 alkyl, C10-20 alkenyl, C10-20 alkynyl is optionally substituted by one or more halogen, hydroxyl, thiol; the carbon atoms in the C10-20 alkyl, C10-20 alkenyl, C10-20 alkynyl are optionally substituted by oxygen atoms or sulfur atoms;

[0085] Preferably, R 1 Selected from C15-20 alkyl, C15-20 alkenyl, C15-20 alkynyl; the C15-20 alkyl, C15-20 alkenyl, C15-20 alkynyl are optionally substituted by one or more halogen, hydroxyl, thiol; the carbon atoms in the C15-20 alkyl, C15-20 alkenyl, C15-20 alkynyl are optionally substituted by oxygen atoms or sulfur atoms;

[0086] More preferably, R 1 is selected from C17 alkyl, C17 alkenyl, C17 alkynyl; the C17 alkyl, C17 alkenyl, C17 alkynyl are optionally substituted by one or more halogens; the carbon atoms in the C17 alkyl, C17 alkenyl, C17 alkynyl are optionally substituted by oxygen atoms or sulfur atoms;

[0087] For example, R 1 Selected from -(CH2) 16 CH3.

[0088] According to an embodiment of the present invention, R 2 is selected from C3-15 alkyl, C3-15 alkenyl, C3-15 alkynyl; the C3-15 alkyl, C3-15 alkenyl, C3-15 alkynyl are optionally substituted by one or more halogen, hydroxyl, thiol; the carbon atoms in the C3-15 alkyl, C3-15 alkenyl, C3-15 alkynyl are optionally substituted by oxygen atoms or sulfur atoms;

[0089] Preferably, R 2 is selected from C5-13 alkyl, C5-13 alkenyl, C5-13 alkynyl; the C5-13 alkyl, C5-13 alkenyl, C5-13 alkynyl are optionally substituted by one or more halogen, hydroxyl, thiol; the carbon atoms in the C5-13 alkyl, C5-13 alkenyl, C5-13 alkynyl are optionally substituted by oxygen atoms or sulfur atoms;

[0090] More preferably, R 2 Selected from C5-13 alkyl, C5-13 alkenyl; the C5-13 alkyl, C5-13 alkenyl is optionally substituted by one or more halogens;

[0091] For example, R 2 Selected from C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C5 alkenyl, C6 alkenyl, C7 alkenyl, C8 alkenyl, C9 alkenyl, C10 alkenyl, C11 alkenyl, C12 alkenyl, C13 alkenyl substituted by one or more halogens.

[0092] According to an embodiment of the present invention, Y is selected from C1-6 alkyl, which is optionally substituted with one or more halogens;

[0093] Preferably, Y is selected from C1-3 alkyl, which is optionally substituted with one or more halogens;

[0094] Further preferably, Y is selected from C2 alkyl, which is optionally substituted by one or more halogens;

[0095] For example, Y is selected from ethyl.

[0096] According to an embodiment of the present invention, Z is selected from -OH, -NH2, -SH, -NR 3 R 4 ; R 3 , R 4 The same or different, each independently selected from C1-3 alkyl, the C1-3 alkyl is optionally substituted by one or more halogens;

[0097] Preferably, Z is selected from -OH, -NH2, -SH;

[0098] Further preferably, Z is selected from -OH.

[0099] According to an embodiment of the present invention, C is selected from hydrogen, C1-3 alkyl, said C1-3 alkyl being optionally substituted by one or more halogens;

[0100] Preferably C is selected from hydrogen.

[0101] According to an embodiment of the present invention, W, X, R 1 , R 2 In the group, at least one group is substituted by one or more fluorine atoms;

[0102] Preferably, the R 1 , R 2 In the group, at least one group is substituted by one or more fluorine atoms;

[0103] More preferably, the R 2 substituted with one or more fluorine atoms;

[0104] For example, R 2 Substituted by 1-30 fluorines, substituted by 3-28 fluorines, substituted by 5-24 fluorines.

[0105] According to an embodiment of the present invention, the fluorinated modified ionizable lipid is selected from the following F-L319 or the compound shown in the following formula II-1:

[0106]

[0107] Wherein, R is selected from the following groups:

[0108]

[0109] According to an embodiment of the present invention, the first ionizable lipid is the same as or different from the second ionizable lipid.

[0110] According to an embodiment of the present invention, the first ionizable lipid is a non-fluorinated modified ionizable lipid.

[0111] According to an embodiment of the present invention, the molar ratio of the first ionizable lipid to the second ionizable lipid is 1:99-99:1, preferably 2:8-8:2, such as 1:4, 1:2, 1:1, 2:1, 4:1, and more preferably 1:1.

[0112] According to an embodiment of the present invention, the first ionizable lipid is selected from 8-[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl)amino]octanoic acid (heptadecan-9-yl) ester (SM102), [(4-hydroxybutyl)azepine diyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin MC3DMA), 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dione (cKK-E12), 9-(4-(dimethylamino)butyryloxy)heptadecanedioic acid di((Z)-non-2-en-1-yl) ester (L-319), N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecene-1-yl-1,3-dioxolane-4-ethylamine (DLin-KC2-DMA), 8-[(2-hydroxyethyl)(8-nonyloxy-8-oxooctyl)amino]octanoic acid (heptadecanedioic acid) ester (Lipid 5), 1,1'-[(2-{4-[2-({2-[bis(2-hydroxydodecyl)amino]ethyl}(2-hydroxydodecyl)amino)ethyl]piperazin-1-yl}ethyl)azadialkyl]bis(dodecan-2-ol) (C12-200), (2,3-dioleoylpropyl)trimethylammonium chloride (DOTAP), dimethyldioctadecylammonium bromide (DDAB), tetrakis(8-methylnonyl) 3',3',3",3"-{[(methylazadialkyl)bis(propane-3,1-diyl)]bis(azatriyl)}tetrapropionate (306Oi10) or more thereof;

[0113] Preferred are 8[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl)amino]octanoic acid (heptadecan-9-yl) ester (SM102) or 9-(4-(dimethylamino)butanoyloxy)heptadecanedioic acid di((Z)-non-2-en-1-yl) ester (L-319).

[0114] The present invention provides lipid nanoparticles comprising the above-mentioned complex ionizable lipid.

[0115] According to an embodiment of the present invention, the lipid nanoparticles include a carrier and an encapsulated nucleic acid, wherein the carrier includes the above-mentioned complex ionizable lipid, auxiliary phospholipid, cholesterol-like substance and pegylated lipid.

[0116] According to an embodiment of the present invention, the nucleic acid is selected from one or more of RNA and DNA.

[0117] According to an embodiment of the present invention, the nucleic acid includes but is not limited to one or more of mRNA, circular RNA, siRNA, microRNA, antisense nucleic acid and plasmid.

