Lipid nanoparticle as well as preparation method and application thereof

Abstract

The invention provides lipid nanoparticles as well as a preparation method and application thereof. The preparation method of the lipid nanoparticles provided by the invention is simple and convenient to operate, the mRNA skeleton can be dehydrated to inhibit the mRNA degradation caused by moisture in the LNP, the empty inclusion rate can be reduced, and the preparation method is suitable for enhancing the stability of the lipid nanoparticles.

Description

Technical Field

[0001] The present application relates to the field of lipid nanoparticles (LNP), and specifically provides a lipid nanoparticle (LNP), a preparation method thereof, and an application thereof. Background Art

[0002] A one-step ultrafiltration method is often used to prepare mRNA LNPs. The composite solution system in this one-step ultrafiltration method contains a small amount of ethanol (less than 5%). Lipids are more soluble in organic solvents than in water. The addition of a neutral buffer increases the pH of the system, weakening the ionization of cationic lipids. Some lipids are soluble in ethanol, resulting in uneven lipid distribution within the solution. Lipid retention in ethanol, uneven lipid distribution, and decreased nucleic acid binding capacity of cationic lipids can lead to the formation of empty LNPs, with an empty fraction reaching up to 60%. This high empty fraction increases production costs, reduces potency, and may also increase toxic side effects during clinical use. The empty fraction is related to the lipid type. Compared with mRNA LNPs prepared by the two-step ultrafiltration method, mRNA LNPs prepared by the one-step ultrafiltration method have a higher empty fraction and poorer stability at 4°C (mRNA is easily degraded), which is unfavorable for global transportation of mRNA LNP preparations.

[0003] To prevent mRNA LNP from losing activity during long-term storage at room temperature, sucrose is typically added to the mRNA LNP solution and stored at -80°C to prevent degradation. However, ultra-low temperature storage at -80°C requires a high level of cold chain transport, making it difficult to transport and preserve mRNA LNP products over long distances and increasing the difficulty and cost of product delivery. The WHO hopes that mRNA LNP vaccines can be stored at 4°C for one month to facilitate global transportation.

[0004] The two-step ultrafiltration method developed by CSPC can reduce the empty package rate and significantly improve the stability of mRNA LNP at 4°C (mRNA integrity). In the first step of ultrafiltration, pH 4.0 citric acid buffer is used to remove ethanol, and in the second step, pH 7.6 Tris buffer is used to replace the pH 4.0 citric acid buffer, which reduces the empty package rate and improves the cold storage stability of mRNA LNP. This shows that the presence of ethanol is an interference factor in the orderly formation of the structure of mRNA LNP. Ethanol has a certain lipophilicity. If a certain amount of "extraction solvent" is added to the system to free the lipids from the interference of ethanol, the purpose of reducing the empty package rate and optimizing the structure of mRNA LNP can be achieved.

[0005] Ionic liquids are liquid mixtures of organic cations and inorganic or organic anions in a specific stoichiometric ratio. They are easy to synthesize and are often used in pharmaceutical preparations to increase the solubility of poorly soluble (lipid-soluble) drugs. Ionic liquids that are completely miscible with water have the potential to serve as solvents for separating and purifying drugs.

[0006] The core of mRNA LNPs contains a significant amount of water (24%). The majority of the mRNA chain resides within the LNP core, surrounded by a column of water and ionizable cationic lipids. mRNA is susceptible to hydrolysis and loss of activity when stored in aqueous solution for extended periods. The liquid water environment within LNPs is the primary cause of mRNA instability under refrigerated conditions (2-8°C).

[0007] Single-stranded mRNA can fold back on itself to form a variety of double-stranded structures, and double-stranded structures of single-stranded mRNA exist widely. At the same time, ionic liquid choline amino acid salts have been shown to have an affinity for RNA. Ionic liquid choline amino acid salts can hydrogen bond with the minor groove base groups of torulomyces RNA and enhance the thermal stability of torulomyces RNA. The third-generation ionic liquid (natural product) choline chloride can also be completely miscible with water and has similar solvent properties to ethanol. It can be used as a competitive lipid solvent in the synthetic system, eliminating the interference of ethanol on LNP formation. In addition, the interaction between choline salts and RNA may cause dehydration of the RNA backbone, produce a minor groove dehydration effect on RNA, prevent RNA hydrolysis, and enhance RNA stability.

