Three-component lipid nanoparticle and composition for delivering genetic material into cell

Abstract

Provided are a new gene drug delivery system based on a three-component lipid nanoparticle (LNP), and a preparation method therefor and the use thereof. The three-component LNP is composed of three ingredients: a cationic lipid, a structural lipid and a polymer-conjugated lipid. By means of precise ratio optimization, the three-component LNP maintains an efficient delivery performance while achieving component simplification. Compared with traditional four-component LNPs, the three-component LNP reduces the types of chemical components during preparation, thereby significantly reducing the complexity and cost of the production process, and making the three-component LNP more suitable for large-scale industrial production. In addition, due to the reduction in the types of components, the three-component LNP exhibits improved biocompatibility, reduced potential immunogenicity and toxicity risks, and higher safety.

Description

Three-component lipid nanoparticles and compositions for delivering genetic material into cells TECHNICAL FIELD

[0001] The present invention belongs to the field of biological medicine, and specifically relates to the formulation composition of lipid nanoparticles and its application in nucleic acid delivery. BACKGROUND

[0002] Nucleic acid drugs as a new type of therapeutic means, because of its wide application prospect in the field of tumor, tissue regeneration, wound healing, pulmonary fibrosis, inflammatory diseases, microbial infection and other fields. However, nucleic acid drugs after injection into the human body, face the challenge of how to deliver to the lesion site efficiently and safely, while also need to avoid damage to normal cells. In order to solve these problems, the development of efficient and safe drug delivery system, especially lipid nanoparticle (LNP) technology, which can protect the nucleic acid drugs at the same time, realize its targeted delivery in vivo.

[0003] The core of lipid nanoparticle technology lies in the design and application of cationic lipids. Cationic lipids have the characteristics of binding to negatively charged nucleic acids, promoting endosome escape, improving in vivo transfection efficiency and pH sensitivity. The structure of cationic lipids contains three important regions: polar head, hydrophobic tail and connecting chain. These domains together determine the delivery efficiency and transfection efficiency of LNP. However, even if the structures of cationic lipids are similar, there may be significant differences in intracellular transfection efficiency, toxicity, expression and sustained expression time of LNP, which indicates that further exploration and optimization of cationic lipid compounds and their LNP delivery systems are needed.

[0004] In the prior art, although a variety of cationic lipids have been developed, such as DOTMA and DODAP, and products based on LNP technology have entered the market, such as siRNA drug Onpattro and mRNA vaccine, how to further optimize the composition of LNP, improve its targeting in specific organs and reduce off-target effects, is still the focus of current research.

[0005] In view of the above, it is necessary to provide a new technical scheme. SUMMARY

[0006] Provided herein is a three-component lipid nanoparticle composition and its application for delivering genetic material into cells. The lipid nanoparticle includes cationic lipids, structural lipids, polymer-conjugated lipids, and nucleic acids encapsulated inside the particle by lipids.

[0007] The lipid nanoparticles of the present application achieve effective encapsulation and protection of nucleic acids by optimizing the proportion of lipid components, while enhancing their stability in vitro and in vivo and intracellular delivery efficiency. Thus, the application potential in the fields of gene therapy and vaccine development is improved.

[0008] The present application can develop a new three-component LNP delivery system by adjusting the non-ionic interaction between cationic lipids and nucleic acids. This system maintains the stability and delivery efficiency of nanoparticles while simplifying the components, improves the safety of mRNA vaccines, and reduces systemic inflammatory response. The necessity of developing three-component lipid nanoparticles lies in improving the delivery efficiency and targeting of nucleic acid drugs, reducing side effects, and expanding the application potential of LNP technology in the treatment of various diseases. Through in-depth research on the composition formula and preparation process of three-component lipid nanoparticles, it is expected to achieve safer and more effective nucleic acid drug delivery.

[0009] The present application relates to a three-component lipid nucleic acid lipid nanoparticle, comprising:

[0010] (a) nucleic acid;

[0011] (b) cationic lipids, which account for 20mol%-65mol% of the total lipids present in the particles in terms of molar percentage;

[0012] (c) structural lipids, including cholesterol or its analogues and derivatives, wherein the cholesterol or its analogues and derivatives account for 25mol%-80mol% of the total lipids present in the particles in terms of molar percentage;

[0013] (d) conjugated lipids that inhibit particle aggregation, which account for 0.5mol%-10mol% of the total lipids present in the particles in terms of molar percentage;

[0014] wherein the total lipids are the sum of cationic lipids, structural lipids and conjugated lipids that inhibit particle aggregation.

[0015] As a preferred embodiment of the present application, the nucleic acid is one or more of deoxyribonucleotides, deoxyribonucleotide analogues, ribonucleotides and ribonucleotide analogues.

[0016] As a preferred embodiment of the present application, the nucleic acid contains one or more natural or artificially synthesized nucleotides.

[0017] As a preferred aspect of the present application, the nucleic acid is one or more of a plasmid, double-stranded DNA, single-stranded DNA, DNA:RNA hybrid, DNA origami nanomedicine, peptide DNA complex, cholesterol DNA complex, oligonucleotide, aptamer, dsRNA, ASO, siRNA, sgRNA, tRNA, miRNA, Antagomir, small activating RNA, circular mRNA, self-replicating mRNA, and mRNA.

[0018] As a preferred aspect of the present application, the mRNA comprises a polypeptide, functional protein, or enzyme that encodes at least one antigen or fragment or epitope thereof.

[0019] As a preferred aspect of the present application, the mRNA is a monocistronic mRNA or a multicistronic mRNA.

[0020] As a preferred aspect of the present application, the polynucleotide unit of the mRNA comprises partial or complete chemical modification.

