Lipid nanoparticles for delivering nucleic acid drug, preparation method therefor, and use thereof

Abstract

A lipid nanoparticle composition for nucleic acid drug delivery, a preparation method therefor, and use thereof. A composition prepared by mixing a steroid-cationic lipid compound with an auxiliary phospholipid and a polyethylene glycol-modified phospholipid has relatively good stability and transfection efficiency. By using lipid nanoparticles for delivering a nucleic acid, such as mRNA, a nucleic acid drug can be efficiently and stably delivered to a target cell or organ. Moreover, such LNPs can be used for the atomized inhalation administration of mRNA and the development of lyophilized formulations of mRNA. A relatively high specific antibody response can be induced in experimental animals, and the compound has better safety.

Description

Lipid nanoparticles for delivering nucleic acid drugs and preparation method and use thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a kind of lipid nanoparticles for delivering nucleic acid drugs and preparation method and use thereof. BACKGROUND

[0002] Nucleic acid drugs mainly refer to compounds containing nucleotides or deoxynucleotides with genetic properties and pharmacological activities, which can be used for the treatment of tumors, tissue regeneration, wound healing, pulmonary fibrosis, inflammatory diseases, microbial infections, etc. After being injected into the human body, nucleic acid drugs need to be delivered to the lesion site by an efficient and safe drug delivery system. The drug delivery system needs to stay long enough to accurately target the lesion site while avoiding damage to normal cells.

[0003] Currently, drug delivery systems can be divided into viral vectors and non-viral vectors. Viral vectors have less application in nucleic acid drugs due to their immunogenicity, tumorigenicity, and limited drug loading capacity. Non-viral vectors, such as polymers (polymer) and lipids (liposome or LNP), can combine nucleic acid drugs with specific ligands to target specific cells, and are widely used in current nucleic acid drugs. LNP is one of the most widely used delivery systems for nucleic acid drugs, and LNP delivery system can safely and effectively deliver nucleic acids. LNP delivery system has the advantages of high nucleic acid encapsulation efficiency, effective cell transfection, strong tissue penetration, low cytotoxicity and immunogenicity, and other advantages for drug delivery. Compared with other drug delivery systems, LNP delivery system has great advantages. Therefore, LNP delivery system has broad development and application prospects.

[0004] In the prior art, LNP delivery systems are often made of ionizable lipids (cationic lipids), steroids, neutral lipids, PEG lipids, nucleic acid drugs, etc. For example: patent document AU2020325221A1 discloses a composition of LNP for target cell delivery, including (i) ionizable lipids; (ii) sterols or other structural lipids; (iii) non-cationic helper lipids or phospholipids; (iv) PEG lipids and (v) encapsulated in LNP and / or associated with the drug (such as nucleic acid molecules), the four components are in a specific ratio to enhance the delivery efficiency of target cells; patent document WO2021 / 250263A1 discloses a composition including ionizable lipids, phospholipids, sterols, PEG lipids and one or more nucleic acids, and discloses specific percentages of less than about 1 mol% C14-PEG2000 lipids and other lipids; patent CN102712935B discloses a lipid particle comprising: cationic lipids; neutral lipids, zwitterionic lipids or anionic lipids; PEG lipids; sterols and nucleic acids, and the above components are assembled into a lipid particle with a solid core, which has a higher coating efficiency; patent document WO2021 / 055849A1 discloses a lipid with the following structure: This structure can improve its safety, effectiveness and specificity; patent document WO2021 / 026358Al discloses a target cell delivery lipid nanoparticle (LNP): including (i) ionizable lipids (ii) sterols or other structural lipids; (iii) non-cationic helper lipids or phospholipids; (iv) effective load; (v) PEG lipids, as a drug delivery system, taking into account safety and effectiveness. In recent years, it has been found that introducing cholesterol into ionizable lipid compounds can also be used to deliver nucleic acid drugs. Patent document US7514099B2 discloses a cholesteric amino lipid compound CLinDMA This compound can be used to deliver siRNA with phospholipids, cholesterol, PEG lipids to form four-component LNP or with phospholipids, DMOBA lipids, cholesterol and PEG lipids to form five-component LNP; patent CN112424214A discloses an ionizable cationic lipid compound formed by cholesterol and linear olefins (3) The compound is constructed into a lipid nanoparticle with cholesterol, DPPC, DOPE and DMG-PEG200 to deliver nucleic acid, 5 kinds of lipid excipients are required, the construction is complex, and the PDI is -0.292, the polydispersity coefficient is poor. In order to adapt to different application scenarios, it is also particularly important to develop different delivery systems suitable for different drug delivery routes, such as using atomization inhalation for drug delivery to treat lung-related diseases or prevent respiratory-related pathogenic bacterial infections. At present, the stable storage of mRNA-LNP is still a big difficulty, and the stable storage of mRNA-LNP at 2-8℃ or even room temperature can be realized by freeze-drying. It can be seen that, by optimizing each component in the composition and optimizing the cationic lipid, in order to further obtain a more safe, effective and stable LNP delivery system which is simple to construct and can be used for different drug delivery routes, further development of the components and the structure of each component is needed.

[0005] Traditional LNP includes 4 components: ionizable cationic lipid, amphiphilic phospholipid, pegylated lipid and cholesterol. Due to the characteristics of mRNA itself, the storage and transportation of mRNA packaged by traditional LNP must be in an ultra-low temperature environment. According to the current research, the Pfizer mRNA vaccine can be stored in a -70℃ ultra-low temperature freezer for 6 months, in a Pfizer special insulation transport box with continuous addition of dry ice for 15 days, and in a normal 2 to 8℃ hospital refrigerator for only 5 days. -70℃ is a very low temperature that is common in research laboratories, but many medical centers do not have such equipment, and developing countries and economically underdeveloped areas lack such equipment. There is no ready-made capacity in the field of nucleic acid vaccine transportation that can reach -70℃ conditions in China at present. Therefore, the problem of nucleic acid vaccine storage needs to be solved.

[0006] SUMMARY

[0007] The present application provides a lipid nanoparticle composition. Different from the traditional 4-component LNP, the ionizable cationic lipid is covalently combined with a steroid to form a 3-component lipid nanoparticle composition with the auxiliary lipid and the pegylated lipid, which has good freeze-drying stability and can meet the storage requirement at 2-8℃.

[0008] In a first aspect, the present application provides a lipid nanoparticle composition for nucleic acid drug delivery, comprising: a steroid-cationic lipid compound, an auxiliary lipid, and a pegylated lipid.

[0009] In one embodiment, the molar ratio of the steroid-cationic lipid: helper lipid: polyethylene glycol lipid is 30-80:20-80:0.5-20; preferably, the molar ratio of the steroid-cationic lipid: helper lipid: polyethylene glycol lipid is 30-80:30-80:0.5-20.

[0010] In one embodiment, the polyethylene glycol lipid is selected from 2[(polyethylene glycol)-2000] N,N-tetracosanoylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycero- methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[ammonia(polyethylene glycol)] (PEG-DSPE), PEG-disteraryl glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerol amide (PEG-DAG), PEG-dipalmitoyl phosphatidyl ethanolamine (PEG-DPPE), or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).

[0011] In one embodiment, the helper lipid is selected from a neutral lipid, a zwitterionic lipid, or an anionic lipid; preferably, the helper lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidyl ethanolamine (POPE), phosphocholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-eicosenoyl-sn-glycero-3-phosphocholine (DEPC), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, distearoyl phosphatidyl ethanolamine (DSPE), or lysophosphatidyl ethanolamine.

[0012] In one embodiment, the composition further comprises a nucleic acid drug; preferably, the nucleic acid drug is DNA or RNA; more preferably, the RNA is selected from antisense RNA, saRNA, mRNA, IncRNA, miRNA, siRNA, piRNA, gRNA, tsRNA, circRNA, and self-replicating mRNA; more preferably, the nucleic acid drug is mRNA; more preferably, the composition is in the form of nucleic acid-lipid particles.