[0008] When preparing mRNA LNPs, a one-step ultrafiltration method results in a high empty packet rate and poor mRNA LNP stability at 4°C. While a two-step ultrafiltration method can reduce the empty packet rate and enhance the stability of mRNA LNPs at 4°C, it adds additional process steps, making it unsuitable for large-scale industrial production. There is a need in the art for a method that reduces the empty packet rate and enables stable storage of mRNA LNP products at 2-8°C, thereby reducing product transportation difficulties and storage costs. Summary of the Invention

[0009] In order to improve the above technical problems, the present invention provides a method for preparing lipid nanoparticles, which comprises:

[0010] 1) preparing an aqueous phase comprising a nucleic acid drug and a quaternary ammonium ion represented by the following formula;

[0011]

[0012] wherein R is a C1-C18 straight or branched chain alkyl group, preferably a C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17 or C18 alkyl group;

[0013] 2) preparing an organic phase, wherein the organic phase comprises a lipid material, wherein the lipid material comprises an ionizable cationic lipid, a structural lipid, a neutral lipid, and a PEGylated lipid, wherein the molar ratio of the ionizable cationic lipid, the structural lipid, the neutral lipid, and the PEGylated lipid is (10-70):(10-50):(4-25):(0.1-10), preferably 50:38.5:10:1.5;

[0014] 3) Preparation of lipid nanoparticles.

[0015] In one embodiment of the present invention, the quaternary ammonium ion in step 1) is a choline ion.

[0016] In one embodiment of the present invention, the step 1) comprises dissolving the nucleic acid drug in a citrate buffer containing 32-75 nM choline ions.

[0017] In one embodiment of the present invention, the aqueous phase contains 32-75 nM of choline chloride, choline glutamate and choline aspartate; preferably, the aqueous phase contains 32-75 nM of choline chloride; more preferably, the aqueous phase contains 40-60 nM of choline chloride.

[0018] In one embodiment of the present invention, the organic phase is an ethanol solution.

[0019] In one embodiment of the present invention, the method further comprises a step of ultrafiltration purification; preferably, the ethanol-containing citric acid buffer is replaced with a Tris buffer by ultrafiltration.

[0020] In one embodiment of the present invention, the citric acid buffer is a 20 mM citric acid buffer with a pH of 4.0.

[0021] In one embodiment of the present invention, the Tris buffer is a 25 mM Tris buffer with a pH of 7.5.

[0022] In one embodiment of the present invention, in step 3), the aqueous phase and the organic phase are mixed in a volume ratio of 3:1 using a microfluidic device.

[0023] In one embodiment of the present invention, the method further comprises a freeze-drying step; preferably, a freeze-protectant such as polysaccharide, protein or amino acid is added before freeze-drying.