[0021] As a preferred aspect of the present application, the mRNA is chemically modified at the polynucleotide unit, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-amino-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0022] As a preferred aspect of the present application, the three-component nucleic acid lipid nanoparticle, wherein the compound of the cationic lipid comprises at least one general structure of the following Formula (I-1):

[0023] or the compound is a pharmaceutically acceptable salt, prodrug, or isomer thereof; wherein m1, m2, m3, and m4 are each independently selected from an integer between 1-18;

[0024] or the compound is a pharmaceutically acceptable salt, prodrug, or isomer thereof;

[0025] wherein,

[0026] m1, m2, m3, and m4 are each independently selected from an integer between 1-18;

[0027] L 1a , L 1band L 1c are each independently selected from the group consisting of -H, -S-SR1, -SR1, -C(=O)OR1, -OC(=O)R1, -N(R2)R1, -C(=O)N(R2)R1, -N(R2)C(=O)R1, -C(=O)NR1, -OR1;

[0028] R1and R2are each independently selected from the group consisting of -H, aliphatic, wherein n1, n2, x1, x2, and o are independently selected from an integer from 0 to 12, and y is independently selected from 1, 2, 3, or 4;

[0029] L2is independently selected from the group consisting of -R3XR4M, -R3XR4Y-H, -R3M, -R3YR5, -M, -R3XR4(Z)M; R3, R4, and R5are each independently selected from the group consisting of C1-C5alkane, C2-C5alkene;

[0030] X is independently selected from the group consisting of -O-, -N(R x )-, -S-, -S-S-, -OC(=O)-, -C(=O)O-, -OP(=O)O-, -OS(=O)O-, -C(=O)N-, -N(C(=O))-;

[0031] R x is independently selected from the group consisting of -H, -(CH2) m -OH, -(CH2) m -CN, -(CH2) m H, and aliphatic, wherein m is independently selected from 0, 1, 2, 3, 4, or 5;

[0032] M is independently selected from the group consisting of -(CH2) m -OH, -(CH2) m -CN, -(CH2) m -N(R**)R* wherein m is independently selected from 0, 1, 2, 3, 4, or 5;

[0033] Y is independently selected from the group consisting of -(CH2) m -G-(CH2) n -, wherein m and n are each independently selected from 0, 1, 2, 3, 4, or 5;

[0034] G is independently selected from a benzene ring or a heterocycle;

[0035] R*and R**are independently selected from the group consisting of H, C1-C3alkane, -(CH2) m Q, wherein m is independently selected from 1, 2, 3, or 4;

[0036] Q is independently selected from the group consisting of: -OH, -CN,

[0037] Q is independently selected from the group consisting of: -OH, -CN, a benzene ring, a heterocyclic compound;

[0038] R A R B R C R is independently selected from the group consisting of: -H, -(CH2) m -H; m is independently selected from 1, 2, and 3;

[0039] R D R is independently selected from the group consisting of: -H, -(CH2) n -H; m is independently selected from 1, 2, and 3;

[0040] Z is independently selected from the group consisting of: -(CH2) m -OH, -(CH2) m -CN, -(CH2) m -N(R**)R*, -(CH2) m -G-(CH2) n -H, wherein m and n are independently selected from 0, 1, 2, 3, 4, or 5;

[0041] G is independently selected from a benzene ring or a heterocyclic compound;

[0042] R* and R** are independently selected from the group consisting of: H, C1-C3 alkane, -(CH2) a Q, wherein a is independently selected from 1, 2, 3, or 4;

[0043] Q is independently selected from the group consisting of: -OH, -CN,

[0044] Q is independently selected from the group consisting of: -OH, -CN, a benzene ring, a heterocyclic compound;

[0045] R A R B R C R is independently selected from the group consisting of: -H, -(CH2) m -H; m is independently selected from 1, 2, and 3;

[0046] R d R is independently selected from the group consisting of: -H, -(CH2) n-H-CN, -NO2, -NH2, -N(CH3)CH3; n is independently selected from 1, 2 and 3.

[0047] As a preferred embodiment of the present application, in the structure of the cationic lipid compound, the structure L 1a、 L 1b and L 1c may be exchanged or replaced with the position connected with L2, and / or the structure L2may be exchanged or replaced with the position connected with L 1a、 L 1b or L 1c ; the number of L2in the general structure is at least one and at most two.

[0048] As a preferred embodiment of the present application, in the structure of the cationic lipid compound, R x is an alicyclic hydrocarbon, R x is an alicyclic hydrocarbon having at least one of the following structural formulae (II-1), (II-2), (II-3) and (II-4), respectively:

[0049] or R x is a pharmaceutically acceptable salt, prodrug or isomer of the structural formula (II-1), (II-2), (II-3) or (II-4);

[0050] wherein, the bond in the structural formula (II-1), (II-2), (II-3) and (II-4) is a single bond or a double bond.

[0051] As a preferred embodiment of the present application, in the structure of the cationic lipid compound, R1is an alicyclic hydrocarbon, R1is vitamin E succinate, adamantane or its derivative;

[0052] R2is an alicyclic hydrocarbon, R2is vitamin E succinate, adamantane or its derivative.

[0053] As a preferred embodiment of the present application, in the structure of the cationic lipid compound, G is a heterocycle, G is an azacycle, an oxacycle or a thiacycle compound.

[0054] As a preferred embodiment of the present application, the cationic lipid compound can be specifically as follows: ​

[0055] As a preferred embodiment of the present application, the cationic lipid is present in a molar percentage of 20-65 mol% of the total lipid present in the particle.

[0056] Further preferred, the cationic lipid is present in a molar percentage of 30-49.5 mol% of the total lipid present in the particle.

[0057] As a preferred embodiment of the present application, the structural lipid is cholesterol or an analog and derivative thereof, including stereoisomers, tautomers, and pharmaceutically acceptable salts thereof, wherein the sterol is selected from one or more of avenasterol, beta-sitosterol, campesterol, ergocalciferol, linolesterol, cholestanol, cholesterol, oxidized forms of cholesterol, reduced forms of cholesterol, coprostanol, desmosterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, epicoprostanol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol; lanosterol, photosterols, algal sterols, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, and / or lithocholic acid alkyl esters.

[0058] As a preferred embodiment of the present application, the structural lipid is present in a molar percentage of 25-80 mol% of the total lipid present in the particle.

[0059] As a preferred embodiment of the present application, the structural lipid is present in a molar percentage of 45-65 mol% of the total lipid present in the particle.

[0060] As a preferred embodiment of the present application, the conjugated lipid that inhibits particle aggregation is a polymer-conjugated lipid.

[0061] Further preferred, the polymer-conjugated lipid can be one or more of a polyethylene glycol-conjugated lipid, a polyinosinic acid-conjugated lipid, and a polyzwitterion-conjugated lipid.