[0118] According to an embodiment of the present invention, the nucleic acid selected in the present invention includes, for example, at least one of CpG-FAM and mFLuc (mRNA encoding firefly luciferase).

[0119] According to an embodiment of the present invention, the complex ionizable lipid accounts for 20 mol% to 70 mol% of the total lipids in the LNP, such as 30 mol%, 40 mol%, 50 mol%, 60 mol%.

[0120] According to an embodiment of the present invention, the auxiliary phospholipid accounts for 2 mol% to 20 mol% of the total lipids in the LNP, for example 4 mol%, 8 mol%, 10 mol%, 12 mol%, 4 mol%, 16 mol%, 18 mol%.

[0121] According to an embodiment of the present invention, the auxiliary phospholipid includes but is not limited to 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylcholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanolamine (DOPE), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylethanolamine (POPE), 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine (DSPE), 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylethanolamine (DPPE), preferably DSPC.

[0122] According to an embodiment of the present invention, the cholesterol-like substance accounts for 10 mol% to 60 mol% of the total lipids in the LNP, for example, 20 mol%, 30 mol%, 40 mol%, 50 mol%.

[0123] According to an embodiment of the present invention, the cholesterol-like substances include but are not limited to one or more of cholesterol, β-sitosterol, cholestanol, cholestanone, cholestenone, 7β-hydroxycholesterol, 7α-hydroxycholesterol, preferably cholesterol.

[0124] According to an embodiment of the present invention, the PEGylated lipid accounts for 0.3 mol% to 30 mol% of the total lipid in the LNP, preferably 0.5 mol% to 2.5 mol%, such as 1.0 mol%, 1.5 mol%, 2.0 mol%.

[0125] According to an embodiment of the present invention, the PEGylated lipids include but are not limited to one or more of 1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-rac-glyceryl-3-methoxypolyethylene glycol (DSG-PEG), 1,2-dipalmitoyl-rac-glyceryl-3-methoxypolyethylene glycol (DPG-PEG), and 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine-methoxypolyethylene glycol (DSPE-PEG), preferably DMG-PEG.

[0126] In the present invention, the above-mentioned "total lipids" refers to the sum of complex ionizable lipids, auxiliary phospholipids, cholesterol substances and PEGylated lipids.

[0127] According to an embodiment of the present invention, the encapsulation rate of the nucleic acid is 20-99%, preferably 70%-90%, such as 70%, 75%, 80%, 85%, 90%.

[0128] According to an embodiment of the present invention, the molar ratio of the nitrogen element contained in the complex ionizable lipid to the phosphorus element contained in the nucleic acid is (1-50):1; preferably (5-10):1, and exemplarily 5.67:1.

[0129] According to an embodiment of the present invention, the hydrated particle size of the composite lipid nanoparticles is less than 500 nm, preferably less than 300 nm, further preferably less than 250 nm, such as 80-180 nm.

[0130] According to an embodiment of the present invention, the polydispersity coefficient of the composite lipid nanoparticles is less than 0.4, preferably less than 0.3, and further preferably less than 0.2, for example, 0.119, 0.102, or 0.093.

[0131] In some embodiments, the molar ratio of the complex ionizable lipid: auxiliary phospholipid: cholesterol substance: polyethylene glycol lipid is 20-80:5-20:20-60:0.1-5, preferably 30-70:8-15:30-50:0.5-3, for example 50:10:38.5:1.5.

[0132] According to an embodiment of the present invention, the composite lipid nanoparticle is capable of:

[0133] 1) Improve nucleic acid expression efficiency, and / or

[0134] 2) Improve the ability of nucleic acids to enter cells, and / or

[0135] 3) Improve the spleen targeting of nucleic acids.

[0136] The present invention also provides a fluorinated modified ionizable lipid as shown in formula II-1:

[0137]

[0138] Wherein, R is selected from the following groups:

[0139]

[0140] The present invention provides lipid nanoparticles comprising the fluorinated modified ionizable lipid represented by the above formula II or the fluorinated modified ionizable lipid represented by formula II-1.

[0141] According to an embodiment of the present invention, the lipid nanoparticles include a carrier and an encapsulated nucleic acid, wherein the carrier includes a fluorinated modified ionizable lipid shown in the above formula II, an auxiliary phospholipid, a cholesterol-like substance and a pegylated lipid.

[0142] According to an embodiment of the present invention, the nucleic acid is selected from one or more of RNA and DNA.

[0143] According to an embodiment of the present invention, the nucleic acid includes but is not limited to one or more of mRNA, circular RNA, siRNA, microRNA, antisense nucleic acid and plasmid.

[0144] According to an embodiment of the present invention, the fluorinated ionizable lipid represented by Formula II accounts for 20 mol% to 70 mol% of the total lipids in the LNP, for example, 30 mol%, 40 mol%, 50 mol%, 60 mol%.

[0145] According to an embodiment of the present invention, the auxiliary phospholipid accounts for 2 mol% to 20 mol% of the total lipids in the LNP, for example 4 mol%, 8 mol%, 10 mol%, 12 mol%, 4 mol%, 16 mol%, 18 mol%.

[0146] According to an embodiment of the present invention, the auxiliary phospholipid includes but is not limited to 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylcholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanolamine (DOPE), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylethanolamine (POPE), 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine (DSPE), 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylethanolamine (DPPE), preferably DSPC.

[0147] According to an embodiment of the present invention, the cholesterol-like substance accounts for 10 mol% to 60 mol% of the total lipids in the LNP, for example, 20 mol%, 30 mol%, 40 mol%, 50 mol%.

[0148] According to an embodiment of the present invention, the cholesterol-like substances include but are not limited to one or more of cholesterol, β-sitosterol, cholestanol, cholestanone, cholestenone, 7β-hydroxycholesterol, 7α-hydroxycholesterol, preferably cholesterol.

[0149] According to an embodiment of the present invention, the PEGylated lipid accounts for 0.3 mol% to 30 mol% of the total lipid in the LNP, preferably 0.5 mol% to 2.5 mol%, such as 1.0 mol%, 1.5 mol%, 2.0 mol%.

[0150] According to an embodiment of the present invention, the PEGylated lipids include but are not limited to one or more of 1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-rac-glyceryl-3-methoxypolyethylene glycol (DSG-PEG), 1,2-dipalmitoyl-rac-glyceryl-3-methoxypolyethylene glycol (DPG-PEG), and 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine-methoxypolyethylene glycol (DSPE-PEG), preferably DMG-PEG.

[0151] In the present invention, the above-mentioned "total lipids" refers to the sum of the fluorinated ionizable lipids, auxiliary phospholipids, cholesterol substances and pegylated lipids shown in Formula II.