[0024] In one embodiment of the present invention, the ionizable cationic lipid is selected from 1,2-dioctadecenyloxy-3-methylammonium propane (chloride), (2,3-dioleoyl-propyl)-trimethylammonium chloride, dimethyldioctadecyl ammonium bromide, N-[1-(2,3-dioleoyl)propyl]-N-(arginine base amide)ethyl-N,N-dimethylammonium trifluoroacetate, N,N-dihydroxyethyl-N-methyl-N-2-(cholesteryloxycarbonylamino)ethylammonium bromide, ethylphosphatidylcholine, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol, (2S)-2,5-bis(3-aminopropylamino)-N-[2-(dioctadecylamino)acetyl]pentanamide, N1- (2-{(1S)-1-[(3-aminopropyl)amino]-4-[bis(3-aminopropyl)amino]butylcarboxamido}ethyl)-3,4-bis(oleoyloxy)-benzamide, N4-cholesterol-spermine, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, 4-(N,N-dimethylamino)butyric acid (dilinoleyl)methyl ester, 9-(4-(dimethylamino)butyryloxy)heptadecanedioic acid di((Z)-non-2-en-1-yl) ester, 8-[(2-hydroxyethyl)(8-nonyloxy-8-oxooctyl)amino]octanoic acid (heptadecan-9-yl) ester, 8-[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl)amino]octanoic acid (heptadecan-9-yl) ester ester, [(4-hydroxybutyl) azadiyl] bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 1,1'-[(2-{4-[2-({2-[bis(2-hydroxydodecyl)amino]ethyl}(2-hydroxydodecyl)amino)ethyl]piperazin-1-yl}ethyl) azadialkyl]bis(dodecan-2-ol), tetrakis(8-methylnonyl) 3,3',3",3"'-{[(methyl azadialkyl)bis(propane-3,1-diyl)]bis(azatriyl)}tetrapropionate, 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dione, 3,6-bis(4-{bis[(9Z,12Z)-2-hydroxyoctadecyl-9,1 2-dien-1-yl]amino}butyl)piperazine-2,5-dione, {[(3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl)]bis(azatriyl)}tetra(ethane-2,1-diyl)(9Z,9'Z,9"Z,9"'Z,12Z,12'Z,12"Z,12"'Z)-tetra(octadec-9,12-dienoate), {[(3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl)]bis(azatriyl)}tetra(butane-4,1-diyl)(9Z,9'Z,9"Z,9"'Z,12Z,12'Z,12"Z,12"'Z)-tetra(octadec-9,12-dienoate), N1, N3,One or more of N5-tris[3-(didodecylamino)propyl]benzene-1,3,5-tricarboxamide (TT3), 9,9',9",9"',9"",9""'-{[(benzo-1,3,5-triamido)tris(propane-3,1-diyl)]triazatriyl}hexanonanoic acid hexa(octane-3-yl) ester (FTT5), preferably one or more of 4-(N,N-dimethylamino)butyric acid (dilinoleyl)methyl ester, SM-102, ALC-0315, MC3, 8-[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl)amino]octanoic acid (heptadecan-9-yl) ester or [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate);

[0025] The neutral lipid is selected from one or more of DOPE, DOPC, DOPS and DMPC, preferably DSPC and / or DOPE;

[0026] The PEGylated lipid is selected from one or more of 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide, 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DSG-PEG2000), DMG-PEG2000, ALC-0159 or n-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine sodium salt, preferably DMG-PEG2000 and / or ALC-0159.

[0027] In one embodiment of the present invention, the structured lipid is selected from cholesterol and cholesterol derivatives, preferably cholesterol or plant sterols.

[0028] In one embodiment of the present invention, the nucleic acid drug includes any one of RNA drug, DNA drug, and plasmid, preferably RNA drug, and more preferably siRNA and mRNA.

[0029] In another aspect, the present invention provides lipid nanoparticles obtained by the above method.

[0030] In one embodiment of the present invention, the average particle size of the lipid nanoparticles is 50-150 nm.

[0031] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned lipid nanoparticles and a pharmaceutically acceptable carrier.

[0032] In one embodiment of the present invention, the administration of the lipid nanoparticles and pharmaceutical composition includes subcutaneous injection or intramuscular injection.

[0033] On the other hand, the present invention provides the use of the aforementioned lipid nanoparticles and pharmaceutical compositions in preventing or treating tumors, infectious diseases, skin and musculoskeletal diseases, digestive system diseases, respiratory system diseases, immune system diseases, genitourinary system diseases, endocrine and metabolic diseases, and nervous system diseases.

[0034] On the other hand, the present invention provides the use of the aforementioned lipid nanoparticles and pharmaceutical compositions in the preparation of drugs for preventing or treating tumors, infectious diseases, skin and musculoskeletal diseases, digestive system diseases, respiratory system diseases, immune system diseases, genitourinary system diseases, endocrine and metabolic diseases, and nervous system diseases.