[0062] As a preferred embodiment of the present application, the polyethylene glycol conjugated lipid described in the present application 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-disterosylglycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearoyl, PEG-diacrylglycerol amide (PEG-DAG), PEG-dipalmitoyl phosphatidyl ethanolamine (PEG-DPPE), or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).

[0063] As a preferred embodiment of the present application, the molar percentage of the polymer conjugated lipid described in the present application in the total lipid present in the particle is 0.5mol% to 10mol%.

[0064] As a preferred embodiment of the present application, the molar percentage of the polymer conjugated lipid described in the present application in the total lipid present in the particle is 2.05mol% to 5.5mol%.

[0065] As a preferred embodiment of the present application, the mass ratio of total lipid to nucleic acid is 2 to 200. Preferably, the mass ratio of total lipid to nucleic acid is 5 to 100. More preferably, the mass ratio of total lipid to nucleic acid is 10 to 50.

[0066] As a preferred embodiment of the present application, the nitrogen to phosphorus ratio (N / P) of the cationic lipid to nucleic acid is 0.5:1 to 30:1. Preferably, the nitrogen to phosphorus ratio of the cationic lipid to nucleic acid is 2:1 to 15:1. More preferably, the nitrogen to phosphorus ratio of the cationic lipid to nucleic acid is 3:1 to 8:1.

[0067] As a preferred embodiment of the present application, the average size of the nucleic acid lipid nanoparticle is 40 to 300nm. Preferably, the average size of the nucleic acid lipid nanoparticle is 60 to 150nm.

[0068] As a preferred embodiment of the present application, the polydispersity index (PDI) of the nucleic acid lipid nanoparticle is 0.02 to 0.4. Preferably, the polydispersity index of the nucleic acid lipid nanoparticle is 0.05 to 0.2.

[0069] As a preferred embodiment of the present application, the encapsulation efficiency of the nucleic acid encapsulated in the lipid nanoparticle is 75% to 100%. Preferably, the encapsulation efficiency of the nucleic acid encapsulated in the lipid nanoparticle is 85% to 100%.

[0070] In one aspect, the present application provides a nanoparticle composition comprising the three-component lipid nanoparticle as described in the above technical solution and a pharmaceutically acceptable carrier thereof.

[0071] As a preferred embodiment of the present application, the nanoparticle composition can transfect cells cultured in vitro to deliver genetic material. Specifically, the cells can be cell lines or primary cells of animals and plants.

[0072] As a preferred embodiment of the present application, the nanoparticle composition can deliver nucleic acids into the cells of the subject, wherein the subject is a vertebrate. Preferably, the subject is a mammal.

[0073] In one aspect, the nanoparticle composition of the present application is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, orally, intrathecally or by inhalation.

[0074] As a preferred embodiment of the present application, the nanoparticle composition containing a nucleic acid dose of about 0.001 mg / kg to about 10 mg / kg is administered to the mammal. BRIEF DESCRIPTION OF DRAWINGS

[0075] Figure 1 is a DLS particle size distribution chart of mRNA-encapsulated three-component LNP;

[0076] Figure 2 is a TEM characterization chart of mRNA-encapsulated three-component LNP;

[0077] Figure 3 is the particle size and monodispersity of three-component nanoparticles encapsulating different nucleic acids;

[0078] Figure 4 is the encapsulation efficiency of three-component nanoparticles encapsulating different nucleic acids;

[0079] Figure 5 is the in vitro screening results of different three-component nanoparticle formulations;

[0080] Figure 6 is the in vivo screening results of different three-component nanoparticle formulations;

[0081] Figure 7 is the particle size and monodispersity of three-component nanoparticle scale-up formulations;

[0082] Figure 8 is the encapsulation efficiency of three-component nanoparticle scale-up formulations;

[0083] Figure 9 is the change in particle size and PDI of three-component LNP before and after freeze-drying and rehydration;

[0084] Figure 10 is the change in state and encapsulation efficiency of three-component LNP before and after freeze-drying and rehydration;

[0085] Figure 11 is the change in body temperature and body weight of mice after administration of three-component LNP;

[0086] Figure 12 is the change in liver function of mice after administration of three-component LNP;

[0087] Figure 13 shows the neutralizing antibody titers against SARS-CoV-2 in mice immunized with three-component LNPs.

[0088] Figure 14 shows the ELISOT plaque results in mice immunized with three-component LNPs. DETAILED DESCRIPTION

[0089] The present application relates to a novel gene drug delivery system based on three-component lipid nanoparticles (LNPs) and its preparation method and application. The three-component LNP is composed of three kinds of lipids, cationic lipid, structural lipid and polymer conjugated lipid. Through precise ratio optimization, it realizes the simplification of components while maintaining high delivery performance. Compared with traditional four-component LNPs, the three-component LNP reduces the types of chemical components in the preparation process, thereby significantly reducing the complexity and cost of the production process, making it more suitable for large-scale industrial production. In addition, due to the reduction of component types, the biocompatibility of the three-component LNP is improved, the potential immunogenicity and toxicity risk is reduced, and the safety is higher.

[0090] TERMS

[0091] Lipid Nanoparticle: In one aspect, nanoparticle compositions comprising the lipid compounds described herein are described herein. In particular embodiments, the nanoparticle compositions comprise the compositions of nucleic acid lipid nanoparticles described in the technical solutions above.

[0092] In some embodiments, the maximum size of the nanoparticle compositions provided herein is 1 pm or less (e.g., < 1 pm, < 900 nm, < 800 nm, < 700 nm, < 600 nm, < 500 nm, < 400 nm, < 300 nm, < 200 nm, < 175 nm, < 150 nm, < 125 nm, < 100 nm, < 75 nm, < 50 nm, or less) when measured, e.g., by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method. In one embodiment, the lipid nanoparticles provided herein have at least one dimension in the range of about 40 nm to about 200 nm. In one embodiment, the at least one dimension is in the range of about 40 nm to about 100 nm.

[0093] The characteristics of a nanoparticle composition can depend on its components. For example, a nanoparticle composition comprising cholesterol as a structural lipid can have different characteristics than a nanoparticle composition comprising a different structural lipid. Similarly, the characteristics of a nanoparticle composition can depend on the absolute or relative amounts of its components. For example, a nanoparticle composition comprising a higher molar fraction of cholesterol can have different characteristics than a nanoparticle composition comprising a lower molar fraction of cholesterol. The characteristics can also vary depending on the method and conditions of preparation of the nanoparticle composition.