[0152] According to an embodiment of the present invention, the nucleic acid selected in the present invention includes, for example, at least one of CpG-FAM and mFLuc (mRNA encoding firefly luciferase).

[0153] According to an embodiment of the present invention, the encapsulation rate of the nucleic acid is 20-99%, preferably 70%-90%, such as 70%, 75%, 80%, 85%, 90%.

[0154] According to an embodiment of the present invention, the molar ratio of nitrogen contained in the fluorinated ionizable lipid to phosphorus contained in the nucleic acid is (1-50):1; preferably (5-10):1, and exemplarily 5.67:1.

[0155] According to an embodiment of the present invention, the hydrated particle size of the lipid nanoparticles is less than 500 nm, preferably less than 300 nm, further preferably less than 250 nm, such as 80-180 nm.

[0156] According to an embodiment of the present invention, the polydispersity coefficient of the lipid nanoparticles is less than 0.4, preferably less than 0.3, further preferably less than 0.2, for example, 0.119, 0.102, or 0.093.

[0157] In some embodiments, the molar ratio of the fluorinated ionizable lipid: auxiliary phospholipid: cholesterol substance: polyethylene glycol lipid is 20-80:5-20:20-60:0.1-5, preferably 30-70:8-15:30-50:0.5-3, for example 50:10:38.5:1.5.

[0158] According to an embodiment of the present invention, the fluorinated lipid nanoparticles are capable of:

[0159] 1) Improve nucleic acid expression efficiency, and / or

[0160] 2) Improve the ability of nucleic acids to enter cells, and / or

[0161] 3) Improve the spleen targeting of nucleic acids.

[0162] The present invention also provides a method for preparing the lipid nanoparticles, which comprises: preparing the lipid nanoparticles by blending an aqueous phase containing nucleic acids and a lipid organic phase through a blending method or a microfluidics method.

[0163] According to an embodiment of the present invention, the preparation method of the lipid organic phase is as follows: ionizable lipids, auxiliary phospholipids, cholesterol substances and pegylated lipids are dissolved in an organic solvent in proportion to obtain an organic phase; preferably, the organic solvent is an organic solvent that can dissolve the above substances, such as ethanol (anhydrous ethanol).

[0164] According to an embodiment of the present invention, the preparation method of the aqueous phase containing nucleic acids is as follows: dissolving the nucleic acids in a buffer solution to obtain an aqueous phase; preferably, the buffer solution can be a buffer solution with a pH of 4 to 6 (such as a sodium citrate buffer solution), for example, a sodium citrate buffer solution with a pH of 4 or 5.5.

[0165] According to an embodiment of the present invention, in the aqueous phase, the concentration of nucleic acid is 0.01 mg / mL-1 mg / mL, preferably 0.1 mg / mL-0.5 mg / mL, for example 0.17 mg / mL.

[0166] According to an embodiment of the present invention, the blending method comprises the following steps: adding the aqueous phase containing nucleic acid to the lipid organic phase, mixing, and then dialyzing the resulting product to obtain the lipid nanoparticles.

[0167] According to an embodiment of the present invention, the dialyzation is performed in a volume greater than 1000 times of 1X PBS buffer solution.

[0168] According to an embodiment of the present invention, the dialysis time is more than 4 hours.

[0169] According to an embodiment of the present invention, the volume ratio of the aqueous phase to the lipid organic phase is 1-6:1, for example 3:1.

[0170] According to an embodiment of the present invention, the blending method specifically comprises the following steps: using a pipette to absorb the aqueous phase containing nucleic acids, quickly adding it to the lipid organic phase, and then rapidly blowing the mixed liquid for more than 30 times, dialyzing the resulting product, for example, dialyzing in a 1X PBS buffer solution with a volume greater than 1000 times for more than 4 hours to obtain the lipid nanoparticles.

[0171] The present invention also provides the use of the lipid nanoparticles in the preparation of biological preparations.

[0172] According to an embodiment of the present invention, the biologic is an injectable biologic.

[0173] In some embodiments, the biologic is a vaccine, preferably an mRNA vaccine.

[0174] According to an embodiment of the present invention, the administration method of the biological agent is tail vein injection.

[0175] The present invention also provides a biological preparation, which comprises the lipid nanoparticles.

[0176] Beneficial effects:

[0177] The invention provides a series of highly fluorinated ionizable lipid molecules with novel structures, and the preparation method has the advantages of simple synthesis and high yield.

[0178] The present invention provides an LNP comprising a fluorinated ionizable lipid or a composite ionized lipid. The LNP has good particle size dispersibility, a high encapsulation rate for nucleic acids, and low toxicity in vivo. It can efficiently deliver nucleic acid drugs so that they have better cell entry efficiency and nucleic acid expression efficiency, and has spleen targeting, and has great application prospects in immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0179] Figure 1 : The chemical structure of the fluorinated lipids used in the examples of the present invention.

[0180] Figure 2 : The synthetic route and structural characterization results in Example 1 of the present invention.

[0181] Figure 3 : Particle size of the four-component LNP of Example 2B.

[0182] Figure 4 : Particle size of the five-component LNP of Example 2D.

[0183] Figure 5 : Electron microscopy characterization of LNP of Example 2D.

[0184] Figure 6 : Cell expression screening results of Example 4A.

[0185] Figure 7 : Example 5 Test results of the cell entry efficiency of five-component LNP.

[0186] Figure 8 : Test results of membrane fusion ability of five-component LNP in Example 6.

[0187] Fig. 9 : Example 7 Test results of ALNP mRNA expression efficiency in vivo (a) small animal imaging results, (b) bioluminescence intensity at 4 hours, 12 hours, and 24 hours.

[0188] Fig.10 : Example 7 of the present invention is the imaging result of the main organs of small animals.

[0189] Fig.11 : In Example 7B, the test results of the mRNA expression efficiency of LNP in vivo.

[0190] Fig.12 : Cytotoxicity detection results in Example 8 of the present invention.

[0191] Fig.13 : The encapsulation efficiency of the five-component LNP containing mRNA of the present invention. DETAILED DESCRIPTION

[0192] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0193] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0194] The auxiliary phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine, abbreviated as DSPC; the polyethylene glycol lipid is 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000, abbreviated as DMG-PEG2000.