[0013] In one embodiment, the steroid-cationic lipid compound in the composition has the structure shown in Formula (I):

[0014] or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof, wherein,

[0015] R1, R2are each independently selected from H, OR’, wherein R’ is selected from H, C1-C9alkyl, cycloalkyl, C2-C9alkenyl, C2-C9alkynyl, C3-8 cycloalkyl, C 3-8 Cycloalkenyl or C 3-8 Cycloacetic group;

[0016] L1 and L2 are each independently selected from chemical bonds (-), -O-, -S-, -O(C=O)O-, and -(C=O)NR. a -、-NR a (C=O)-, -O(C=O)-, -(C=O)O-, -SS-, -S(O) x -、-OS(O) x O-, -C(=O)S-, -SC(=O)-, -NR a C(=O)NR b -、-OC(=O)NR a -、-NR a C(=O)O-, -OC(=O)S-, -SC(=O)O-, -P(O)(OR a )O-、-OP(O)(OR a O- or C 1-12 A combination of one or more alkylene groups;

[0017] x is selected from 0, 1, or 2;

[0018] R a R b Each is independently selected from H and C. 1-12 Alkyl, C 2-12 alkenyl or C 2-12 alkynyl group;

[0019] R3 is selected from naturally occurring or non-naturally occurring steroids;

[0020] R4 is selected from C 6-24 Alkyl, C 6-24 alkenyl, C 6-24 Alkynes or C 6-24 Alkoxy;

[0021] m, n, o, p, and q are each independently selected from any integer between 0 and 12;

[0022] The condition is that it does not contain compounds:

[0023] In some embodiments, R1 and R2 are each independently selected from H, OH, and C1-C9 alkoxy groups;

[0024] L1 and L2 are each independently selected from chemical bonds (-), -O-, -S-, -O(C=O)O-, and -(C=O)NR. a -、-NRa (C=0)-, -0(C=0)-, -(C=0)0-, -S-S-, -S(0) x -, -OS(O) x O-, -C(=0)S-, -SC(=0)-, -NR a C(=0)NR b -, -OC(=0)NR a -, -NR a C(=0)0-, -OC(=0)S-, -SC(=0)0-; preferably, L1, L2are each independently selected from -0-, -S-, -0(C=0)0-, -(C=0)NR a -, -NR a (C=0)-, -0(C=0)-, -(C=0)0-, -S-S-, -S(0) x -, -OS(O) x O-, -C(=0)S-, -SC(=0)-; more preferably, L1, L2are each independently selected from -0-, -S-, -0(C=0)0-, -0(C=0)-, -(C=0)0-, -S-S-;

[0025] x is selected from 0, 1 or 2;

[0026] R a , R b are each independently selected from H, C 1-12 alkyl, C 2-12 alkenyl or C 2-12 alkynyl;

[0027] R3is selected from a naturally occurring or non-naturally occurring steroidal;

[0028] R4is selected from an optionally substituted C 6-24 alkyl, C 6-24 alkenyl; preferably, R4is selected from an optionally substituted C 6-24 alkyl;

[0029] m, n, o, p, q are each independently selected from any integer between 0-12.

[0030] In some embodiments, said R3is a sterol;

[0031] Preferably, said sterol is an animal sterol, or an oxidized or reduced form thereof; and / or, said sterol is a plant sterol, or an oxidized or reduced form thereof; and / or, said sterol is a synthetic sterol, or an oxidized or reduced form thereof;

[0032] More preferably, the sterol is selected from cholesterol, an oxidized form of cholesterol, a reduced form of cholesterol, an alkyl lithocholate, stigmasterol, stigmastanol, campesterol, ergosterol, or sitosterol;

[0033] More preferably, the sterol is an oxidized form of cholesterol, a reduced form of cholesterol, an alkyl lithocholate, stigmasterol, stigmastanol, campesterol, ergosterol, or sitosterol;

[0034] More preferably, the sterol is selected from avenasterol, beta-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, fucosterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol; lanosterol, photosterols, phaeosterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, or lithocholic acid;

[0035] More preferably, the sterol has the following structural formula:

[0036] wherein R" is C 1-20 alkyl.

[0037] In some embodiments, the R4 is selected from the following structure:

[0038] Preferably, the R4 is selected from the following structure:

[0039] In some embodiments, the lipid compound of Formula (I) described herein is a lipid compound of Formula (II-1), Formula (II-2), Formula (II-3), or Formula (II-4):

[0040] or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof;

[0041] wherein, m, n, o, p, q are each independently selected from any integer between 0-12;

[0042] R1, R2 are each independently H, OR5, wherein R5 is selected from H, C1-C6 alkyl, cycloalkyl, alkenyl, alkynyl;

[0043] L1, L2 are each independently selected from a combination of one or more of a chemical bond (-), -O-, -S-, -O(C=O)O-, -O(C=O)-, -(C=O)O-;

[0044] R4 is selected from C6-24 Alkyl, C 6-24 alkenyl, C 6-24 Alkynes or C 6-24 Alkyl group.

[0045] In some implementations, L1 is selected from any one of -O-, -O(C=O)O-, -O(C=O)-, and -(C=O)O-; preferably, L1 is selected from -(C=O)O-.

[0046] In some implementations, L2 is selected from any one of -O-, -O(C=O)O-, -O(C=O)-, -(C=O)O-, and -SS-; preferably, L2 is selected from -O(C=O)-.

[0047] In some implementations, R4 is selected from Preferably, R4 is selected from More preferably, R4 is selected from Most preferably, R4 is selected from

[0048] In some implementations, m is selected from 2, 3, or 4; preferably, m is selected from 3.

[0049] In some implementations, p and q are each independently selected from 1, 2, 3, 4, 5, 6, 7 or 8; preferably, p is selected from 4, 5, 6, 7 or 8.

[0050] In some embodiments, in the compound of formula II-1 described herein, L1 is selected from -(C=O)O-; L2 is selected from -O(C=O)-; R4 is selected from... m is selected from 3; p is selected from 2; q is selected from any integer between 4 and 8.

[0051] In some embodiments, the present invention provides compounds selected from the following or pharmaceutically acceptable salts thereof.

[0052] In some embodiments, the nitrogen-to-phosphorus ratio of the lipid nanoparticles is (1-15):1, including but not limited to 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1 or 15:1.

[0053] In some embodiments, the lipid nanoparticle has a diameter of 15 nm to 300 nm; preferably, the lipid nanoparticle has a diameter of 60 nm to 102 nm; more preferably, the lipid nanoparticle has a diameter of 80 nm to 90 nm.

[0054] In some embodiments, the dosage form of the lipid nanoparticle composition is a liquid formulation or a lyophilized powder.

[0055] In a second aspect, the present application provides a method for preparing the lipid nanoparticle described herein, comprising dissolving the steroid-cationic lipid compound, the neutral phospholipid and the polyethylene glycol lipid described herein in a solvent, and mixing with the nucleic acid drug to obtain.

[0056] In some embodiments, the nucleic acid drug is mRNA.

[0057] In a third aspect, the present application provides use of the lipid nanoparticle composition described herein in the preparation of a bioactive substance delivery system.

[0058] In some embodiments, the bioactive substance delivery system is an aerosol inhalation delivery system.

[0059] In some embodiments, the dosage form of the aerosol inhalation delivery system is a liquid formulation or a lyophilized powder.

[0060] In some embodiments, the aerosol inhalation delivery system is a liquid formulation, which is administered by aerosolization, and / or is administered by inhalation or is administered by oral inhalation; or the aerosol inhalation is administered by mucosal administration, aerosolization of a liquid formulation. Preferably, the aerosol inhalation is administered by inhalation or is administered by oral inhalation mucosal administration.

[0061] In one embodiment, the aerosol inhalation delivery system is a liquid formulation, which is administered by intramuscular injection.

[0062] In one embodiment, the delivery system further comprises a pharmaceutically acceptable excipient, preferably, the excipient comprises one or more of sodium acetate, tromethamine, potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate, sucrose.

[0063] In one embodiment, the delivery system is a vaccine.

[0064] In one embodiment, the vaccine is a vaccine for preventing cancer, viral infection, bacterial infection or fungal infection; preferably, the virus is selected from the group consisting of norovirus, Ebola virus, coronavirus, cytomegalovirus, dengue virus, Zika virus, coxsackievirus, enterovirus, hepatitis virus, herpes simplex virus, human papillomavirus, influenza virus, Marburg virus, measles virus, poliovirus, rabies virus, rotavirus or rubella virus.

[0065] Advantages of the present application:

[0066] The steroid-cationic lipid compound prepared in the present application has better stability and transfection efficiency. The lipid nanoparticles are used for delivering nucleic acid drugs, such as mRNA, which can efficiently and stably deliver nucleic acid drugs to target cells or organs, and the LNP can be used for aerosol inhalation administration of mRNA. It can induce a higher specific antibody response in experimental animals, and has better safety. BRIEF DESCRIPTION OF DRAWINGS

[0067] Figure 1 shows the expression of Luc-mRNA-LNP in Hep3B cells detected by an enzyme marker;

[0068] Figure 2 shows the animal live imaging and ex vivo imaging of mice after intramuscular injection of mRNA-LNP;

[0069] Figure 3 shows the ELISA detection of serum antigen-specific antibody titers of mice immunized with mRNA-LNP;

[0070] Figure 4a shows the ICS detection of TNFα+CD8 + T cell immune map of mice immunized with mRNA-LNP;

[0071] Figure 4b shows the ICS detection of IFNγ+CD8 + T cell immune map of mice immunized with mRNA-LNP;

[0072] Figure 4c shows the ICS detection of IL2+CD8 + T cell immune map of mice immunized with mRNA-LNP;

[0073] Figure 5 shows the ELISA detection of cytokine IL-6 expression in serum of CD1 mice 4-6 hours after intramuscular injection of 30ug mRNA-LNP;

[0074] Figure 6 shows the expression level of mRNA-LNP in Hep3B cells before and after aerosolization detected by bioluminescence;

[0075] Figure 7 shows the bioluminescence detection of the expression distribution of firefly luciferase in mice after tracheal inhalation of mRNA-LNP. DETAILED DESCRIPTION

[0076] The technical solutions in the present application will be described clearly and completely below in combination with the drawings of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all. It should be noted that the methods used in the present application are conventional methods unless otherwise specified, and the reagents used in the present application are commercially available products. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0077] Definitions

[0078] As used in this specification, unless the context dictates the contrary, the following words and phrases are generally intended to have the meanings set forth below.

[0079] As used herein, the term “lipid nanoparticle”, “Lipid Nanoparticle” or “LNP” refers to a particle having a size in the nanometer range, e.g., 1 nm to 1,000 nm, which comprises one or more types of lipid molecules.