[0094] Nanoparticle characterization: Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure a variety of characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

[0095] Size: The average size of a nanoparticle composition can be between tens of nanometers and hundreds of nanometers. For example, the average size can be about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of a nanoparticle composition can be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In certain embodiments, the average size of a nanoparticle composition can be about 70 nm to about 100 nm. In some embodiments, the average size can be about 80 nm. In other embodiments, the average size can be about 100 nm.

[0096] PDI: The nanoparticle composition can be relatively homogeneous. A polydispersity index can be used to indicate the homogeneity of the nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A smaller (e.g., less than 0.3) polydispersity index generally indicates a narrower particle size distribution. The polydispersity index of the nanoparticle composition can be about 0 to about 0.25, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition can be about 0.10 to about 0.20.

[0097] Encapsulation efficiency (EE%): The encapsulation efficiency of a nucleic acid describes the amount of nucleic acid encapsulated or otherwise associated with the nanoparticle composition after preparation relative to the initial amount provided. The encapsulation efficiency desirably is high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of nucleic acid in a solution containing the nanoparticle composition before and after disruption of the nanoparticle composition with one or more organic solvents or surfactants. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., mRNA) in solution. For the nanoparticle compositions described herein, the encapsulation efficiency of the therapeutic and / or prophylactic agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0098] Zeta potential: The zeta potential of a nanoparticle composition can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of the nanoparticle composition. Nanoparticle compositions having a relatively low positive or negative charge are generally desirable because highly charged species can undesirably interact with cells, tissues, and other components in the body. In some embodiments, the zeta potential of a nanoparticle composition can be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0099] Nitrogen to phosphorus ratio (N / P ratio): The nitrogen to phosphorus ratio refers to the ratio of the number of moles of nitrogen atoms in the lipid molecules to the number of phosphate groups in the RNA molecules. In certain examples, the formulation of the nanoparticle can include one or more types of RNA. The selection of these RNAs, and the lipid species and amounts paired with them, are made to achieve a particular nitrogen to phosphorus (N / P) ratio. Generally, lower N / P ratios are more desirable. By careful selection of the RNA and lipid species and amounts thereof, N:P ratios of about 0.5: 1 to 30: 1 can be achieved, and specific values can be 0.5: 1, 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 12: 1, 14: 1, 16: 1, 18: 1, 20: 1, 22: 1, 24: 1, 26: 1, 28: 1, or 30: 1. In certain specific cases, the N:P ratio can be between 2: 1 and 8: 1. In other cases, the N:P ratio can be between 5: 1 and 8: 1. Specifically, the N:P ratio can be 5.0: 1, 5.5: 1, 5.67: 1, 6.0: 1, 6.5: 1, or 7.0: 1. For example, a typical N:P ratio can be 5.67: 1.

[0100] Lyophilization: Lyophilization is a process that removes water from a formulation to extend its stability. In the LNP lyophilization process, it includes pre-freezing, primary drying (sublimation drying), and secondary drying (desorption drying). In the pre-freezing stage, LNP is frozen at low temperature to ensure that water is completely converted into ice crystals. Next, in the primary drying stage, ice crystals are sublimated to remove most of the water in a vacuum environment. Finally, through the secondary drying stage, residual bound water is further removed to ensure the stability of LNP. After lyophilization, LNP usually appears as a white fluffy cake, and can quickly recover to a milky white liquid without solid residues after re-dissolving in water.

[0101] Liver function markers: ALT (alanine aminotransferase) and AST (aspartate aminotransferase) are two important liver function markers commonly used to assess liver health. ALT mainly exists in liver cells, and its level will significantly increase when the liver is damaged. AST exists in various tissues, including the heart, liver, muscle and kidney, so its increase can not only indicate liver problems, but also be related to heart or muscle damage. The levels of ALT and AST are usually determined by blood tests.

[0102] Neutralizing antibody titer detection: Neutralizing antibody detection is a laboratory technique used to evaluate the effectiveness of vaccines and the immune response after viral infection. It assesses the neutralizing ability of antibodies to viruses by measuring the titer of neutralizing antibodies in serum samples. Neutralizing antibodies can recognize and bind to viruses, preventing them from binding to host cell receptors and thus preventing viral infection. This detection is important for understanding the level of immune protection after vaccination and the recovery status after viral infection. Common detection methods include cell-based micro-neutralization tests (such as Vero cell neutralization test), and neutralization tests using pseudovirus systems, which are widely used due to their high safety and ease of operation. In addition, enzyme-linked immunosorbent assay (ELISA) and colloidal gold test strips are also rapid methods for detecting neutralizing antibodies. These techniques help evaluate the immune effect of vaccines and the immune protection status of individuals against viruses.

[0103] Enzyme-Linked Immuno Spot Assay (ELISpot): is a highly sensitive immunological detection technique that can detect and quantify cells secreting specific cytokines or antibodies at the single cell level. The principle of ELISpot technology is to capture cytokines or antibodies secreted by cells using specific antibodies, and then form visible spots on the solid surface through enzyme-linked color development reaction. Each spot represents a single cell secreting cytokines, so by counting the number of spots, the frequency of cells secreting specific cytokines can be determined.

[0104] Example 1 Preparation and characterization of three-component lipid nanoparticles

[0105] The cationic lipid, the structural lipid and the polymer-conjugated lipid provided herein are dissolved in ethanol at certain molar ratios and the mRNA is diluted in 10-50 mM citrate buffer pH=4. The LNP is prepared at a total lipid to mRNA weight ratio of about 10:1 to 30:1 by mixing the lipid ethanol solution with the mRNA aqueous solution at a volume ratio of 1:3 using a microfluidic device at a total flow rate in the range of 6-30 mL / min. The ethanol is removed using dialysis and replaced with PBS. Finally, the lipid nanoparticles are filtered through a 0.22 pm sterile filter.

[0106] The lipid nanoparticle size is determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) using a 90° scattering detection mode. The encapsulation efficiency of the lipid nanoparticle is determined using a Quant-it-Ribogreen-RNA quantification assay kit (Thermo Fisher Scientific, UK) according to the manufacturer’s instructions.