[0195] Example 1 Synthesis of F-L319

[0196] Preparation of 19-1,18-diene-10-ol (2): Mg powder (0.90 g, 37.5 mmol, 3 e.q.) and a magnetite were added to a flame-vacuum dried 250 mL three-necked flask. A 25 mL dropping funnel was added to the side port, and a reflux condenser was installed in the middle port. The entire device was sealed and gas exchanged with nitrogen three times. 10 mL of dry tetrahydrofuran was added to the three-necked flask, 9-bromo-1-nonene (1) (2.50 g, 12.5 mmol, 1 e.q.) was dissolved in 10 mL of tetrahydrofuran and then injected into the dropping funnel. Stirring and heating were started until the solution boiled. When the temperature was close to reflux, about 1 mL of 9-bromo-1-nonene tetrahydrofuran solution was slowly added dropwise, and a grain of iodine tetrahydrofuran solution was added at the same time. The halogenated hydrocarbon was slowly added dropwise (about one drop per second) until it was complete. The reaction was refluxed for 12 hours, cooled with a cold water bath (water temperature about 20°C), ethyl formate (0.4g, 5.4mmol, 0.95eq) was dissolved in 5mL THF and transferred to a dropping funnel, stirred and added dropwise to the reaction mixture. After the addition was completed, the reaction solution was reacted at reflux temperature for 12h. After the reaction was completed, 20mL of saturated ammonium chloride solution was added to quench, filtered, and the obtained filtrate was vacuum-removed tetrahydrofuran, extracted twice with ethyl acetate (20mL), extracted once with petroleum ether (20mL), and the organic phases were combined and dried by spin drying; the filter residue was washed twice with hot (about 50°C) ethyl acetate (20mL x 2), and once with dichloromethane (about 30°C, 20mL). The obtained solution was spin dried, combined and distilled under reduced pressure, and the product at 178-180°C (vacuum gauge reading 2mmHg) was collected to obtain 0.702g of white solid, with a yield of 41.0%, mp38°C; R f =0.38 (PE / EA=9:1, v / v).

[0197] Preparation of 4-bromobutyric acid-1,18-diene-10-nonadecanyl ester (3): A magnetic bar was added to a 250 mL eggplant-shaped bottle after flame vacuum drying, and a 25 mL dropping funnel was installed. The entire apparatus was gas-exchanged with nitrogen three times. 19-1,18-diene-10-ol (2) (16.50 g, 58.82 mmol, 1 e.q.), 100 mL of dichloromethane and 4 mL of triethylamine were added, and 4-bromobutyryl chloride (14.54 mL, 118 mmol, 2 e.q.) was added dropwise at a rate of 2 drops per second under an ice-water bath. After the addition, the mixture was reacted at room temperature for 20 hours, and then extracted twice with a saturated sodium bicarbonate solution (100 mL), and the organic phase was collected. The organic phase was extracted once with a saturated sodium chloride solution (100 mL), and the organic phase was collected. After drying with anhydrous sodium sulfate, the solvent was removed in vacuo, and the mixture was separated by column chromatography. Gradient elution was performed, and pure PE was first used, and the polarity was gradually increased to PE:EtOAc=30:1 for separation, to obtain 18.202 g of a light yellow oily liquid with a yield of 72.0%, R f =0.52 (PE / EtOAc=9:1, v / v).

[0198] Preparation of 9-((4-bromobutyryl)oxy)heptadecanedioic acid (4): 4-bromobutyric acid-1,18-diene-10-nonadecanyl ester (1.00 g, 2.33 mmol, 1 e.q.) was added to a 250 mL eggplant-shaped bottle, 40 mL of dichloromethane, 40 mL of acetonitrile, and ruthenium trichloride trihydrate (30 mg, 0.0233 mmol, 10% mol) was added thereto. Sodium periodate (4.98 g, 23.3 mmol, 10 e.q.) was added to a dropping funnel and dissolved in 50 mL of distilled water. In a water bath slightly below room temperature, sodium periodate solution was added dropwise (1 drop per 1 second). The solution was bright orange-yellow. The reaction was continued for 4 hours. After the plate test showed that no raw material was left, the reaction was stopped. At this time, the color of the reaction solution was bright yellow to dark green. The reaction system was extracted three times with saturated sodium thiosulfate solution (80 mL) until the aqueous phase was almost colorless. The organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed in vacuo to obtain 0.962 g of a black oily liquid with a yield of 85.6%. f =0.62 (DCM:MeOH=20:1, v / v). The product was used in the next step without purification.

[0199] Preparation of 9-((4-(dimethylamino)butyryl)oxy)heptadecanedioic acid (5): 9-((4-bromobutyryl)oxy)heptadecanedioic acid (4) (465.43 mg, 1 mmol, 1 e.q.), 2M dimethylamine tetrahydrofuran solution (5 mL, 10 mmol), and potassium carbonate (304.05 mg, 2.2 mmol, 2.2 eq) were added to a pressure tube, and reacted at 50°C for 8 hours. After cooling to room temperature, acetic acid was added dropwise with stirring until the white precipitate disappeared, 5 mL of water was added, and the mixture was extracted with 10 mL of dichloromethane three times. The organic phase was collected, and then the organic phase was extracted once with a saturated sodium chloride solution. The organic phases were collected and combined, and the solvent was removed under reduced pressure to obtain 356 mg of a yellow oily liquid with a yield of 82.9%. R f =0.21 (DCM: MeOH = 10: 1, v / v). The obtained product can be used for the next step reaction without purification.

[0200] Preparation of 9-((4-(dimethylamino)butyryl)oxy)heptadecanedioic acid di(2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexafluorononyl) ester (F-L319): 9-((4-(dimethylamino)butyryl)oxy)heptadecanedioic acid (5) (42.96 mg, 0.1 mmol, 1 e.q.), 5 mL of dichloromethane, 4-(N, N-dimethylamino) pyridine (6.11 mg, 0.05 mmol, 0.5 eq), stirred at room temperature for 30 minutes, then added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (42.17 mg, 0.22 mmol, 2.2 eq), continued stirring for 15 minutes, added 1H,1H,9H-hexafluorononanol (95.06 mg, 0.22 mmol, 2.2 eq), and stirred overnight. The reaction solution was turbid, and then extracted once with 10 mL of ammonium chloride solution, water, sodium bicarbonate, and saturated brine. The organic phase was collected, dried, and separated by column chromatography. The developing solvent was DCM: MeOH: Et3N = 100: 10: 1. After collecting until no organic flow out, the solvent was removed in vacuo, and impurities were removed at 60 ° C for 2 hours using an oil pump to obtain a pure product. The product was a yellow oily liquid of 38 mg, with a yield of 30%, R f =0.86 (DCM).

[0201] The synthetic route and structural characterization of F-L319 are as follows: Figure 2 A and Figure 2 As shown in B-2C.