[0080] As used herein, the term “gene medicine” generally consists of a carrier or delivery system containing an engineered gene construct, the active ingredient of which can be DNA, RNA, genetically modified viruses, bacteria or cells, by introducing exogenous genes into target cells or tissues, replacing, compensating, blocking, correcting specific genes, to achieve the purpose of treating and preventing diseases.

[0081] As used herein, the term “nucleic acid” refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single- or double-stranded form, and includes DNA, RNA and their hybrids.

[0082] As used herein, the term “lipid compound” or “lipid” refers to a group of organic compounds, which includes but is not limited to esters of fatty acids, and is generally characterized by being poorly soluble in water but soluble in many organic solvents. The solvent of the present application includes but is not limited to: water, benzene, toluene, pentane, hexane, methanol, ethanol, isopropanol, diethyl ether, ethyl acetate, acetone, carbon tetrachloride. In some embodiments of the present application, the solvent is ethanol.

[0083] As used herein, the term “alkyl” refers to a saturated straight-chain or branched-chain hydrocarbon group. As used herein, C 1-9 Alkyl includes straight-chain or branched-chain alkyl groups containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 carbon atoms, for example including but not limited to C 1- 8, C 2-7 , C 2-8 , C 3-6 or C 4-7 alkyl; C 6-24Alkyl includes straight-chain or branched-chain alkyl groups containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, for example including but not limited to C 6- 8, C 6-17 , C 6-22 , C 10-16 , or C 14-17 alkyl; C 1-5 Alkyl includes straight-chain or branched-chain alkyl groups containing 1, 2, 3, 4, or 5 carbon atoms, for example including but not limited to C 1-4 , C 2-4 , C 2-3 , C 3-5 , or C 4-5 alkyl; C 1-20 Alkyl includes straight-chain or branched-chain alkyl groups containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, for example including but not limited to C 1-8 , C 1-10 , C 6-22 , C 10-16 , or C 14-17 alkyl.

[0084] As used herein, the term "alkenyl" refers to an unsaturated straight-chain or branched-chain hydrocarbon group containing one or more unsaturated carbon-carbon double bonds. The unsaturated carbon-carbon double bond can be present at any stable point along the chain. As used herein, C 2-9 Alkenyl includes straight-chain or branched-chain alkenyl groups containing 2, 3, 4, 5, 6, 7, 8, or 9 carbon atoms, for example including but not limited to C 1-8 , C 2- 7, C 2-8 , C 3-6 , or C 4-8 straight-chain or branched-chain alkenyl; C 6-24 Alkenyl includes straight-chain or branched-chain alkenyl groups containing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms, for example including but not limited to C 6-8 , C 6-17 , C 6-22 , C 10-16 , or C 14-17 alkenyl.

[0085] As used herein, the term "alkynyl" refers to an unsaturated straight-chain or branched-chain hydrocarbon group containing one or more unsaturated carbon-carbon triple bonds. As used herein, C 2-9 Alkynyl includes straight-chain or branched-chain alkynyl groups containing 2, 3, 4, 5, 6, 7, 8, or 9 carbon atoms, for example including but not limited to C1-8 , C 2-7 , C 2-8 , C 3-6 or C 4-7 alkynyl.

[0086] As used herein, one or more carbons of other positions of the removed end group position of alkyl, alkenyl, alkynyl can be replaced with a heteroatom such as nitrogen, oxygen, sulfur and oxides thereof such as nitric oxide, carbonyl, (sul)fur groups, for example, -(CH2) m -O-(CH2) n -, -(CH2) m -S-(CH2) n -, -(CH2) m -S-S-(CH2) n -, -(CH2) m -CO-(CH2) n -, -(CH2) m -OCO-(CH2) n -, -(CH2) m -OCOO-(CH2) n - (wherein m, n can be an integer from 1 to 9) can be unsubstituted or substituted with one or more heteroatom substituents as described herein. In certain embodiments, no heteroatoms are included in the alkyl, alkenyl, or alkynyl group.

[0087] As used herein, the term "alkylene" refers to a saturated, divalent linear or branched hydrocarbon group. As used herein, C 1-9 Alkylene includes straight chain or branched chain alkylene containing 1, 2, 3, 4, 5, 6, 7, 8, or 9 carbon atoms, for example, including but not limited to C 1-8 , C 2-7 , C 2-8 , C 3-6 or C 4-7 alkylene; C 1-12 Alkylene includes straight chain or branched chain alkylene containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, for example, including but not limited to C 1-8 , C 2-7 , C 2-8 , C 3-6 or C 4-12 alkylene; C 1-10 Alkylene includes straight chain or branched chain alkylene containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, for example, including but not limited to C 1-8 , C 2-7 , C 2-8 , C 3-6 or C 4-10 alkylene; C1-6 Alkylene includes straight-chain or branched-chain alkylene groups containing 1, 2, 3, 4, 5, or 6 carbon atoms, for example including but not limited to C 1-5 , C 2-4 , C 3-6 , or C 4-6 alkylene groups. As used herein, an alkylene, alkenylene, or alkynylene group can include one or more cyclic aliphatic groups and / or one or more heteroatoms such as oxygen, nitrogen, or sulfur, and can be optionally substituted with one or more substituents such as alkyl, halo, alkoxy, hydroxyl, amino, aryl, ether, ester, or amide. One or more carbons other than the terminal group positions can be replaced with a heteroatom such as nitrogen, oxygen, sulfur, and oxides thereof such as nitrogen oxides, carbonyl, sulfone, or sulfoxide groups. In certain embodiments, the alkylene, alkenylene, or alkynylene group is unsubstituted. In certain embodiments, the alkylene, alkenylene, or alkynylene group does not include any heteroatoms.

[0088] As used herein, the term "alkoxy" refers to an "alkyl-O-" group, wherein alkyl is as defined herein. As used herein, C 6-24 Alkoxy includes straight-chain or branched-chain alkoxy groups containing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms, for example including but not limited to C 6-8 , C 6-17 , C 6-22 , C 10-16 , or C 14-17 alkoxy groups.

[0089] As used herein, the term "cycloalkyl" refers to a saturated cyclic hydrocarbon group. As used herein, C 3-8 Cycloalkyl includes cycloalkyl groups containing 3, 4, 5, 6, 7, or 8 carbon atoms, for example including but not limited to C 3-7 , C 4-7 , or C 3-6 cycloalkyl groups.

[0090] As used herein, the term "cycloalkenyl" refers to a cyclic hydrocarbon group that includes at least one carbon-carbon double bond. As used herein, C 3-8 Cycloalkenyl includes cycloalkenyl groups containing 3, 4, 5, 6, 7, or 8 carbon atoms, for example including but not limited to C 3-7 , C 4- , or C 3-6 cycloalkenyl groups.

[0091] As used herein, the term "cycloalkynyl" refers to a cyclic hydrocarbon group that includes at least one carbon-carbon triple bond. As used herein, C 3-8Cycloalkynyl groups include cycloalkynyl groups containing 3, 4, 5, 6, 7, or 8 carbon atoms, for example including but not limited to C 3-7 , C 4-7 , or C 3-6 alkynyl groups.

[0092] As used herein, the term "cationic lipid" refers to a lipid molecule that is positively charged in response to environmental pH or hydrogen ion activity.

[0093] As used herein, the term "helper lipid" refers to a lipid that is not positively charged under environmental pH conditions, which includes neutral lipids that are not charged, zwitterionic lipids, and negatively charged anionic lipids.

[0094] As used herein, the term "polyethylene glycol lipid" refers to a lipid molecule that comprises a lipid moiety and a polyethylene glycol moiety.

[0095] As used herein, the term "delivery system" refers to a formulation or composition that modulates the spatial, temporal, and dosage distribution of a biologically active ingredient within a biological organism.

[0096] Examples

[0097] Example 1 Synthesis of compound 4

[0098] Step 1: Synthesis of compound la

[0099] Dissolve 2-hexyldecanoic acid (12.8 g, 50 mmol) in 30 mL of dichloromethane, add 6-bromo-hexanol (9.0 g, 50 mmol), DMAP (2.1 g, 20 mmol), triethylamine (6.2 g, 60 mmol), and stir to dissolve. Add a solution of EDC.HCL (11.0 g, 60 mmol) in dichloromethane dropwise, and stir at room temperature for 16 hours after the addition is complete. Quench with water, add dilute hydrochloric acid, adjust the pH to 1-3, and separate the layers. Wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to give compound la (14.5 g, pale yellow oil) in 70% yield.

[0100] MS m / z (ESI): 419.3 [M+1] + .

[0101] Step 2: Synthesis of compound lb

[0102] Compound 1a (12.6 g, 30 mmol) was added to a mixture of 25 mL of tetrahydrofuran and 50 mL of acetonitrile at room temperature, and then 1-(3- aminopropyl)imidazole (15.0 g, 120 mmol), potassium carbonate (8.5 g, 60 mmol), and potassium iodide (0.5 g, 3 mmol) were sequentially added. The mixture was stirred at 40-50°C for 16 h, and the reaction progress was monitored. After compound 1a was completely consumed, the mixture was cooled to room temperature, and the solvent was removed at 45°C. The crude product was dissolved in dichloromethane, washed with saturated brine twice, dried over anhydrous sodium sulfate, concentrated, and then separated and purified by column chromatography on silica gel (DCM / MeOH 60:1-40:1) to obtain compound 1b (7.2 g, yellowish oil) at a yield of 52%.