[0035] Beneficial effects

[0036] The method provided by the present disclosure is simple to operate, and liquid exchange can be completed in a single ultrafiltration step; it can dehydrate the mRNA backbone, reduce the water content in the mRNA LNP core, inhibit mRNA degradation caused by water in the LNP, and compete with the interfering solvent ethanol to form the lipids required for LNP, thereby reducing the empty packaging rate; and is suitable for enhancing the stability of mRNA LNPs with different lipid formulations under refrigerated conditions (2-8°C). DETAILED DESCRIPTION

[0037] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0038] definition

[0039] As used herein, "pharmaceutical composition" refers to a variety of preparations. The preparation containing a therapeutically effective amount of the nucleic acid molecules provided herein is in the form of a sterile liquid solution, liquid suspension or lyophilized form, optionally containing a stabilizer or excipient.

[0040] It will be understood that the aforementioned lipid nanoparticles will be administered together with suitable pharmaceutically acceptable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, etc. A large number of suitable formulations can be found in the pharmacopoeia known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, Pa. (1975)), in particular Chapter 87 of Blaug and Seymour. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (e.g., Lipofectin, TMSM102, DOPE, cholesterol, and PEG 1000-DMG), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, emulsions of polyethylene glycol (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycol. Any of the foregoing mixtures may be suitable for use in the treatment or therapy according to the present application, provided that the active ingredient in the formulation is not inactivated by the formulation and that the formulation is physiologically compatible and tolerated for the route of administration.

[0041] As used herein, "treating" an individual suffering from a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing the underlying disease and / or preventing the worsening of symptoms or disease progression. Treatment also includes any pharmaceutical use of any of the nucleic acid molecules or vectors provided, as well as the compositions provided herein.

[0042] The lipid nanoparticles and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (e.g., by powder, ointment, cream and / or drops), transmucosal, nasal, oral, sublingual; by intratracheal instillation, bronchial instillation and / or inhalation; and / or as oral spray, nasal spray and / or aerosol. Particularly contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration by blood and / or lymphatic supply, and / or direct administration to the affected part. Typically, the most appropriate route of administration will depend on various factors, including the nature of the medicament (e.g., stability in the gastrointestinal environment), and / or the condition of the subject (e.g., whether the subject can tolerate oral administration). In some embodiments, the lipid nanoparticles or pharmaceutical compositions provided by the invention are suitable for topical administration to the eyes of the subject.

[0043] As used herein and unless otherwise specified, the terms "comprises," "includes," "has," "contains," and their grammatical equivalents should generally be understood as open-ended and non-limiting, e.g., not excluding other unlisted elements or steps.

[0044] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0045] Example 1: Preparation of mRNA LNPs using an aqueous phase containing choline chloride

[0046] The test was conducted using three lipid formulations already on the market:

[0047] Lipid Prescription 1,

[0048] SM-102, cholesterol, DSPC, DMG-PEG2000 (the molar ratio of SM-102, cholesterol, DSPC, and DMG-PEG2000 is 50:38.5:10:1.5);

[0049] Lipid Prescription 2,

[0050] ALC-0315, cholesterol, DSPC, ALC-0159 (the molar ratio of ALC-0315, cholesterol, DSPC, and ALC-0159 is 50:38.5:10:1.5);

[0051] Lipid Prescription 3,

[0052] MC3, cholesterol, DSPC, DMG-PEG2000 (the molar ratio of MC3, cholesterol, DSPC and DMG-PEG2000 is 50:38.5:10:1.5).

[0053] The mRNA LNPs were prepared using the three lipid formulations described above and tested using the following steps:

[0054] (1) mRNA was dissolved in 20 mM, pH 4.0 citric acid buffer, and choline chloride was added to make the concentration of choline chloride 40 nM, 50 nM and 60 nM, respectively. Then, the aqueous phase for mRNA LNP synthesis was prepared, and 20 mM, pH 4.0 citric acid buffer without choline chloride was used as a control.

[0055] (2) The three lipid formulations were dissolved in ethanol to obtain three different lipids, and then the aqueous phase in step (1) was used to synthesize mRNA LNPs using a microfluidic device, Inano E. The average particle size of the obtained lipid nanoparticles was 50-150 nm.