[0202] Example 2 Preparation of lipid nanoparticles LNP

[0203] Example 2A

[0204] Four-component lipid nanoparticles (referred to as four-component LNPs) were prepared as follows:

[0205] (1) dissolving a nucleic acid drug with FAM fluorescently modified CPG DNA (FAM-CPG) in a 50 mM sodium citrate buffer solution at pH = 4 to form an aqueous phase; the concentration of the nucleic acid drug is 0.17 mg / mL;

[0206] (2) The fluorinated ionizable lipid F-L319, auxiliary phospholipid DSPC, cholesterol and pegylated lipid DMG-PEG2000 prepared in Example 1 were dissolved in anhydrous ethanol at a molar ratio of 50:38.5:10:1.5 to form an organic phase; the molar ratio of nitrogen in the fluorinated ionizable lipid F-L319 to phosphorus in the entrapped nucleic acid drug was 5.67:1;

[0207] (3) The aqueous phase and the organic phase were mixed by a blending method, with the volume ratio of the aqueous phase to the organic phase being 3:1. After the mixing was completed, the resulting product was dialyzed using a PBS buffer solution to prepare four-component lipid nanoparticles LNP.

[0208] Example 2B

[0209] The preparation method of the four-component lipid nanoparticles of this example is basically the same as that of Example 2A, except that the nucleic acid drug is mRNA encoding firefly luciferase (mFLuc).

[0210] Example 2C

[0211] The preparation method of the five-component lipid nanoparticles of this embodiment is basically the same as that of Example 2A, except that the fluorinated ionizable lipid is a mixture of L319 and F-L319 (referred to as five-component LNP), and the molar ratios of L319 and F-L319 are 1:0, 2:1, 1:1, and 0:1.

[0212] Example 2D

[0213] The preparation method of the five-component lipid nanoparticles of this example is basically the same as that of Example 2B, except that the fluorinated ionizable lipid is a mixture of L319 and F-L319, and the molar ratios of L319 and F-L319 are 1:0, 2:1, 1:1, and 0:1.

[0214] Example 2E

[0215] The preparation method of the four-component lipid nanoparticles of this embodiment is basically the same as that of embodiment 2B, except that the fluorinated ionizable lipid F-L319 is replaced by SM102, C5F5, C5F8, C6F9, C7F9, C7F12, C8F13, C9F13, C9F17, C10F19, C11F21, C12F23, and C13F24. Among them, C5F5, C5F8, C6F9, C7F9, C7F12, C8F13, C9F13, C9F17, C10F19, C11F21, C12F23, and C13F24 are all fluorinated SM102 (CAS: 2089251-47-6), and its structural formula is as follows Figure 1 shown.

[0216] Example 3 Characterization of Hydrated Particle Size of Lipid Nanoparticles

[0217] 60 μL of the lipid nanoparticles prepared in Examples 2A to 2E were taken and the hydrated particle size of the lipid nanoparticles in 1x PBS buffer solution was measured using dynamic light scattering. Figure 3 is the particle size of the five-component LNP of Example 2D, wherein Figure 4 is the particle size of the four-component LNP of Example 2E.

[0218] Take 10 μL of the five-component lipid nanoparticles prepared in Example 2D (the molar ratio of L319 to F-L319 is 2:1), and use a transmission electron microscope to photograph the morphology of the lipid nanoparticles. The results are as follows: Figure 5 shown.

[0219] The hydrated particle sizes of the lipid nanoparticles prepared in Examples 2A to 2E are all less than 250 nm, and the polydispersity coefficient is less than 0.2, indicating that the preparation method of the present invention can form stable lipid nanoparticles with uniform particle size.

[0220] Example 4 Screening of five-component LNPs using in vitro transfection

[0221] Using the mFLuc-loaded LNPs prepared in Implementation 2D, four-component LNPs were selected as a control group to screen different ratios of F-L319 and L319 to act synergistically as ionizable lipids.

[0222] The experimental steps are as follows:

[0223] DC2.4 cells were incubated overnight in a 96-well plate at 20,000 cells per well. When the cell confluence reached 50%, the LNPs loaded with mFLuc prepared in Example 2D were added, wherein L319:F-L319=1:0 (denoted as L319-LNP) was used as the control group, L319:F-L319=2:1 (denoted as Hybrid-LNP1), L319:F-L319=1:1 (denoted as Hybrid-LNP2), and L319:F-L319=0:1 (denoted as F-L319-LNP) were used as the experimental group, and cultured in a saturated humidity incubator at 37°C and 5% CO2 concentration for 12 hours. Half of the culture medium was removed, and cell lysate and firefly luciferase substrate were added. After shaking for 5 minutes, the bioluminescence value was read using an ELISA reader. The test results are shown in Figure 6 .

[0224] Depend on Figure 6 It can be seen that the five-component LNP (Hybrid-LNP2) with the synergistic effect of L319 and F-L319 at a molar ratio of 1:1 has the highest mRNA expression efficiency.

[0225] Example 5 Verification of the Cell-Entering Ability of Five-Component LNP

[0226] The mFLuc-encapsulated five-component LNP prepared in Example 2D was used to detect the cell-entering ability of the five-component LNP.

[0227] The experimental steps are as follows: DC2.4 cells were incubated overnight in a 96-well plate at 20,000 cells per well. When the cell confluence reached 50%, L319:F-L319=1:0 (denoted as L319-LNP) in Example 2D was added as a control group and L319:F-L319=2:1 (denoted as Hybrid-LNP1), L319:F-L319=1:1 (denoted as Hybrid-LNP2), and L319:F-L319=0:1 (denoted as F-L319-LNP) were added as experimental groups. A negative control group (i.e., only culture medium and cells were added to the well plate, denoted as Blank) and an mRNA control group (i.e., culture medium, cells and corresponding concentrations of mRNA were added to the well plate, denoted as FreeDNA) were also set up and incubated at 37°C for 4 hours. After the co-incubation, the cells were digested with 0.25% trypsin, collected, centrifuged, washed three times with 1x PBS, and analyzed by flow cytometry. Figure 7 .

[0228] Depend on Figure 7It can be seen that the five-component LNPs (Hybrid-LNP1, Hybrid-LNP2) with a 2:1 molar ratio of L319 and F-L319 and a 1:1 molar ratio of L319 and F-L319 have higher mRNA entry into cells.

[0229] Example 6 Verification of the membrane fusion ability of the five-component LNP

[0230] The membrane fusion ability of the five-component LNPs containing mFLuc prepared in Example 2D was tested.

[0231] The experimental steps are as follows: mouse red blood cells are placed in a transparent 96-well plate, and a sodium citrate buffer solution of pH 5.5 and a PBS buffer solution of pH 7.4 are added respectively, and L319:F-L319=1:0 (denoted as L319-LNP) in Example 2D is added as a control group and L319:F-L319=2:1 (denoted as Hybrid-LNP1), L319:F-L319=1:1 (denoted as Hybrid-LNP2), and L319:F-L319=0:1 (denoted as F-L319-LNP) are added as experimental groups, and a negative control group (i.e., only buffer solution and red blood cells are added to the well plate, denoted as Untreated cells) and a positive control group (i.e., buffer solution, red blood cells and 0.1% Triton X-100 are added to the well plate, denoted as 0.1% Triton X-100) are incubated at 37°C for 1 hour. After the co-incubation is completed, the supernatant is centrifuged and the absorbance of the supernatant is measured at 540nm. The degree of hemolysis of red blood cells is determined by the absorbance value. For the test results, see Figure 8 .