[0107] The apparent pKa of the LNP formulation is correlated with the efficiency of the LNP for nucleic acid delivery in vivo. The apparent pKa ranges between about 5 and about 7. The apparent pKa of each formulation is determined using an assay based on fluorescence of 2-(p-toluidinyl)-6-naphthalene sulfonic acid (TNS). LNP formulations comprising cationic lipid / structural lipid / polymer-conjugated lipid in PBS are prepared as described above. A 300 mM stock solution of TNS in distilled water is prepared. The LNP formulation is diluted to 0.1 mg / mL total lipid in 3 mL of a buffer solution containing 50 mM sodium citrate, 50 mM sodium phosphate, 50 mM sodium borate and 30 mM sodium chloride with a pH value in the range of 3 to 9. An aliquot of the TNS solution is added to give a final concentration of 0.1 mg / mL and after vortex mixing, the fluorescence intensity is measured in a Molecular Devices Spectramax iD3 spectrometer at room temperature using an excitation wavelength of 325 nm and an emission wavelength of 435 nm. A sigmoidal curve best fit analysis is applied to the fluorescence data and the pKa value is measured as the pH value that gives half the maximum fluorescence intensity.

[0108] Figure 1 is a comparison chart of the detailed DLS characterization results of the three-component lipid nanoparticles of A36 lipid and the four-component lipid nanoparticles (cationic lipid is SM102), which results show that the hydrated particle size of the three-component A36 lipid nanoparticles is about 75 nm, and the PDI is below 0.1, which exhibits better size and good monodispersity compared to the four-component lipid nanoparticles.

[0109] Transmission electron microscopy characterization of lipid nanoparticles

[0110] Freshly prepared three-component lipid nanoparticles were concentrated (total lipid 20-25 mg / mL) and added to glow-discharged copper grids (3-5 μL) and flash frozen using a FEI Mark IV Vitrobot (FEI, Hillsboro, OR, USA) to form vitreous ice. The grids were moved into a Gatan 70° cryo-transfer system pre-equilibrated to -180 °C and the samples were imaged. All samples (unless otherwise stated) were imaged at 55,000x magnification with a nominal underfocus of 1-2 μm to enhance contrast.

[0111] Figure 2 is the TEM characterization result of three-component lipid nanoparticles (cationic lipid is A26), which shows that the three-component lipid nanoparticles have a uniform monodisperse spherical particle morphology under dark field, and the measured particle size is about 50 nm.

[0112] Example 3: Packaging of different types of nucleic acids by three-component lipid nanoparticles

[0113] The packaging capacity of the three-component formulation for different nucleic acids was tested. The experimental group used the cationic lipids described in the specification, and the formulation used cationic lipid (A26): structural lipid: polymer conjugated lipid = 36 mol%: 61.5 mol%: 2.5 mol%, N / P = 5.67 to prepare three-component LNP. siRNA, circRNA, mRNA, saRNA, and pcDNA were packaged using the preparation method described in Example 1, and were detected by the characterization method described in Example 1. Figure 3 is the particle size and monodispersity of three-component nanoparticles encapsulating different nucleic acids, and Figure 4 is the encapsulation efficiency. From the test results, the three-component LNP can effectively encapsulate different types of nucleic acids, and the encapsulation efficiency is > 90%.

[0114] Example 4: Lipid screening of three-component lipid nanoparticles

[0115] The effects of different cationic lipid three-component nanoparticle formulations were evaluated using particle size, PDI, encapsulation efficiency, and in vitro bioluminescence. The experimental group used the cationic lipids described in the specification, and the prescription used cationic lipids: structural lipids: polymer-conjugated lipids = 36 mol%: 61.5 mol%: 2.5 mol%, N / P = 5.67 to prepare 3-component LNP. The control group used Dlin-MC3-DMA and SM-102 as cationic lipids, and the prescription used cationic lipids: phospholipids: cholesterol: structural lipids: polymer-conjugated lipids = 50 mol%: 10%: 38.5 mol%: 1.5 mol%, N / P = 5.67 to prepare control LNP. The above LNP was prepared by the preparation method described in Example 1, and the physicochemical properties were characterized by the method described in Example 1.

[0116] Further, the LNP was transfected into HEK293T (ATCC No. CRL-3216), and luciferase expression was analyzed 6 hours after transfection. Briefly, cells were plated at 60-70% confluency and incubated at 37°C with 5% CO2 in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and 1x penicillin-streptomycin (Pen-Strep). During transfection, cells were transfected at a transfection dose of 100 ng / well. After 6 hours of transfection, the LNP-containing medium was carefully removed, and the cells were gently rinsed once with PBS. Luciferase expression was measured using a luciferase assay kit (Bright-Glo® (Promega), Catalog No. RG051S) following the manufacturer's protocol. TM ) Catalog No. RG051S) following the manufacturer's protocol.

[0117] The test results of the test lipid nanoparticles measured from the test group are listed in Table 2 below. Table 2 shows the particle size, PDI, encapsulation efficiency, and in vitro F-Luc activity detection results of different cationic lipids encapsulating nucleic acids.

[0118] Table 2: Comparison table of properties of three-component lipid nanoparticles

[0119] Note: A, B, C, and D represent the normalized results of the luminescence flux of the compound multiplied by MC3, and the multiplication range is as follows:

[0120] A: ≥ 2

[0121] B: ≥ 1 and < 2

[0122] C: ≥ 0.1 and < 1

[0123] D: <0.1.

[0124] Example 5 In vitro formulation screening of three-component lipid nanoparticles

[0125] The molar ratio of cationic lipid, structural lipid, polymer-conjugated lipid and the N:P ratio in the lipid component of the nanoparticle composition were changed to further optimize the formulation. The preferred compound in Example 4 was selected as the cationic lipid, and cholesterol was selected as the structural lipid. Analogues or derivatives of cholesterol can also be used. PEG 2000 A series of three-component lipid nanoparticles were prepared using DMG as the polymer-conjugated lipid, and other polymer-conjugated lipids can also be used. The size, encapsulation efficiency, Luc or hEPO expression level, and cytokine distribution of the compositions were evaluated. The ratio design is shown in Table 3, and the above LNP was prepared using the preparation method described in Example 1, and the evaluation method of in vitro transfection is shown in Example 4. Figure 5 shows the in vitro F-Luc activity detection results of different three-component LNP formulations.