[0056] (3) The mRNA LNP solution in step (2) was ultrafiltered and the ethanol-containing citrate buffer was replaced with 25 mM Tris buffer (pH 7.5).

[0057] (4) After the treatment in step (3), the empty package rate of mRNA LNP prepared in aqueous phase containing different concentrations of choline chloride was detected.

[0058] (5) The mRNA LNPs treated in step (3) were stored at 4°C for 180 days, and the mRNA integrity of the LNPs prepared in aqueous phase with different concentrations of choline chloride was detected using a capillary electrophoresis instrument, Angilent 5200 Fragment Analyzer.

[0059] The following is the experimental data of the test results.

[0060] ①Lipid prescription 1: SM-102, DSPC, cholesterol, DMG-PEG2000

[0061] Table 1. Empty package rate of mRNA LNPs prepared with lipid formulation 1 and aqueous phases containing different concentrations of choline chloride

[0062] Choline chloride concentration (mM) Empty package rate (%) 0 12.8 40 4.3 50 4.5 60 4.1

[0063] Table 2. Properties of mRNA LNPs prepared from lipid formulation 1 and aqueous solutions with different choline chloride concentrations

[0064]

[0065] ②Lipid Prescription 2: ALC-0315, DSPC, cholesterol, ALC-0159

[0066] Table 3. Empty package rate of mRNA LNPs prepared with lipid formulation 2 and aqueous choline chloride at different concentrations

[0067] Choline chloride concentration (mM) Empty package rate (%) 0 33.0 40 15.2 50 14.8 60 14.4

[0068] Table 4. Properties of mRNA LNPs prepared with lipid formulation 2 and aqueous choline chloride at different concentrations

[0069]

[0070] ③Lipid prescription 3: MC3, DSPC, cholesterol, DMG-PEG2000

[0071] Table 5. Empty package rate of mRNA LNPs prepared with lipid formulation 3 and different concentrations of choline chloride aqueous phase

[0072] Choline chloride concentration (mM) Empty package rate (%) 0 17.4 40 5.8 50 6.2 60 6.7

[0073] Table 6. Properties of mRNA LNPs prepared with lipid formulation 3 and aqueous choline chloride at different concentrations

[0074]

[0075] As shown in the data in Tables 1-6, mRNA LNPs prepared using the three lipid formulations described above exhibited a reduced empty mRNA LNP rate after adding 40-60 mM choline chloride to the aqueous phase. Furthermore, the integrity of the encapsulated mRNA remained stable even after 180 days of storage at 4°C, effectively preventing mRNA degradation in the LNPs during long-term refrigeration. However, mRNA encapsulated without choline chloride exhibited a significant loss of integrity after 180 days of storage at 4°C.

[0076] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A method for preparing lipid nanoparticles, comprising: 1) preparing an aqueous phase comprising a nucleic acid drug and a quaternary ammonium ion represented by the following formula; wherein R is a C1-C18 straight or branched chain alkyl group, preferably a C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17 or C18 alkyl group; 2) preparing an organic phase, wherein the organic phase comprises a lipid material, wherein the lipid material comprises an ionizable cationic lipid, a structural lipid, a neutral lipid, and a PEGylated lipid, wherein the molar ratio of the ionizable cationic lipid, the structural lipid, the neutral lipid, and the PEGylated lipid is (10-70):(10-50):(4-25):(0.1-10), preferably 50:38.5:10:1.5; 3) Preparation of lipid nanoparticles.

2. The preparation method according to claim 1, wherein the quaternary ammonium ion in step 1) is a choline ion; Preferably, the step 1) comprises dissolving the nucleic acid drug in a citrate buffer containing 32-75 nM choline ions; Preferably, the aqueous phase contains 32-75 nM of choline chloride, choline glutamate and choline aspartate; Preferably, the aqueous phase contains 32-75 nM choline chloride; more preferably, the aqueous phase contains 40-60 nM choline chloride.