[0232] Depend on Figure 8 It can be seen that the membrane fusion ability of the five-component LNP of the present invention is slightly lower than that of the non-fluorinated LNP (L319-LNP), but it does not affect its application.

[0233] Example 7 Verification of mRNA expression efficiency of NP in vivo

[0234] Example 7A Verification of mRNA expression efficiency of five-component LNP in vivo

[0235] The five-component LNPs loaded with mFLuc prepared in Example 2D, L319:F-L319=1:0 (denoted as L319-LNP) were used as the control group and L319:F-L319=2:1 (denoted as Hybrid-LNP1), L319:F-L319=1:1 (denoted as Hybrid-LNP2), and L319:F-L319=0:1 (denoted as F-L319-LNP) were used as experimental groups to detect the mRNA expression efficiency of the five-component LNPs in vivo.

[0236] The experimental steps are as follows: female C57BL / 6 mice aged 6-8 weeks were selected, and 2 μg of mRNA LNP was administered to each mouse in each group by intramuscular injection. 200 μL of 200 mg / mL firefly luciferin was injected intraperitoneally into each mouse 4 hours, 12 hours and 24 hours after administration. After 15 minutes, the bioluminescence intensity of the mice was detected by small animal live imaging. After 24 hours of imaging, the mice were killed, and the heart, liver, spleen, lungs, and kidneys were dissected and the luminescence intensity was detected by small animal live imaging.

[0237] The experimental results are as follows Figure 9-10 As shown. Fig. 9 It can be seen that after using the five-component LNPs (Hybrid-LNP1, Hybrid-LNP2) with a 2:1 molar ratio of L319 and F-L319 and a 1:1 molar ratio of L319 and F-L319, the injection site had the strongest bioluminescence intensity at 4 hours, 12 hours, and 24 hours. Fig. 9 In the figure, (a) is the small animal imaging result, and (b) is the bioluminescence intensity at 4 hours, 12 hours, and 24 hours. Fig.10 These are the imaging results of the main organs of small animals. The results show that LNPs (Hybrid-LNP1, Hybrid-LNP2) with a molar ratio of 2:1 between L319 and F-L319 and a molar ratio of 1:1 between L319 and F-L319 are mainly expressed in the spleen.

[0238] It can be seen from this that the five-component LNP of the present invention has a high mRNA expression efficiency in vivo and has a high degree of spleen targeting.

[0239] Example 7B Verification of mRNA expression efficiency of four-component LNP in vivo

[0240] The mRNA expression efficiency in vivo was determined using the four-component LNPs encapsulating mFLuc prepared in Example 2E, with SM-102 as the control group and C5F5, C5F8, C6F9, C7F9, C7F12, C8F13, C9F13, C9F17, C10F19, C11F21, C12F23, and C13F24 as the experimental groups.

[0241] The experimental steps refer to Example 7A. The test results refer to Fig.11 .

[0242] Depend on Fig.11It can be seen that C5F5, C5F8, C6F9, C7F9, C7F12, C8F13, C9F13, C9F17, C10F19, C11F21, C12F23, and C13F24 fluoroliposome nanoparticles are mainly expressed in the spleen.

[0243] Example 8 Verification of toxicity detection of five-component LNP

[0244] Referring to Example 2D, LNPs containing mRNA at different concentrations, the mRNA concentrations were 0, 0.05, 0.25, 2.50, and 5.00 μg / mL.

[0245] DC 2.4 cells were cultured with LNPs containing different concentrations of mRNA for 12 h, and then the medium containing LNPs was replaced with culture medium. Cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay according to the manufacturer's instructions (Vazyme). The results were measured by a microplate reader (BioTek Synergy H1) at a wavelength of 450 nm. The test results are shown in Fig.12 .

[0246] Depend on Fig.12 It can be seen that the five-component LNP encapsulating mRNA of the present invention has low toxicity in vivo.

[0247] Example 9 Verification of the encapsulation efficiency of the five-component LNP

[0248] Take L319:F-L319=1:0 (referred to as L319-LNP) in Example 2D as the control group and L319:F-L319=2:1 (referred to as Hybrid-LNP1), L319:F-L319=1:1 (referred to as Hybrid-LNP2), L319:F-L319=0:1 (referred to as F-L319-LNP) as the experimental group, add RiboGreen detection reagent. Set 485nm as the absorption wavelength and 525nm as the emission wavelength, and read the fluorescence intensity.

[0249] Depend on Fig.13 It can be seen that the five-component LNP for encapsulating mRNA of the present invention has a good encapsulation efficiency.

[0250] The above is a description of the exemplary embodiments of the present invention. However, the protection scope of the present application is not limited to the above embodiments. Any modification, equivalent substitution, improvement, etc. made by those skilled in the art within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. The fluorinated modified ionizable lipid shown in Formula I: in, N represents the nitrogen element; M, P, and Q are the same or different, and are independently selected from biodegradable functional groups; preferably, including but not limited to ester groups, amide groups, acetal groups, ether bonds, and thioether bonds; L1, L2, L3 are the same or different, and are independently selected from C1-20 alkyl; the C1-20 alkyl is optionally substituted by one or more halogens, hydroxyls, and mercapto groups; the carbon atoms in the C1-20 alkyl are optionally substituted by oxygen atoms or sulfur atoms; R1 and R2 are the same or different and are independently selected from C1-6 alkyl; the C1-6 alkyl is optionally substituted by one or more halogens, hydroxyls, and mercapto groups; the carbon atoms in the C1-6 alkyl are optionally substituted by oxygen atoms or sulfur atoms; R3 and R4 are the same or different and are independently selected from C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl; the C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl are optionally substituted by one or more halogen, hydroxyl, thiol; the carbon atoms in the C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl are optionally substituted by oxygen atoms or sulfur atoms; Among the M, P, Q, L1, L2, L3, R1, R2, R3, and R4 groups, at least one group is substituted by one or more fluorine atoms; Preferably, M, P, and Q are the same or different, and are independently selected from an ester group, an ether bond, and a thioether bond; Preferably, L1 is selected from C1-10 alkyl; the C1-10 alkyl is optionally substituted by one or more halogen, hydroxyl, thiol; Preferably, L2 and L3 are the same or different and are independently selected from C1-10 alkyl; the C1-10 alkyl is optionally substituted by one or more halogen, hydroxyl, or mercapto; Preferably, R1 and R2 are the same or different and are independently selected from C1-3 alkyl; Preferably, R3 and R4 are the same or different and are independently selected from C5-15 alkyl, C5-15 alkenyl, C5-15 alkynyl; the C5-15 alkyl, C5-15 alkenyl, C5-15 alkynyl are optionally substituted with one or more halogen, hydroxyl, thiol; Preferably, at least one of the L1, L2, L3, R1, R2, R3, and R4 groups is substituted by one or more fluorine atoms; Preferably, the fluorinated modified ionizable lipid is selected from the following compounds:

2. A lipid nanoparticle comprising the fluorinated ionizable lipid of formula I according to claim 1; Preferably, the lipid nanoparticles include a carrier and an encapsulated nucleic acid, wherein the carrier includes a fluorinated ionizable lipid, an auxiliary phospholipid, a cholesterol-like substance and a pegylated lipid as shown in the above formula I; Preferably, the nucleic acid is selected from one or more of RNA and DNA; Preferably, the fluorinated ionizable lipid accounts for 20 mol% to 70 mol% of the total lipids in the LNP; Preferably, the auxiliary phospholipid accounts for 2 mol% to 20 mol% of the total lipids in the LNP; Preferably, the auxiliary phospholipid includes but is not limited to one or more of 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylcholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanolamine (DOPE), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylethanolamine (POPE), 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine (DSPE), and 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylethanolamine (DPPE); Preferably, the cholesterol-like substance accounts for 10 mol% to 60 mol% of the total lipids in the LNP; Preferably, the cholesterol-like substances include, but are not limited to, one or more of cholesterol, β-sitosterol, cholestanol, cholestanone, cholestenone, 7β-hydroxycholesterol, and 7α-hydroxycholesterol; Preferably, the PEGylated lipid accounts for 0.3 mol% to 30 mol% of the total lipid in the LNP; Preferably, the PEGylated lipids include but are not limited to one or more of 1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-rac-glyceryl-3-methoxypolyethylene glycol (DSG-PEG), 1,2-dipalmitoyl-rac-glyceryl-3-methoxypolyethylene glycol (DPG-PEG), 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine-methoxypolyethylene glycol (DSPE-PEG); Preferably, the molar ratio of the nitrogen element contained in the fluorinated ionizable lipid to the phosphorus element contained in the nucleic acid is (1-50):1; Preferably, the hydrated particle size of the fluorinated lipid nanoparticles is less than 500 nm; Preferably, the polydispersity coefficient of the fluorinated lipid nanoparticles is less than 0.4; Preferably, the molar ratio of the fluorinated ionizable lipid: auxiliary phospholipid: cholesterol substance: polyethylene glycol lipid is 20-80:5-20:20-60:0.1-5; Preferably, the fluorinated lipid nanoparticles are capable of: 1) Improve nucleic acid expression efficiency, and / or 2) Improve the ability of nucleic acids to enter cells, and / or 3) Improve the spleen targeting of nucleic acids.

3. A composite ionizable lipid, comprising a first ionizable lipid and a second ionizable lipid; The second ionizable lipid is selected from the fluorinated modified ionizable lipid of formula I as described in claim 1 or 2 or the fluorinated modified ionizable lipid of formula II as shown below: in, N represents the nitrogen element; W and X are the same or different, and are independently selected from C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl; the C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl are optionally substituted by one or more halogen, hydroxyl, thiol; the carbon atoms in the C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl are optionally substituted by oxygen atoms or sulfur atoms; A and B are the same or different, and are independently selected from biodegradable functional groups; preferably, including but not limited to ester groups, amide groups, acetal groups, ether bonds, and thioether bonds; R 1 , R 2 are the same or different, and are independently selected from C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl; the C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl are optionally substituted by one or more halogen, hydroxyl, thiol; the carbon atoms in the C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl are optionally substituted by oxygen atoms or sulfur atoms; Y is selected from C1-10 alkyl, the C1-10 alkyl being optionally substituted with one or more halogens; Z is selected from -OH, -NH2, -SH, -NR 3 R 4 ; R 3 , R 4 The same or different, each independently selected from C1-5 alkyl, the C1-5 alkyl is optionally substituted by one or more halogens; C is selected from hydrogen, C1-5 alkyl, the C1-5 alkyl being optionally substituted by one or more halogens; C, Y, W, X, A, B, Z, R 1 , R 2 In the group, at least one group is substituted by one or more fluorine atoms.

4. The complex ionizable lipid according to claim 3, characterized in that W and X are the same or different and are independently selected from C3-10 alkyl and C3-10 alkenyl; the C3-10 alkyl and C3-10 alkenyl are optionally substituted by one or more halogens, hydroxyls and mercapto groups; the carbon atoms in the C3-10 alkyl and C3-10 alkenyl are optionally substituted by oxygen atoms and sulfur atoms; Preferably, A and B are selected from ester groups; Preferably, R 1 Selected from C10-20 alkyl, C10-20 alkenyl, C10-20 alkynyl; the C10-20 alkyl, C10-20 alkenyl, C10-20 alkynyl is optionally substituted by one or more halogen, hydroxyl, thiol; Preferably, R 2 is selected from C3-15 alkyl, C3-15 alkenyl, C3-15 alkynyl; the C3-15 alkyl, C3-15 alkenyl, C3-15 alkynyl are optionally substituted by one or more halogen, hydroxyl, thiol; the carbon atoms in the C3-15 alkyl, C3-15 alkenyl, C3-15 alkynyl are optionally substituted by oxygen atoms or sulfur atoms; Preferably, Y is selected from C1-6 alkyl, which is optionally substituted with one or more halogens; Preferably, Z is selected from -OH, -NH2, -SH, -NR 3 R 4 ; Preferably, R 3 , R 4 The same or different, each independently selected from C1-3 alkyl, the C1-3 alkyl is optionally substituted by one or more halogens; Preferably, C is selected from hydrogen, C1-3 alkyl, said C1-3 alkyl being optionally substituted by one or more halogens; Preferably, W, X, R 1 , R 2 In the group, at least one group is substituted by one or more fluorine atoms; Preferably, the fluorinated modified ionizable lipid is selected from the following F-L319 or the compound shown in the following formula II-1: Wherein, R is selected from the following groups: Preferably, the first ionizable lipid is the same as or different from the second ionizable lipid; Preferably, the first ionizable lipid is a non-fluorinated modified ionizable lipid; Preferably, the molar ratio of the first ionizable lipid to the second ionizable lipid is 1:99 to 99:1; Preferably, the first ionizable lipid is selected from 8-[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl)amino]octanoic acid (heptadecan-9-yl) ester (SM-102), [(4-hydroxybutyl)azepinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin MC3 DMA), 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dione (cKK-E12), 9-(4-(dimethylamino)butyryloxy)heptadecanedioic acid di((Z)-non-2-en-1-yl) ester (L319), N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecen-1-yl-1,3-dioxolane-4-ethylamine (DLin-KC2-DMA), 8-[(2-hydroxyethyl)(8-nonyloxy-8-oxooctyl)amino]octanoic acid (heptadecanedioic acid) ester (Lipid5) , 1,1'-[(2-{4-[2-({2-[bis(2-hydroxydodecyl)amino]ethyl}(2-hydroxydodecyl)amino)ethyl]piperazin-1-yl}ethyl)azadialkyl]bis(dodecan-2-ol) (C12-200), (2,3-dioleoylpropyl)trimethylammonium chloride (DOTAP), dimethyldioctadecylammonium bromide (DDAB), tetrakis(8-methylnonyl) 3',3',3",3"-{[(methylazadialkyl)bis(propane-3,1-diyl)]bis(azatriyl)}tetrapropionate (306Oi10) or one or more thereof.