[0103] MS m / z (ESI): 464.4 [M+1] + .

[0104] Step 3: Synthesis of compound 1c

[0105] Cholesterol (11.6 g, 30 mmol) was added to 60 mL of dichloromethane at room temperature, and then acrylic acid (4.3 g, 60 mmol) and dicyclohexyl carbodiimide (12.4 g, 60 mmol) were sequentially added. Triethylamine (6.1 g, 60 mmol) was added dropwise to the system, and the mixture was stirred for 16 h. After cholesterol was completely consumed, the mixture was cooled to room temperature, and the dichloromethane was evaporated. Compound 1c (9.2 g, white solid) was obtained by crystallization using a mixture of methyl tert-butyl ether and acetonitrile at a yield of 67%.

[0106] MS m / z (ESI): 455.4 [M+1] + .

[0107] Step 4: Synthesis of compound 4

[0108] Compound 1b (7.0 g, 15 mmol) and compound 1c (8.2 g, 18 mmol) were added to a mixture of 100 mL of tetrahydrofuran and 100 mL of acetonitrile at room temperature, and then the mixture was stirred at 40-50°C for 16 h. The solvent was removed at 45°C, and the crude product was dissolved in dichloromethane, washed with saturated brine twice, dried over anhydrous sodium sulfate, concentrated, and then separated and purified by column chromatography on silica gel (DCM / MeOH 100:1-50:1) to obtain compound 5 (6.8 g, yellowish oil) at a yield of 50%.

[0109] MS m / z (ESI): 905.6 [M+1]+;

[0110] 1 H NMR (300 MHz, CDC13): δ 7.51 (s, 1H), 7.08 (s, 1H), 6.95 (s, 1H), 5.38-5.39 (m, 1H), 4.62-4.66 (m, 1H), 4.09 (t, 2H, J = 6.6 Hz), 4.01 (t, 2H, J = 6.9 Hz), 2.75-2.77 (m, 2H), 2.32-2.40 (m, 9H), 1.90-2.01 (m, 7H), 0.88-1.62 (m, 71H), 0.70 (s, 3H).

[0111] Synthesis of compound 7 of example 2

[0112] Step 1: Synthesis of compound 2a

[0113] Dissolve 2-hexyldecanoic acid (12.8 g, 50 mmol) in 30 mL of dichloromethane, add 8-bromo-1-octanol (10.5 g, 50 mmol), DMAP (2.4 g, 20 mmol), and stir to dissolve. Add a solution of EDC-HCL (11.5 g, 60 mmol) in dichloromethane dropwise, and stir at room temperature for 16 hours after the addition is complete. Quench with water, add dilute hydrochloric acid, adjust the pH to 1-3, and separate the layers. Wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to give compound 2a (14.1 g, pale yellow oil) in 63% yield.

[0114] MS m / z (ESI): 447.3 [M+1] + .

[0115] Step 2: Synthesis of compound 2b

[0116] Dissolve 2-hexyldecanoic acid (12.8 g, 50 mmol) in 30 mL of dichloromethane, add 8-bromo-1-octanol (10.5 g, 50 mmol), DMAP (2.4 g, 20 mmol), and stir to dissolve. Add a solution of EDC-HCL (11.5 g, 60 mmol) in dichloromethane dropwise, and stir at room temperature for 16 hours after the addition is complete. Quench with water, add dilute hydrochloric acid, adjust the pH to 1-3, and separate the layers. Wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to give compound 2a (14.1 g, pale yellow oil) in 63% yield.

[0117] MS m / z (ESI): 447.3 [M+1]+ .

[0118] Step 3: Synthesis of compound 7

[0119] Compound 2b (7.4 g, 15 mmol), compound 1c (8.0 g, 18 mmol) were added into 15 mL tetrahydrofuran, 30 mL acetonitrile mixed solution at room temperature, heated to 70-80 °C, stirred for 16 h, 45 °C to remove the solvent, dissolved in dichloromethane, washed with saturated brine twice, the organic phase was dried over anhydrous sodium sulfate, concentrated to give the crude product, then separated by silica gel column chromatography (DCM / MeOH 100:1-50:1), compound 9 (6.3 g, light yellow oil), yield 45%.

[0120] MS m / z (ESI): 932.8 [M+1] + ;

[0121] 1 H NMR (300 MHz, CDC13): δ 7.51 (s, 1H), 7.08 (s, 1H), 6.95 (s, 1H), 5.33-5.35 (m, 1H), 4.58-4.69 (m, 1H), 4.08 (t, 2H, J = 6.6 Hz), 4.01 (t, 2H, J = 6.9 Hz), 2.77 (br, 2H), 2.32-2.42 (m, 9H), 1.86-1.93 (m, 8H), 0.87-1.64 (m, 74H), 0.70 (s, 3H).

[0122] Synthesis of compound 8 of Example 3

[0123] Step 1: Synthesis of compound 3a

[0124] 2-hexyl decanoic acid (12.8 g, 50 mmol) was dissolved in 30 mL dichloromethane, 4-bromo-1-butanol (7.7 g, 50 mmol), DMAP (2.4 g, 20 mmol) were added and stirred to dissolve. A solution of EDC-HCL (11.5 g, 60 mmol) in dichloromethane was added dropwise, and after the addition was completed, it was stirred at room temperature for 16 hours. Quench with water, add dilute hydrochloric acid, adjust the pH to 1-3, separate the organic phase. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give compound 3a (13.0 g, light yellow oil), yield 66%.

[0125] MS m / z (ESI): 391.2 [M+1] + .

[0126] Step 2: Synthesis of compound 3b

[0127] Compound 3a (11.7 g, 30 mmol) was added into 25 mL of tetrahydrofuran, 50 mL of acetonitrile mixed solution at room temperature, then 1-(3-aminopropyl)imidazole (15 g, 120 mmol), potassium carbonate (8.3 g, 60 mmol), potassium iodide (0.5 g, 3 mmol) were added in turn, and the temperature was raised to 40-50 °C, stirred for 16 h, and the reaction progress was monitored. After compound 3a was completely consumed, the temperature was lowered to room temperature, and the solvent was removed at 45 °C. The crude product was dissolved in dichloromethane, washed with saturated brine twice, and the organic phase was dried over anhydrous sodium sulfate. The crude product was concentrated, separated and purified by silica gel column chromatography (DCM / MeOH 60:1-40:1), to obtain compound 3b (7.0 g, light yellow oil), with a yield of 53%.

[0128] MS m / z (ESI): 436.4 [M+1] + .

[0129] Step 3: Synthesis of compound 8

[0130] Compound 3b (7.0 g, 15 mmol) and compound 1c (8.0 g, 18 mmol) were added into 15 mL of tetrahydrofuran, 30 mL of acetonitrile mixed solution at room temperature, and the temperature was raised to 70-80 °C, stirred for 16 h, and the solvent was removed at 45 °C. The crude product was dissolved in dichloromethane, washed with saturated brine twice, and the organic phase was dried over anhydrous sodium sulfate. The crude product was concentrated, separated and purified by silica gel column chromatography (DCM / MeOH 100:1-50:1), to obtain compound 10 (6.6 g, light yellow oil), with a yield of 50%.

[0131] MS m / z (ESI): 876.5 [M+1] + ;

[0132] 1 H NMR (300 MHz, CDC13): δ 7.52 (s, 1H), 7.08 (s, 1H), 6.95 (s, 1H), 5.33-5.35 (m, 1H), 4.58- 4.69 (m, 1H), 4.09 (t, 2H, J = 6.6 Hz), 4.00 (t, 2H, J = 6.9 Hz), 2.76 (br, 2H), 2.32-2.44 (m, 9H), 1.86-1.92 (m, 7H), 0.87-1.64 (m, 67H), 0.70 (s, 3H).

[0133] Synthesis of compound 9 of Example 4

[0134] Step 1: Synthesis of compound 4a

[0135] Dissolve 1,3-dibromopropane (2.16 g, 10.0 mmol) in 30 mL of n-butanol, add cholesterols (1.94 g, 5.0 mmol) and sodium hydroxide (1.20 g, 30 mmol), stir at reflux for 5 h. After the reaction is completed, add dilute hydrochloric acid to adjust the pH to neutral. Remove n-butanol by rotary evaporation, add 20 mL of dichloromethane for dilution, then wash with 30 mL of saturated brine, dry the organic phase with anhydrous sodium sulfate, filter and concentrate, and separate by column chromatography to obtain the product 4a (1.25 g, light yellow oil), with a yield of 48%.

[0136] MS m / z (ESI): 521.3 [M+1] + .

[0137] Step 2: Synthesis of compound 9

[0138] Dissolve compound 4a (463 mg, 1.0 mmol) in tetrahydrofuran, add acetonitrile, compound 1b (625 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), potassium iodide (332 mg, 2.0 mmol), and stir at 83°C for 16-20 h. Cool to room temperature, filter, wash the filter residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract twice with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, filter and concentrate, separate by column chromatography to obtain the product 11 (434 mg, light yellow oil), with a yield of 48%.