[0126] Table 3 Comparison of formulation design of different three-component LNP

[0127] Example 6 In vivo formulation screening of three-component lipid nanoparticles

[0128] The preferred three-component LNP formulation of Example 6 was evaluated in vivo, and the preferred LNP was administered at a dose of 0.25 mpk by intramuscular injection. The test animals were 6-8-week-old female SPF BALB / c mice (n = 3) selected and raised in a SPF animal room with a temperature of 20-26°C, a humidity of 40-70%, and sufficient feed and water. Commercial lipids were used in the control group for in vivo effect comparison. Six hours after administration, 200 μL of D-luciferin potassium salt with a concentration of 15 mg / mL was injected intraperitoneally, and live imaging was performed 5 minutes after D-luciferin potassium salt injection. Figure 6 shows the in vivo F-Luc activity detection results of different three-component LNP formulations, and compared with the commercial four-component prescription, the three-component LNP formulations all have a certain degree of improvement.

[0129] Example 7 Scale-up preparation of three-component lipid nanoparticles

[0130] The mRNA working solution was mixed with the lipid working solution in a volume ratio of 1:3 using a microfluidic instrument (I-Nano-P, Miannar (Shanghai) Instrument Technology Co., Ltd.) and a matching R-MDM chip to prepare an LNP solution encapsulating mRNA. The preparation volume was 10 L, and the prepared LNP solution was diluted with 25 times the volume of 0.5x PBS (containing 6% sucrose, pH 7.2-7.4) buffer solution, and then concentrated and purified by tangential flow ultrafiltration (TFF) to remove the ethanol solution in the system. The mRNA content in the LNP solution was detected using the Ribogreen method described above, and an appropriate amount of dilution solution was added to adjust the final mRNA concentration in the LNP solution to 50 μg / mL to obtain the final LNP preparation. FIG. 7 shows the particle size and monodispersity of the three-component LNP prepared in different preferred formulations, and FIG. 8 shows the encapsulation efficiency of the preparation. Different formulations of different lipids were used for scale-up, and the particle size of the three-component LNP was between 50-100 nm, the PDI was <0.1, and the encapsulation efficiency was >90%, indicating that the three-component LNP has good process scale-up migration.

[0131] Example 8 Freeze-drying storage of three-component lipid nanoparticles

[0132] The LNP preparation in Example 7 was loaded into 6R vaccine bottles, 2 mL per bottle, and freeze-dried according to the process flow of pre-freezing → primary sublimation → secondary sublimation → nitrogen backfilling → plugging → capping. The freeze-drying equipment (Beijing Songyuan, LGJ-50FYQ) was used. The process parameters were selected as follows: the pre-freezing temperature was -45°C, and the temperature was maintained for 6 hours. The primary freeze-drying temperature was -40°C for 24 hours, and -20°C for 24 hours. The secondary freeze-drying temperature was 4°C for 24 hours. The vacuum degree during freeze-drying was 10 bar. The results are shown in FIG. 10. After freeze-drying, the samples were stored at 25°C or 2-8°C. When used, 2 mL of sterile water for injection was added, and the freeze-dried preparation was quickly reconstituted within 10 seconds after slight shaking, and then used. FIG. 9 shows the changes in particle size and PDI of different three-component LNPs before and after freeze-drying and rehydration, and FIG. 10 shows the changes in encapsulation efficiency and drug loading concentration of different three-component LNPs before and after freeze-drying and rehydration. The particle size change of the three-component LNP before and after freeze-drying was <10%, and the encapsulation efficiency after freeze-drying and rehydration was >85%, indicating that the three-component LNP is also suitable for the current freeze-drying process.

[0133] Example 9 Safety of three-component lipid nanoparticles

[0134] The three-component lipid nanoparticles described in the application were administered by intramuscular injection at a dose of 0.25 / 2.5 mpk, and the test animals were 6-8-week-old female SPF BALB / c mice; and the body temperature and body weight were measured according to the observation periods of 0d, 1d, 7d, 14d, 15d, 21d, and 28d. The test results are shown in Figure 11. Compared with the PBS blank group, all the administration groups did not produce obvious weight loss, and the body temperature was normally fluctuated within the acceptable range. At the same time, the above-mentioned compound was administered by intramuscular injection at a dose of 2.5 mpk, and the test animals were 6-8-week-old female SPF BALB / c mice; and the biochemical indicators of ALT and AST were detected according to the observation periods of 1d, 7d, 15d, and 21d. The results are shown in Figure 12. The ALT and AST of all administration groups were within the normal value range within 21 days after administration. And there was no acute ALT / AST increase within 24 hours after injection. It shows that the three-component LNP described in the application has good safety.

[0135] Example 10 Immunological efficacy of three-component lipid nanoparticles

[0136] The three-component lipid nanoparticles described in the application were administered by intramuscular injection at a dose of 0.15 mpk, and immunized according to the immunization procedures of 0d and 7d. The mouse serum was taken 14 days after the booster immunization, and the S protein-specific neutralizing antibody was detected by the pseudovirus neutralization test. The specific operation is as follows: after water bath inactivation of the serum, centrifugation was performed, and the supernatant was collected. The inactivated serum was diluted with serum-free DMEM medium. Diluted serum and pseudovirus were added to a 96-well plate, and incubated at 37°C for 1 hour. After incubation, 293T-ACE2-p2A-mTag BFP2 cells were added to the 96-well plate. The neutralizing antibody detection was performed according to the instructions of the Novizhan pseudovirus detection kit. Figure 13 shows the neutralizing antibody titers of PEDV-S protein in mice immunized with different three-component LNPs. The test results show that compared with the four-component LNP, the three-component LNP can induce a stronger neutralizing reaction and has a stronger humoral immune efficacy at the same immunization dose.