5. A lipid nanoparticle comprising the complex ionizable lipid according to claim 3 or 4; Preferably, the lipid nanoparticle comprises a carrier and an encapsulated nucleic acid, wherein the carrier comprises the complex ionizable lipid, auxiliary phospholipid, cholesterol-like substance and PEGylated lipid as described in claim 3 or 4; Preferably, the nucleic acid is selected from one or more of RNA and DNA; Preferably, the complex ionizable lipid accounts for 20 mol% to 70 mol% of the total lipids in the LNP; Preferably, the auxiliary phospholipid accounts for 2 mol% to 20 mol% of the total lipids in the LNP; Preferably, the auxiliary phospholipid includes but is not limited to one or more of 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylcholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanolamine (DOPE), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylethanolamine (POPE), 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine (DSPE), and 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylethanolamine (DPPE); Preferably, the cholesterol-like substance accounts for 10 mol% to 60 mol% of the total lipids in the LNP; Preferably, the cholesterol-like substances include, but are not limited to, one or more of cholesterol, β-sitosterol, cholestanol, cholestanone, cholestenone, 7β-hydroxycholesterol, and 7α-hydroxycholesterol; Preferably, the PEGylated lipid accounts for 0.3 mol% to 30 mol% of the total lipid in the LNP; Preferably, the PEGylated lipids include but are not limited to one or more of 1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-rac-glyceryl-3-methoxypolyethylene glycol (DSG-PEG), 1,2-dipalmitoyl-rac-glyceryl-3-methoxypolyethylene glycol (DPG-PEG), 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine-methoxypolyethylene glycol (DSPE-PEG); Preferably, the molar ratio of the nitrogen element contained in the composite ionizable lipid to the phosphorus element contained in the nucleic acid is (1-50):1; Preferably, the hydrated particle size of the composite lipid nanoparticles is less than 500 nm; Preferably, the polydispersity coefficient of the composite lipid nanoparticles is less than 0.4; Preferably, the molar ratio of the composite ionizable lipid: auxiliary phospholipid: cholesterol substance: polyethylene glycol lipid is 20-80:5-20:20-60:0.1-5; Preferably, the composite lipid nanoparticles are capable of: 1) Improve nucleic acid expression efficiency, and / or 2) Improve the ability of nucleic acids to enter cells, and / or 3) Improve the spleen targeting of nucleic acids.

6. Fluoro-modified ionizable lipids represented by formula II-1: in, R is selected from the following groups:

7. A lipid nanoparticle comprising the fluorinated modified ionizable lipid of formula II according to claim 3 or the fluorinated modified ionizable lipid of formula II-1 according to claim 6; Preferably, the lipid nanoparticles include a carrier and an encapsulated nucleic acid, wherein the carrier includes a fluorinated modified ionizable lipid as shown in formula II of claim 3 or a fluorinated modified ionizable lipid as shown in formula II-1 of claim 6, an auxiliary phospholipid, a cholesterol-like substance and a pegylated lipid; Preferably, the nucleic acid is selected from one or more of RNA and DNA; Preferably, the fluorinated ionizable lipid represented by formula II accounts for 20 mol% to 70 mol% of the total lipids in the LNP; Preferably, the auxiliary phospholipid accounts for 2 mol% to 20 mol% of the total lipids in the LNP; Preferably, the auxiliary phospholipid includes but is not limited to one or more of 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylcholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glyceryl-3-phosphatidylethanolamine (DOPE), 2-oleoyl-1-palmitoyl-sn-glyceryl-3-phosphatidylethanolamine (POPE), 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine (DSPE), and 1,2-dipalmitoyl-sn-glyceryl-3-phosphatidylethanolamine (DPPE); Preferably, the cholesterol-like substance accounts for 10 mol% to 60 mol% of the total lipids in the LNP; Preferably, the cholesterol-like substances include, but are not limited to, one or more of cholesterol, β-sitosterol, cholestanol, cholestanone, cholestenone, 7β-hydroxycholesterol, and 7α-hydroxycholesterol; Preferably, the PEGylated lipid accounts for 0.3 mol% to 30 mol% of the total lipid in the LNP; Preferably, the PEGylated lipids include but are not limited to one or more of 1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-rac-glyceryl-3-methoxypolyethylene glycol (DSG-PEG), 1,2-dipalmitoyl-rac-glyceryl-3-methoxypolyethylene glycol (DPG-PEG), 1,2-distearoyl-sn-glyceryl-3-phosphatidylethanolamine-methoxypolyethylene glycol (DSPE-PEG); Preferably, the molar ratio of the nitrogen element contained in the fluorinated ionizable lipid to the phosphorus element contained in the nucleic acid is (1-50):1; Preferably, the hydrated particle size of the lipid nanoparticles is less than 500 nm; Preferably, the polydispersity coefficient of the lipid nanoparticles is less than 0.4; Preferably, the molar ratio of the fluorinated ionizable lipid: auxiliary phospholipid: cholesterol substance: polyethylene glycol lipid is 20-80:5-20:20-60:0.1-5; Preferably, the lipid nanoparticles are capable of: 1) Improve nucleic acid expression efficiency, and / or 2) Improve the ability of nucleic acids to enter cells, and / or 3) Improve the spleen targeting of nucleic acids.

8. A method for preparing lipid nanoparticles according to any one of claims 2, 5 or 7, comprising: The lipid nanoparticles are prepared by blending the aqueous phase containing nucleic acid and the lipid organic phase through a blending method or a microfluidics method.

9. Use of the lipid nanoparticles according to any one of claims 2, 5 or 7 in the preparation of biological preparations; Preferably, the biologic is an injectable biologic; Preferably, the biological agent is a vaccine; Preferably, the biological agent is administered by tail vein injection.

10. A biological preparation comprising the lipid nanoparticles according to any one of claims 2, 5 or 7.

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