[0139] MS m / z (ESI): 903.9 [M+1] + ;

[0140] 1 H NMR (300 MHz, CDC13): δ 8.02 (s, 1H), 7.45 (s, 1H), 7.12 (s, 1H), 6.96 (s, 1H), 5.40 (t, 1H, J = 5.4 Hz), 4.10-3.92 (m, 2H), 3.35 (t, 2H, J = 5.4 Hz), 3.28-3.01 (m, 7H), 2.50-2.42 (m, 4H), 2.10-0.85 (m, 81H), 0.70 (s, 3H).

[0141] Example 5 Synthesis of compound 11

[0142] Step 1: Synthesis of compound 5a

[0143] To a solution of 2-(oxirane-2-yl)ethan-1-ol (2.64 g, 30 mmol) in 80 mL of dichloromethane was added 4-dimethylaminopyridine (7.3 g, 60 mmol) followed by portion wise addition of p-nitrophenyl chloroformate (6.6 g, 33 mmol) and the reaction stirred at room temperature for 3 h. To this reaction was added cholesterol (13.5 g, 35 mmol) and the mixture stirred at room temperature overnight. After completion of the reaction as indicated by TLC, the reaction was diluted with 50 mL of dichloromethane and washed with 60 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give the product 5a (9.3 g, pale yellow oil) in 62% yield.

[0144] MS m / z (ESI): 501.4 [M+1] + .

[0145] Step 2: Synthesis of compound 5b

[0146] To compound 5a (7.5 g, 15 mmol) was added 1-(3-aminopropyl)imidazole (7.5 g, 60 mmol) at room temperature and the reaction was heated to 70-80 °C and stirred for 16 h. After completion of the reaction as indicated by monitoring the consumption of compound 5a, the reaction was cooled to room temperature. The crude product was dissolved in dichloromethane and washed with saturated brine twice. The organic phase was dried over anhydrous sodium sulfate, concentrated to give the crude product which was purified by column chromatography on silica gel (DCM / MeOH 40:1-20:1) to give compound 5b (3.8 g, pale yellow oil) in 41% yield.

[0147] MS m / z (ESI): 626.5 [M+1] + .

[0148] Step 3: Synthesis of compound 11

[0149] To 5b (3.1 g, 5 mmol) in tetrahydrofuran was added acetonitrile, 1a (2.5 g, 6 mmol), potassium carbonate (2.75 g, 20 mmol) and potassium iodide (1.66 g, 10 mmol) and the reaction stirred at 40-50 °C for 16-20 h. The reaction was cooled to room temperature, filtered and the filtrate was washed with saturated sodium bicarbonate solution and extracted with dichloromethane twice. The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The product was purified by column chromatography on silica gel (DCM / MeOH 40:1-30:1) to give compound 13 (2.2 g, pale yellow oil) in 46% yield.

[0150] MS m / z (ESI): 964.8 [M+1] + .

[0151] Synthesis of compound 14 of Example 6

[0152] Step 1: Synthesis of compound 6a

[0153] Dissolve 2-hexyldecanoic acid (12.8 g, 50 mmol) in 30 mL of dichloromethane, add 5-hexen-1-ol (5.0 g, 50 mmol), DMAP (2.4 g, 20 mmol), and stir to dissolve. Add a solution of EDC.HCL (11.5 g, 60 mmol) in dichloromethane dropwise, and stir at room temperature for 16 hours after the addition is complete. Quench with water, add dilute hydrochloric acid, adjust the pH to 1-3, and separate the layers. Wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to give compound 6a (11.9 g, pale yellow oil) in 70% yield.

[0154] MS m / z (ESI): 339.3 [M+1] + .

[0155] Step 2: Synthesis of compound 6b

[0156] Dissolve compound 6a (10.2 g, 30 mmol) in 30 mL of dichloromethane, and cool to 5-10 °C in an ice bath. Add a solution of m-CPBA (7.8 g, 45 mmol) in dichloromethane dropwise, remove the ice bath after the addition is complete, and stir at room temperature for 16 hours. Monitor the reaction progress, and stop stirring when compound 6a is consumed completely. Remove the solvent at 45 °C, dissolve the crude product in ethyl acetate, wash twice with saturated sodium bicarbonate, and wash once with saturated sodium chloride. Dry the organic phase over anhydrous sodium sulfate, concentrate to give the crude product, and separate compound 6b (9.6 g, pale yellow oil) by silica gel column chromatography (n-hexane / ethyl acetate 60:1-40:1) in 90% yield.

[0157] MS m / z (ESI): 355.3 [M+1] + .

[0158] Step 3: Synthesis of compound 6c

[0159] Compound 6b (8.9 g, 25 mmol), was added to 1-(3-aminopropyl)imidazole (12.5 g, 100 mmol) at room temperature, warmed to 70-80 °C, stirred for 16 h, monitored the reaction progress, compound 6b was consumed completely, cooled to room temperature, dissolved in dichloromethane, washed with saturated brine twice, dried the organic phase over anhydrous sodium sulfate, concentrated to get the crude product, separated by silica gel column chromatography (DCM / MeOH 40:1-20:1), compound 6c (6.0 g, light yellow oil), yield 50%.

[0160] MS m / z (ESI): 480.4 [M+1] + .

[0161] Step 4: Synthesis of compound 14

[0162] Compound 6c (6.0 g, 12.5 mmol), compound 1c (6.7 g, 15 mmol) was added to 10 ml tetrahydrofuran, 20 ml acetonitrile mixed solution at room temperature, warmed to 70-80 °C, stirred for 16 h, removed the solvent at 45 °C, dissolved in dichloromethane, washed with saturated brine twice, dried the organic phase over anhydrous sodium sulfate, concentrated to get the crude product, separated by silica gel column chromatography (DCM / MeOH 120:1-100:1), compound 16 (3.9 g, light yellow oil), yield 34%.

[0163] MS m / z (ESI): 920.9 [M+1] + ;

[0164] 1 H NMR (300 MHz, CDC13): δ 7.52 (s, 1H), 7.07 (s, 1H), 6.94 (s, 1H), 5.33-5.35 (m, 1H), 4.58-4.69 (m, 1H), 4.08 (t, 2H, J = 6.6 Hz), 3.95-4.03 (m, 2H), 2.90-3.77 (m, 3H), 2.32-2.77 (m, 10H), 1.82-1.94 (m, 9H), 0.87-1.64 (m, 67H), 0.67 (s, 3H).

[0165] Example 7 Synthesis of compound 16

[0166] Step 1: Synthesis of compound 7a

[0167] Compound 7a (14.51 g, yellowish oil) was obtained in 83% yield by dissolving 9-bromononanoic acid (8.85 g, 40 mmol), 1-heptanol (13.94 g, 120 mmol) in 100 mL of dichloromethane, adding p-toluenesulfonic acid (7.8 mmol), anhydrous sodium sulfate (120 mmol), stirring at room temperature overnight, removing the solvent by rotary evaporation after monitoring the reaction completion by TLC, and purifying the product by silica gel column chromatography (DCM / MeOH 120:1-100:1).

[0168] 1 H NMR (300 MHz, CDC13) δ: 0.86-0.90 (m, 6H), 1.27-1.48 (m, 24H), 1.52-1.64 (m, 6H), 1.85-1.88 (m, 2H), 3.37-3.53 (m, 4H), 3.54-3.59 (m, 2H), 4.44-4.47 (m, 1H).

[0169] Step 2: Synthesis of compound 7b

[0170] Compound 7a (11.7 g, 26.8 mmol) was added to a mixture of 25 mL of tetrahydrofuran and 50 mL of acetonitrile at room temperature, followed by 1-(3- aminopropyl)imidazole (13.4 g, 108 mmol), potassium carbonate (7.3 g, 53 mmol), and potassium iodide (0.5 g, 3 mmol), and the mixture was warmed to 40-50°C, stirred for 16 h, and monitored for the completion of the reaction. After the compound 7a was consumed, the mixture was cooled to room temperature, and the solvent was removed at 45°C. The crude product was dissolved in dichloromethane, washed with saturated brine twice, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography (DCM / MeOH 60:1-40:1) to obtain compound 7b (6.1 g, yellowish oil) in 47% yield.

[0171] MS m / z (ESI): 480.5 [M+1] + .

[0172] Step 3: Synthesis of compound 16

[0173] Compound 7b (7.2 g, 15 mmol), compound 1c (8.0 g, 18 mmol) were added to a mixture of 15 mL of tetrahydrofuran and 30 mL of acetonitrile at room temperature, and the mixture was stirred at 70-80 °C for 16 h. The solvent was removed at 45 °C, and the crude product was dissolved in dichloromethane and washed twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated to give the crude product, which was separated by silica gel column chromatography (DCM / MeOH 100:1-50:1) to give compound 18 (8.4 g, yellowish oil) in 61% yield.

[0174] MS m / z (ESI): 920.8 [M+1] + ;

[0175] 1 H NMR (300 MHz, CDC13): 7.57 (dd, 1 H, J = 1.9, 1.1 Hz), 7.18 (dd, 1 H, J = 3.4, 1.1 Hz), 6.85 (dd, 1 H, J = 3.4, 1.9 Hz), 5.23 (dd, 1 H, J = 7.1, 6.9 Hz), 4.75-4.96 (m, 2 H), 3.72-3.83 (m, 2 H), 3.50-3.61 (m, 4 H), 2.42-2.90 (m, 10 H), 1.18-2.20 (m, 61 H), 1.04 (d, 3 H, J = 6.8 Hz), 0.73-0.93 (m, 17 H), δ 0.66 (q, 2 H, J = 7.2 Hz).