[0137] Further, the detection method of the immunoblotting test is as follows: the mice were euthanized to take the spleen and peripheral blood, and the isolated mouse spleen cells and peripheral blood immune cells after lysis were diluted to 2×10 5cells / mL, then added to a 96-well ELISPOT plate pre-coated with mouse IFN-γ antibody; 0.1 mL of overlapping short peptides (10 μg / mL) was added, and incubated at 37°C for 36 h. Washed with PBS for 5 times. Added 0.1 mL of biotinylated anti-mouse IFN-γ antibody (1 μg / mL), and incubated at room temperature for 2 h. Washed with PBS for 5 times. Added 0.1 mL of streptavidin-HRP, and incubated at room temperature for 1 h, and washed with PBS for 5 times. Developed by adding TMB substrate solution until obvious spots appeared, and washed with pure water to stop the reaction. The number of spots was read by an ELISPOT analyzer, and the number of spot forming cells per 1 x 106 6 cells was calculated. Figure 14 shows the ELISOT spot results of mice immunized with different three-component LNPs. The experimental results show that, compared with four-component LNPs, three-component LNPs can induce stronger ELISpot spot reaction at the same immunization dose, and have stronger cellular immune efficacy.

[0138] Although the embodiments of the present application have been shown and described above, it should be understood by those skilled in the art that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications and variations to the above embodiments within the scope of the present application.

Claims

1. A three-component nucleic acid lipid nanoparticle for delivering genetic material into a cell, characterized in that, The nucleic acid comprises: a cationic lipid at a molar percentage of 20-65 mol% of the total lipid present in the particle; a structural lipid comprising cholesterol or its analogues and derivatives, wherein the cholesterol or its analogues and derivatives are at a molar percentage of 25-80 mol% of the total lipid present in the particle; a conjugated lipid that inhibits particle aggregation at a molar percentage of 0.5-10 mol% of the total lipid present in the particle; wherein the total lipid is the sum of the cationic lipid, the structural lipid, and the conjugated lipid that inhibits particle aggregation.

2. The nucleic acid lipid nanoparticle of claim 1, wherein, The nucleic acid is a deoxyribonucleotide, a deoxyribonucleotide analogue, a ribonucleotide, or a ribonucleotide analogue.

3. The nucleic acid lipid nanoparticle of claim 2, wherein the nucleic acid is, The nucleic acid comprises one or more natural or artificially synthesized nucleotides.

4. The nucleic acid lipid nanoparticle of claim 2, wherein, The nucleic acid is one or more of a plasmid, double-stranded DNA, single-stranded DNA, DNA:RNA hybrid, DNA origami nanomedicine, peptide DNA complex, cholesterol DNA complex, oligonucleotide, aptamer, circRNA, dsRNA, ASO, siRNA, sgRNA, tRNA, miRNA, Antagomir, small activating RNA, circular mRNA, self-replicating mRNA, and mRNA.

5. The nucleic acid lipid nanoparticle of claim 4, wherein, The mRNA comprises a polypeptide, a functional protein, or an enzyme that encodes at least one antigen or a fragment or epitope thereof.

6. The nucleic acid lipid nanoparticle of claim 5, wherein, The mRNA is a single-cistronic mRNA or a multi-cistronic mRNA.

7. The nucleic acid lipid nanoparticle of claim 5, wherein, The polynucleotide unit of the mRNA comprises partial or complete chemical modification.

8. The nucleic acid lipid nanoparticle of claim 7, wherein, The mRNA with chemically modified polynucleotide units, wherein the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-amino-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine.

9. The nucleic acid lipid nanoparticle of any one of claims 1-8, wherein, Compounds in which the cationic lipid comprises at least one structural general formula of the following formula (I-1): or the compound is a pharmaceutically acceptable salt, prodrug, or isomer thereof; wherein m1, m2, m3, and m4 are each independently selected from an integer between 1 and 18; or the compound is a pharmaceutically acceptable salt, prodrug, or isomer thereof; wherein m1, m2, m3, and m4 are each independently selected from an integer between 1 and 18; L 1a , L 1b and L 1c are each independently selected from the following structures: -H, -S-SR1, -SR1, -C(=O)OR1, -OC(=O)R1, -N(R2)R1, -C(=O)N(R2)R1, -N(R2)C(=O)R1, -C(=O)NR1, -OR1; R1and R2are each independently selected from the following structures: -H, aliphatic, wherein n1, n2, x1, x2, and o are independently selected from an integer between 0 and 12, and y is independently selected from 1, 2, 3, or 4; L2 is independently selected from the following structures: -R3XR4M, -R3XR4Y-H, -R3M, -R3YR5, -M, -R3XR4(Z)M; R3, R4, and R5 are each independently selected from the following structures: C1-C5 alkane, C2-C5 alkene; X is independently selected from the following structures: -0-, -N(R x )-, -S-, -S-S-, -OC(=0)-, -C(=0)0-, -OP(=0)0-, -OS(=0)0-, -C(=0)N-, -N(C(=0))-; R x independently selected from the group consisting of -H, -(CH2) m -OH, -(CH2) m -CN, -(CH2) m H and an alicyclic hydrocarbon, wherein m is independently selected from 0, 1, 2, 3, 4 or 5; M is independently selected from the following structures: -(CH2) m -OH, -(CH2) m -CN, -(CH2) m -N(R**)R*, wherein m is independently selected from 0, 1, 2, 3, 4, or 5; Y is independently selected from the following structures: -(CH2) m -G-(CH2) n - wherein m and n are each independently selected from 0, 1, 2, 3, 4, or 5; G is independently selected from a benzene ring or a heterocycle; R* and R** are independently selected from the group consisting of H, C1-C3 alkane, -(CH2) m Q, wherein m is independently selected from 1, 2, 3, or 4; Q is independently selected from the following structures: -OH, -CN, are independently selected from the group consisting of: a benzene ring or a heterocycle compound; R A , R B , R C are independently selected from the group consisting of -H, -(CH2) m -H; m is independently selected from 1, 2 and 3; R D independently selected from the group consisting of: -H, -(CH2) n -H-CN, -NO2, -NH2, -N(CH3)CH3; n is independently selected from 1, 2 and 3; Z is independently selected from the following structures: -(CH2) m -OH, -(CH2) m -CN, -(CH2) m -N(R**)R*, -(CH2) m -G-(CH2) n -H, wherein m and n are independently selected from 0, 1, 2, 3, 4, or 5; G is independently selected from a benzene ring or a heterocycle; a benzene ring or a heterocycle compound; R* and R** are independently selected from the group consisting of H, C1-C3 alkane, -(CH2) a Q, wherein a is independently selected from 1, 2, 3, or 4; Q is independently selected from the following structures: -OH, -CN, are independently selected from the group consisting of: benzene ring, heterocyclic compound; R A , R B , R C are independently selected from the group consisting of -H, -(CH2) m -H; m is independently selected from 1, 2 and 3; R d independently selected from the group consisting of -H, -(CH2) n -H-CN, -NO2, -NH2, -N(CH3)CH3; n is independently selected from 1, 2 and 3.