[0176] Synthesis of compound 17

[0177] Step 1: Synthesis of compound 8a

[0178] 1-bromo-nonadeca-7,10-diene (cis-7,10) (10.3 g, 30 mmol) was added to a mixture of 25 mL of tetrahydrofuran and 50 mL of acetonitrile at room temperature, and then 1-(3- aminopropyl)imidazole (15 g, 120 mmol), potassium carbonate (8.3 g, 60 mmol), and potassium iodide (0.5 g, 3 mmol) were sequentially added. The mixture was stirred at 40-50 °C for 16 h. After the consumption of 1-bromo-nonadeca-7,10-diene was confirmed, the mixture was cooled to room temperature, and the solvent was removed at 45 °C. The crude product was dissolved in dichloromethane and washed twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated to give the crude product, which was separated by silica gel column chromatography (DCM / MeOH 60:1-40:1) to give compound 8a (7.6 g, yellowish oil) in 65% yield.

[0179] MS m / z (ESI): 388.4 [M+1]+ .

[0180] Step 2: Synthesis of compound 17

[0181] Compound 8a (5.8 g, 15 mmol), compound 1c (8.0 g, 18 mmol) were added into 15 mL tetrahydrofuran, 30 mL acetonitrile mixed solution at room temperature, heated to 70-80 °C, stirred for 16 h, 45 °C to remove the solvent, dissolved in dichloromethane, washed with saturated brine twice, the organic phase was dried over anhydrous sodium sulfate, concentrated to give the crude product, then separated and purified by silica gel column chromatography (DCM / MeOH 100:1-50:1) to give compound 19 (7.0 g, yellowish oil), yield 57%.

[0182] MS m / z (ESI): 814.7 [M+1] + ;

[0183] 1 H NMR (300 MHz, CDC13): 7.67 (t, 1H, J = 2.0 Hz), 7.22 (dd, 1H, J = 4.8, 2.0 Hz), 6.94 (dd, 1H, J = 4.8, 2.0 Hz), 5.52-5.74 (m, 3H), 5.23-5.47 (m, 3H), 3.81-3.92 (m, 2H), 2.58-3.01 (m, 12H), 1.11-2.23 (m, 52H), 0.73-1.02 (m, 16H).

[0184] Example 9 Synthesis of compound 18

[0185] Step 1: Synthesis of compound 9a

[0186] 2-hexyl decanoic acid (1.28 g, 5.0 mmol) was dissolved in 20 mL dichloromethane, bis(2-hydroxyethyl) disulfide (1.54 g, 10.0 mmol), DMAP (0.21 g, 2.0 mmol), triethylamine (0.62 g, 6.0 mmol) were added and stirred to dissolve. A solution of EDC.HCL (1.10 g, 6.0 mmol) in dichloromethane was added dropwise, and after the addition was completed, it was stirred at room temperature for 16 hours. Quench with water, add dilute hydrochloric acid, adjust the pH to 1-3, separate the organic phase. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated, and column chromatography gave compound 9a (1.00 g, yellowish oil), yield 51%.

[0187] MS m / z (ESI): 393.2 [M+1] + .

[0188] Step 2: Synthesis of compound 9b

[0189] Compound 9a (1.00 g, 2.5 mmol) was dissolved in 20 mL of dichloromethane, triethylamine (0.76 g, 7.5 mmol) was added, and the solution was cooled to 0 °C in an ice water bath. Methylsulfonyl chloride (0.72 g, 6.2 mmol) was added, and the solution was stirred at room temperature for 16-20 h. The reaction was complete, and the solution was washed twice with saturated sodium bicarbonate and twice with water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 9b (1.00 g, light yellow oil) in 85% yield.

[0190] MS m / z (ESI): 471.2 [M+1] + .

[0191] Step 3: Synthesis of compound 9c

[0192] Compound 9b (0.94 g, 2.0 mmol) was dissolved in 20 mL of acetonitrile, potassium carbonate (0.83 g, 6.0 mmol) and 4-aminobutanol (0.18 g, 2.0 mmol) were added, and the solution was stirred at room temperature for 16-20 h. The reaction was complete, and the solution was filtered over celite. The filtrate was dissolved in dichloromethane, washed twice with saturated sodium bicarbonate and twice with water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 9c (758 mg, light yellow oil) in 76% yield.

[0193] MS m / z (ESI): 500.3 [M+1] + .

[0194] Step 4: Synthesis of compound 18

[0195] Compound 9c (0.75 g, 1.5 mmol) and compound 1c (0.8 g, 1.8 mmol) were added to a mixture of 5 mL of tetrahydrofuran and 30 mL of acetonitrile at room temperature, and the solution was warmed to 70-80 °C and stirred for 16 h. The solvent was removed at 45 °C, and the crude product was dissolved in dichloromethane, washed twice with saturated brine, and dried over anhydrous sodium sulfate. The crude product was separated by silica gel column chromatography (DCM / MeOH 100:1-50:1) to give compound 20 (917 mg, light yellow oil) in 65% yield.

[0196] MS m / z (ESI): 940.7 [M+1] + ;

[0197] 1H NMR (300 MHz, CDC13): 7.57 (dd, 1 H, J = 1.9, 1.1 Hz) 7.18 (dd, 1 H, J = 3.4, 1.1 Hz), 6.85 (dd, 1 H, J = 3.4, 1.9 Hz), 5.23 (dd, 1 H, J = 7.1, 6.9 Hz), 4.89 (m, 1 H), 4.36-4.47 (t, 2 H, J = 5.2 Hz), 3.72-3.83 (m, 2 H), 3.26-3.48 (m, 4 H), 3.08 (t, 2 H, J = 7.1 Hz), 2.34-2.89 (m, 9 H), 1.03-2.17 (m, 54 H), 0.73-0.93 (m, 17 H), δ 0.66 (q, 2 H, J = 7.2 Hz).

[0198] Synthesis of compound 21 of example 10

[0199] Step 1: Synthesis of compound 10a

[0200] Cholesterol (11.6 g, 30 mmol) was added into 60 mL of dichloromethane, followed by 5-bromo-1-pentanoic acid (5.5 g, 30 mmol), DMAP (1.5 g, 12 mmol), and a solution of EDC.HCL (6.9 g, 36 mmol) in dichloromethane dropwise. After the addition was completed, the mixture was stirred at room temperature for 16 h. The reaction was quenched by water, and diluted with dilute hydrochloric acid to adjust the pH value to 1-3. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give compound 10a (10.5 g, yellowish oil) in 64% yield.

[0201] MS m / z (ESI): 549.3 [M+1] + .

[0202] Step 2: Synthesis of compound 21

[0203] Compound 1b (7.0 g, 15 mmol) and compound 10a (9.9 g, 18 mmol) were added into 15 mL of tetrahydrofuran and 30 mL of acetonitrile, and the mixture was stirred at 70-80 °C for 16 h. The solvent was removed at 45 °C, and the crude product was dissolved in dichloromethane, washed with saturated brine twice, and dried over anhydrous sodium sulfate. The crude product was concentrated and purified by silica gel column chromatography (DCM / MeOH 100:1-50:1) to give compound 23 (6.5 g, yellowish oil) in 46% yield.

[0204] MS m / z (ESI): 933.2 [M+1] +;

[0205] 1 H NMR (300MHz, CDCl3): δ7.53(s,1H),7.10(s,1H),6.97(s,1H),5.33-5.35(m,1H),4.58-4.69(m,1H ), 4.08 (t, 4H, J = 6.6Hz), 2.31-2.56 (m, 11H), 1.86-2.01 (m, 8H), 0.87-1.65 (m, 74H), 0.70 (s, 3H).

[0206] Example 11: Encapsulation of mRNA- by the three-component LNP composition and characterization of LNP-mRNA

[0207] Taking Luc mRNA as an example, the construction of a steroid-cationic lipid trivalent LNP-mRNA vaccine is demonstrated: The lipid compound, auxiliary lipid, and polyethylene glycol lipid of this invention are dissolved in an ethanol solution according to the molar ratios in Table 1 to obtain a lipid mixture. The mRNA is dissolved in a pH 4.0 acetate buffer. The lipid mixture and mRNA are then processed at a flow rate ratio of 1:3 using the Ignite nanoparticle preparation instrument from Precision Nanosystems to prepare an mRNA-LNP composition (specific formulations are shown in Table 1). The packaged mRNA-LNP is dialyzed and ultrafiltered to concentrate it in a 10.7 mM Tris 20 mM NaAc pH 7.5 buffer solution. After sterile filtration, samples for subsequent animal experiments are obtained. Encapsulation efficiency, average particle size, PDI, and Zeta potential are measured.

[0208] Table 1. Molar percentage of lipid formulations and nitrogen-phosphorus ratio of the three-component LNPs formed by steroid-cationic lipid compounds.

[0209] Patent CN112424214A Compound (3)

[0210] US patent US7514099B2 compound (CLinDMA):

[0211] Patent CN117069785A Compound 5

[0212] As shown in Table 2, the results indicate that the compositions of the steroid-cationic lipids, auxiliary phosphate esters, polyethylene glycol lipids, and mRNA of the present invention exhibit good physicochemical properties. For example, the average particle size is between 60-100 nm, the PDI is less than 0.15, indicating good polydispersity; the zeta potential is between ±10 mV, indicating low electroweakness; and the encapsulation efficiency is greater than 85%. The PDI and mRNA encapsulation efficiency of the three-component lipid nanoparticle formulation provided by the present invention are superior to the lipid nanoparticle formulations made from lipid compounds provided in CN112424214A and US7514099B2. This demonstrates that the three-component LNPs made from the steroid-cationic lipid compounds provided by the present invention can achieve efficient encapsulation of mRNA.

[0213] Table 2 Characterization parameters of the three-component Luc-mRNA-LNP composition formed by steroid-cationic lipid compounds.

[0214] Example 12: Cellular experiments using a composition of mRNA-LNPs encoding Luc

[0215] The cell density was 6×10 5 Hep3B cells (cells / mL) were seeded at a rate of 200 μL / well in 96-well cell culture plates. After 24 hours, each well was transfected with 100 ng of Luc mRNA-LNP (prepared in Example 11). The cell culture plates were then incubated at 37°C in a 5% CO2 cell culture incubator. The negative control group was transfected with an equal volume of physiological saline. After 24 hours, a Luciferase detection kit was added, and the fluorescence value was detected using a multi-mode microplate reader. The results are shown in Figure 1. The results indicate that the three-component LNP composition formed using the steroid-cationic lipid compound described in this invention can achieve high expression of Luc-mRNA in cells, with a significantly higher expression level than the control group.

[0216] Example 13: In vivo expression and distribution of mRNA-LNPs encoding Luc in mice

[0217] LNP encapsulating 5 pg of Lucifease mRNA was injected intramuscularly into 6-8 weeks old female BALB / c mice (3 per group), and 24 hours after administration, the mice were anesthetized, intraperitoneally injected with D-Luciferin luciferase substrate, and imaged. The mice were dissected, and the heart, liver, spleen, lung, kidney, and pancreas were imaged ex vivo. As shown in FIG. 2, after intramuscular injection of the three-component LNP constructed using the cholesterol cationic lipid compound provided in the present application, the mRNA was mainly expressed at the injection site, but not in the liver, spleen, and other organs. In contrast, a large amount of mRNA entered the liver and spleen in addition to the injection site in the four-component LNP of ALC-0315, indicating that the three-component LNP formed using the cholesterol cationic lipid compound provided in the present application can greatly reduce the exposure of mRNA in other internal organs, thereby improving the safety of the drug.

[0218] Example 14: Animal immunization test of new crown antigen mRNA-LNP composition

[0219] The preparation of the new crown antigen mRNA-LNP was performed according to the lipid formulation in Table 1 in Example 11 using the new crown S protein antigen mRNA. 6-8 weeks old female BALB / c mice were randomly divided into groups of 5, and immunized by intramuscular injection of the hind leg. Immunization was performed on day 0 and day 14, and the immunization dose was 3 pg mRNA-LNP. The negative control group was injected with the same volume of normal saline. On day 28, blood was collected and serum was separated, and the specific antibody titer against the SARS-CoV-2 virus S protein antigen was detected by ELISA. The mice were sacrificed and the spleen tissue was taken, and ICS was performed for antigen-specific (TNF-a, IFN-g, and IL-2 secreting CD8+T cell immune detection). The results are shown in FIGS. 3, 4a, 4b, and 4c. The three-component LNP (LNP1-LNP11) constructed using the cationic lipid compound provided in the present application can induce better humoral immunity, and the geometric mean antibody titer (GMT) is significantly higher than that of the control groups 1, 2, and 3, and is basically consistent with that induced by the commercial ALC-0315 four-component LNP. However, the CD8+T cell immune effect induced by the LNP (LNP1-LNP11) provided in the present application is significantly better than that of the three-component LNP constructed using the control compound, and is significantly better than that of the ALC-0315 four-component LNP. Among them, the LNP1 constructed using compound 5 induced significantly better CD8+T cell immunity than the others. The above results show that the three-component LNP constructed using the compound provided in the present application can induce high humoral immunity when delivering vaccine mRNA, and at the same time, the delivery system can induce higher CD8+T cell immunity, thereby having greater advantages for vaccines that need to induce high CD8+T cell immune response, especially tumor vaccines.

[0220] Example 15 Safety evaluation of cholesterolic cationic compound

[0221] The new coronavirus S protein antigen mRNA-LNP complex was inoculated into CD1 mice by intramuscular injection at a dose of 30 ug, and the negative control group was injected with the same volume of normal saline. The safety of LNP was evaluated by monitoring the systemic inflammatory factor IL-6 in mice. As shown in Figure 5, after high-dose administration, the expression level of systemic inflammatory factor IL-6 caused by the three-component LNP1, LNP5 and LNP6 experimental groups based on compounds 4, 11 and 14 was significantly lower than that of the four-component LNP (control group 4) of the marketed compound, indicating that the LNP delivery mRNA constructed by the compound provided by the present application has low systemic exposure after intramuscular injection, thereby having better systemic safety.

[0222] Example 16 Freeze-drying study of cholesterolic cationic lipid compound three-component LNP

[0223] Taking the Luc-mRNA-LNP complex as an example, the mRNA-LNP was dissolved in a 20 mM Tris-sodium acetate pH 7.5 buffer solution containing 10% mass fraction sucrose, and 300 μL was aliquoted into 3 mL syringes for freeze-drying. The freeze-drying program: pre-freezing stage: -30°C for 2 hours; desorption drying stage: 25°C for 1 hour; sublimation drying stage: -40°C for 2h, -20°C for 2h, the whole process vacuum pressure control ≤10Pa. 2-8°C stability for 1 month. After reconstitution with sterile enzyme-free water, the mRNA encapsulation efficiency, LNP particle size, potential and PDI were detected.

[0224] Table 3 Changes in key quality attributes before and after LNP freeze-drying

[0225] The results are shown in Table 3. The three-component LNP of the cholesterolic cationic lipid compound provided by the present application has small changes in various physicochemical parameters after freeze-drying and rehydration.

[0226] Example 17 Atomization study of cholesterolic cationic lipid compound three-component LNP

[0227] The LNP complex of Luc-mRNA was used, the aerosol droplets were collected after the mRNA-LNP was atomized by the atomizer, the particle size, PDI and potential were detected by the nanoparticle size detector, and the content and encapsulation rate were detected by the Ribogreen detection kit. The experimental results are shown in Table 4 below. After the LNP1, LNP5 and LNP6 samples were atomized, the particle size, PDI and encapsulation rate and other key quality attributes changed little compared with the commercial LNP (control group 4). After atomization, the particles still maintained good particle state, and the encapsulation rate change was also less than 10%, while the particle morphology of the control sample was destroyed after atomization, and the mRNA encapsulation rate could not be detected.

[0228] Table 4 Key quality attribute parameters of LNP before and after atomization

[0229] The atomized samples were transfected into Hep3B cells, 500 ng mRNA-LNP / 10 5 cells / well, and the luciferase detection mRNA expression level was detected after 24 hours, as shown in Figure 6. Compared with the control group formulation after atomization, the cell expression of LNP1, LNP5 and LNP6 three samples after atomization still maintained good cell transfection activity, while the commercial control group 4 after atomization had no cell transfection activity.

[0230] Example 18 Expression in the lung of mice after LNP inhalation

[0231] 7-9 week old C57BL / 6 female mice were selected, and 5ug Luc-mRNA-LNP complex was given by intratracheal atomization with a quantitative atomization needle. After 24 hours, the mice were anesthetized and injected with a fluorescent substrate, and then fluorescence imaging was performed. The results are shown in Figure 7. After the mice inhaled Luc-mRNA-LNP, strong fluorescence expression was detected in the lung, which indicated that the three groups of LNP could realize mRNA expression in the lung of mice after inhalation, and further indicated that the three-component LNP based on the cholesterol cationic lipid compound designed by us could be used in the application of mRNA inhalation therapy.

[0232] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

[0233] It should be further noted that various specific technical features described in the above specific embodiments can be combined in any suitable manner, and in order to avoid unnecessary repetition, the present application will not describe various possible combinations again.

Claims

1. A lipid nanoparticle composition for nucleic acid drug delivery, characterized in that, comprise: a steroid-cationic lipid compound, a helper lipid, and a polyethylene glycol lipid.

2. The lipid nanoparticle composition for nucleic acid drug delivery according to claim 1, characterized by, The molar ratio of the steroid-cationic lipid: helper lipid: polyethylene glycol lipid is 30-80: 20-80: 0.5-20.

3. The lipid nanoparticle composition for nucleic acid drug delivery according to claim 1, characterized by, The molar ratio of the steroid-cationic lipid: helper lipid: polyethylene glycol lipid is 30-80: 30-80: 0.5-20.

4. The lipid nanoparticle composition for nucleic acid drug delivery according to claim 1, characterized by, The polyethylene glycol lipid is selected from 2[(polyethylene glycol)-2000] N,N-tetracosanoylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycero-methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteraryl glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearoyl, PEG-diacylglycerol amide (PEG-DAG), PEG-dipalmitoyl phosphatidyl ethanolamine (PEG-DPPE), or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).