10. The nucleic acid lipid nanoparticle of claim 9, wherein, In the structure of the cationic lipid compound, the structure L 1a、 L 1b and L 1c may be exchanged or replaced with the position connected with L2, and / or the structure L2may be exchanged or replaced with L 1a、 L 1b or L 1c ; the number of L2in the general structure is at least one and at most no more than two.

11. The nucleic acid lipid nanoparticle of claim 9 or 10, wherein, R in the structure of the cationic lipid compound x is an alicyclic hydrocarbon, x is an alicyclic hydrocarbon having at least one of the following structural formulae (II-1), (II-2), (II-3) and (II-4): or R x a pharmaceutically acceptable salt, prodrug, or isomer of structural formula (II-1), (II-2), (II-3), or (II-4); wherein the structure of formula (II-1), (II-2), (II-3) and (II-4) is the bond is a single bond or a double bond.

12. The nucleic acid lipid nanoparticle of claim 9 or 10, wherein, In the structure of the cationic lipid compound, R1 is an alicyclic hydrocarbon, R1 is vitamin E succinate, adamantane or its derivative; R2 is an alicyclic hydrocarbon, R2 is vitamin E succinate, adamantane or its derivative.

13. The nucleic acid lipid nanoparticle of claim 9 or 10, wherein, In the structure of the cationic lipid compound, G is a heterocyclic compound, G is an azacyclic, oxacyclic or thiacyclic compound.

14. The nucleic acid lipid nanoparticle of any one of claims 9-13, wherein, The cationic lipid compound can specifically be of the following structure:

15. The nucleic acid lipid nanoparticle of any one of claims 1-14, wherein, The molar percentage of the cationic lipid in the total lipid present in the particle is 20mol%-65mol%.

16. The nucleic acid lipid nanoparticle of claim 15, wherein, The molar percentage of the cationic lipid in the total lipid present in the particle is 30mol%-49.5mol%.

17. The nucleic acid lipid nanoparticle of any one of claims 1-14, wherein, The structural lipid is cholesterol or its analogues and derivatives, including stereoisomers, tautomers and pharmaceutically acceptable salts thereof, wherein the steroid is selected from: avenasterol, β-sitosterol, campesterol, ergocalciferol, elaeostearol, cholestanol, cholesterol, oxidized forms of cholesterol, reduced forms of cholesterol, coprostanol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, fecosterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol; lanosterol, luminsterol, laminarosterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid and / or lithocholic acid alkyl ester.

18. The nucleic acid lipid nanoparticle of claims 1-14, wherein, The molar percentage of the structural lipid in the total lipid present in the particle is 25mol%-80mol%.

19. The nucleic acid lipid nanoparticle of claim 18, wherein, The molar percentage of the structural lipid in the total lipid present in the particle is 45mol%-65mol%.

20. The nucleic acid lipid nanoparticle of any one of claims 1-14, wherein, The conjugated lipid that inhibits aggregation of the particles is a polymer-conjugated lipid, which includes one or more polymers conjugated to form a polymer-conjugated lipid.

21. The nucleic acid lipid nanoparticle of claim 20, wherein, The polymer-conjugated lipid is a polyethylene glycol-conjugated lipid, a polyinosinic acid-conjugated lipid, a polyzwitterion-conjugated lipid.

22. The nucleic acid lipid nanoparticle of claim 21, wherein, The polyethylene glycol-conjugated lipid is selected from: 2-[(polyethylene glycol)-2000]-N,N-tetracosanoylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterosylglycerol (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).

23. The nucleic acid lipid nanoparticle of claims 1-14, wherein, The molar percentage of the polymer-conjugated lipid in the total lipid present in the particle is 0.5mol%-10mol%.

24. The nucleic acid lipid nanoparticle of claim 23, wherein, The molar percentage of the polymer-conjugated lipid in the total lipid present in the particle is 2.05mol%-5.5mol%.

25. The nucleic acid lipid nanoparticle of any one of claims 1-24, wherein, wherein the total lipid is the sum of the cationic lipid, the structural lipid, and the conjugated lipid that inhibits particle aggregation, and the mass ratio of the total lipid to the nucleic acid is 2-200.

26. The nucleic acid lipid nanoparticle of any one of claims 1-24, wherein, wherein the nitrogen to phosphorous ratio (N / P) of the cationic lipid to the nucleic acid is 0.5:1-30:

1.

27. The nucleic acid lipid nanoparticle of any one of claims 1-24, wherein, wherein the nucleic acid lipid nanoparticle has an average size of 40-300 nm.

28. The nucleic acid lipid nanoparticle of any one of claims 1-24, wherein, wherein the nucleic acid lipid nanoparticle has a monodispersity coefficient (PDI) of 0.02-0.

5.

29. The nucleic acid lipid nanoparticle of any one of claims 1-24, wherein, wherein the nucleic acid is encapsulated in the nanoparticle with an encapsulation efficiency of 65%-100%.

30. A nanoparticle composition, characterized in that, comprising the nucleic acid lipid nanoparticle of any one of claims 1-29 and a pharmaceutically acceptable carrier thereof.

31. The nanoparticle composition of claim 30, wherein, The nanoparticle composition can transfect cells in culture in vitro to deliver genetic material.

32. The nanoparticle composition of claim 29, wherein, The nanoparticle composition can deliver the nucleic acid into cells of the subject, wherein the subject is a vertebrate.

33. The nanoparticle composition of claim 32, wherein, The nanoparticle composition can be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, orally, intrathecally, or by inhalation.

34. The nanoparticle composition of claim 31, wherein, The nanoparticle composition can be administered to the mammal at a dose of about 0.001 mg / kg to about 10 mg / kg of the nucleic acid.

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