5. The lipid nanoparticle composition for nucleic acid drug delivery according to claim 1, wherein, The helper lipid is selected from a neutral lipid, a zwitterionic lipid, or an anionic lipid; preferably, the helper lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), oleoyl phosphatidyl choline (POPC), 1-palmitoyl-2-oleoyl phosphatidyl ethanolamine (POPE), phosphocholine (DOPC), dimyristoyl phosphatidyl choline (DMPC), phosphatidyl choline (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidyl ethanolamine (PE), egg phosphatidyl choline (EPC), dilauroyl phosphatidyl choline (DLPC), dimyristoyl phosphatidyl choline (DMPC), 1-myristoyl-2-palmitoyl phosphatidyl choline (MPPC), 1-palmitoyl-2-myristoyl phosphatidyl choline (PMPC), 1-palmitoyl-2-stearoyl phosphatidyl choline (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidyl choline (SPPC), 1,2-eicosenoyl-sn-glycero-3-phosphocholine (DEPC), lysophosphatidyl choline, dilinoleoyl phosphatidyl choline distearoyl phosphatidyl ethanolamine (DSPE), or lysophosphatidyl ethanolamine.

6. The lipid nanoparticle composition for nucleic acid drug delivery according to claim 1, wherein, The composition further comprises a nucleic acid drug, which is DNA or RNA, the RNA is selected from the group consisting of antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, gRNA, tsRNA, circRNA or self-replicating mRNA; preferably, the nucleic acid drug is mRNA; more preferably, the composition is in the form of nucleic acid drug-lipid nanoparticle.

7. The lipid nanoparticle composition for nucleic acid drug delivery according to any one of claims 1-6, wherein, The steroid-cationic lipid compound has a structure represented by Formula (I): or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof, wherein, R1, R2are each independently selected from H, OR', wherein R' is selected from H, C1-C9alkyl, C2-C9alkenyl, C2-C9alkynyl, C 3-8 cycloalkyl, C 3-8 cycloalkenyl or C 3-8 cycloalkynyl; L1, L2are each independently selected from a chemical bond (-), -0-, -S-, -0(C=0)0-, -(C=0)NR a -, -NR a (C=0)-, -0(C=0)-, -(C=0)0-, -S-S-, -S(0) x -, -OS(O) x O-, -C(=0)S-, -SC(=0)-, -NR a C(=0)NR b -, -OC(=0)NR a -, -NR a C(=0)0-, -OC(=0)S-, -SC(=0)0-, -P(0)(OR a )0-, -OP(0)(OR a )0-, or C 1-12 alkylene; x is selected from 0, 1 or 2; R a , R b are each independently selected from H, C 1-12 alkyl, C 2-12 alkenyl or C 2-12 alkynyl; R3 is selected from a naturally occurring or non-naturally occurring steroid; R4is selected from optionally substituted C 6-24 alkyl, C 6-24 alkenyl, C 6-24 alkyne or C 6-24 alkoxy; m, n, o, p, q are each independently selected from any integer between 0-12; with the proviso that the compound:

8. The lipid nanoparticle composition for nucleic acid drug delivery according to claim 7, wherein, R1, R2 are each independently selected from H, OH, C1-C9 alkoxy; L1, L2are each independently selected from the group consisting of a chemical bond (-), -0-, -S-, -0(C=0)0-, -(C=0)NR a -, -NR a (C=0)-, -0(C=0)-, -(C=0)0-, -S-S-, -S(0) x -, -OS(O) x O-, -C(=0)S-, -SC(=0)-, -NR a C(=0)NR b -, -OC(=0)NR a -, -NR a C(=0)0-, -OC(=0)S-, -SC(=0)0-; preferably, L1, L2are each independently selected from the group consisting of -0-, -S-, -0(C=0)0-, -(C=0)NR a -, -NR a (C=0)-, -0(C=0)-, -(C=0)0-, -S-S-, -S(0) x -, -OS(O) x O-, -C(=0)S-, -SC(=0)-; more preferably, L1, L2are each independently selected from the group consisting of -0-, -S-, -0(C=0)0-, -0(C=0)-, -(C=0)0-, -S-S-; x is selected from 0, 1 or 2; R a , R b are each independently selected from H, C 1-12 alkyl, C 2-12 alkenyl or C 2-12 alkynyl; R3 is selected from a naturally occurring or non-naturally occurring steroid; R4is selected from the group consisting of optionally substituted C 6-24 alkyl, C 6-24 alkenyl; preferably, R4is selected from the group consisting of optionally substituted C 6-24 alkyl; m, n, o, p, q are each independently selected from any integer between 0-12.

9. The lipid nanoparticle composition for nucleic acid drug delivery according to claim 7, wherein, said R3 is a sterol; Preferably, the sterol is an animal sterol, or an oxidized or reduced form thereof; and / or, the sterol is a plant sterol, or an oxidized or reduced form thereof; and / or, the sterol is a synthetic sterol, or an oxidized or reduced form thereof; More preferably, the sterol is selected from the group consisting of cholesterol, an oxidized form of cholesterol, a reduced form of cholesterol, alkyl lithocholates, stigmasterol, stigmastanol, campesterol, ergosterol or sitosterol; More preferably, the sterol is an oxidized form of cholesterol, a reduced form of cholesterol, alkyl lithocholates, stigmasterol, stigmastanol, campesterol, ergosterol or sitosterol; More preferably, the sterol is selected from the group consisting of avenasterol, beta-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, dinosterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, lanosterol, photosterols, phytosterol, poriferasterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid or lithocholic acid; More preferably, the sterol has the structural formula: wherein R" is C 1-20 alkyl.

10. The lipid nanoparticle composition for nucleic acid drug delivery according to any one of claims 7-9, wherein, said R4 is selected from: Preferably, said R4 is selected from:

11. The lipid nanoparticle composition for nucleic acid drug delivery according to any one of claims 7-10, wherein, The steroid-cationic lipid compound is selected from: or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof; wherein L1, L2, R4, m, p and q are as defined in any one of claims 7-10.

12. The lipid nanoparticle composition for nucleic acid drug delivery according to any one of claims 7-11, wherein, L1is selected from any one of -0-, -0(C=0)0-, -0(C=0)-, -(C=0)0-; L2is selected from any one of -0-, -0(C=0)0-, -0(C=0)-, -(C=0)0-, -S-S-; R4is selected from m is selected from 2 or 3 or 4; p and q are each independently selected from 1, 2, 3, 4, 5, 6, 7 or 8.

13. The lipid nanoparticle composition for nucleic acid drug delivery according to claim 11, wherein, The steroid-cationic lipid compound is a compound represented by Formula II-1 or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof; wherein L1is selected from -(C=0)0-; L2is selected from -0(C=0)-; R4is selected from m is selected from 3; p is selected from 2; q is selected from any integer between 4-8.

14. The lipid nanoparticle composition for nucleic acid drug delivery according to claim 7, characterized in that, The compound is selected from: or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof.

15. The lipid nanoparticle composition for nucleic acid drug delivery of any one of claims 1-14, wherein, The lipid nanoparticle has a nitrogen to phosphorous ratio of (1-15): 1; preferably, the lipid nanoparticle has a nitrogen to phosphorous ratio of (1-10):

1.

16. The lipid nanoparticle composition for nucleic acid drug delivery of any one of claims 1-14, wherein, The lipid nanoparticle has a diameter of 15 nm to 300 nm.

17. The lipid nanoparticle composition for nucleic acid drug delivery of any one of claims 1-14, wherein, The dosage form of the composition is a liquid formulation or a lyophilized powder.

18. The method of claim 1-14 for the preparation of a lipid nanoparticle composition for nucleic acid drug delivery, characterized in that, The method comprises dissolving a steroid-cationic lipid compound, a helper lipid, and a polyethylene glycol lipid in a solvent, and mixing the steroid-cationic lipid compound, the helper lipid, and the polyethylene glycol lipid with a nucleic acid drug to prepare a lipid nanoparticle composition for nucleic acid drug delivery.

19. Use of the lipid nanoparticle composition according to any one of claims 1-17 in the preparation of a bioactive substance delivery system.

20. Use according to claim 19, characterized in that, The bioactive substance delivery system is an aerosol inhalation delivery system.

21. Use according to claim 20, characterized in that, The aerosol inhalation delivery system is for administration of a liquid formulation in an aerosol form, and / or the aerosol inhalation delivery system is for transmucosal administration; preferably, the aerosol inhalation delivery system is for nasal inhalation administration or oral inhalation administration.

22. The use according to any one of claims 19-21, characterized in that, The delivery system further comprises a pharmaceutically acceptable excipient, preferably, the excipient comprises one or more of a combination of sodium acetate, tromethamine, potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate, and sucrose.

23. Use according to any one of claims 19-21, characterized in that, The delivery system is a vaccine.

24. Use according to claim 23, characterized in that, The vaccine is a vaccine for preventing cancer, viral infection, bacterial infection, or fungal infection; preferably, the virus is selected from the group consisting of norovirus, Ebola virus, coronavirus, cytomegalovirus, dengue virus, Zika virus, coxsackievirus, enterovirus, hepatitis virus, herpes simplex virus, human papillomavirus, influenza virus, Marburg virus, measles virus, poliovirus, rabies virus, rotavirus, and rubella virus.

25. Use of the lipid nanoparticle composition according to any one of claims 1-16 in the preparation of a lyophilized formulation.

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