3. The preparation method according to claim 1 or 2, wherein the organic phase is an ethanol solution; Preferably, the method further comprises the step of ultrafiltration purification; more preferably, the ethanol-containing citric acid buffer is replaced with Tris buffer by ultrafiltration; Preferably, the citric acid buffer is 20 mM, pH 4.0 citric acid buffer; The Tris buffer is 25 mM Tris buffer with a pH of 7.

5. 4 . The preparation method according to claim 1 , wherein in step 3), the aqueous phase and the organic phase are mixed in a volume ratio of 3:1 using a microfluidic device.

5. The preparation method according to any one of claims 1 to 4, further comprising a freeze-drying step; preferably, a freeze-protectant such as a polysaccharide, protein or amino acid is added before freeze-drying.

6. The preparation method according to any one of claims 1 to 5, wherein the ionizable cationic lipid is selected from 1,2-dioctadecenyloxy-3-methylammonium propane (chloride), (2,3-dioleoyl-propyl)-trimethylammonium chloride, dimethyldioctadecyl ammonium bromide, N-[1-(2,3-dioleoyl)propyl]-N-(arginine amide)ethyl-N,N-dimethylammonium trifluoroacetate, N,N-dihydroxyethyl-N-methyl-N-2-(cholesteryloxycarbonylamino)ethylammonium bromide, ethylphosphatidylcholine, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol, (2S)-2,5-bis(3-aminopropylamino)-N-[2-(dioctadecylamino)acetyl] ] valeramide, N1-(2-{(1S)-1-[(3-aminopropyl)amino]-4-[bis(3-aminopropyl)amino]butylformamido}ethyl)-3,4-bis(oleoyloxy)-benzamide, N4-cholesterol-spermine, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, 4-(N,N-dimethylamino)butyric acid (dilinoleyl)methyl ester, 9-(4-(dimethylamino)butyryloxy)heptadecanedioic acid di((Z)-non-2-en-1-yl) ester, 8-[(2-hydroxyethyl)(8-nonyloxy-8-oxooctyl)amino]octanoic acid (heptadecan-9-yl) ester, 8-[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl)amino]octanoic acid (decane-9-yl) ester 9-heptanediyl) ester, [(4-hydroxybutyl) azadiyl] bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 1,1'-[(2-{4-[2-({2-[bis(2-hydroxydodecyl)amino]ethyl}(2-hydroxydodecyl)amino)ethyl]piperazin-1-yl}ethyl) azadialkyl]bis(dodecan-2-ol), tetrakis(8-methylnonyl) 3,3',3",3"'-{[(methyl azadialkyl)bis(propane-3,1-diyl)]bis(azatriyl)}tetrapropionate, 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dione, 3,6-bis(4-{bis[(9Z,12Z)-2-hydroxyoctadecyl- 9,12-dien-1-yl]amino}butyl)piperazine-2,5-dione, {[(3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl)]bis(azatriyl)}tetra(ethane-2,1-diyl)(9Z,9'Z,9"Z,9"'Z,12Z,12'Z,12"Z,12"'Z)-tetra(octadec-9,12-dienoate), {[(3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl)]bis(azatriyl)}tetra(butane-4,1-diyl)(9Z,9'Z,9"Z,9"'Z,12Z,12'Z,12"Z,12"'Z)-tetra(octadec-9,12-dienoate), N1, N3,One or more of N5-tris[3-(didodecylamino)propyl]benzene-1,3,5-tricarboxamide (TT3), 9,9',9",9"',9"",9""'-{[(benzo-1,3,5-triamido)tris(propane-3,1-diyl)]triazatriyl}hexanonanoic acid hexa(octane-3-yl) ester (FTT5), preferably one or more of 4-(N,N-dimethylamino)butyric acid (dilinoleyl)methyl ester, SM-102, ALC-0315, MC3, 8-[(2-hydroxyethyl)(6-oxo-6-decyloxyhexyl)amino]octanoic acid (heptadecan-9-yl) ester or [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate); The neutral lipid is selected from one or more of DOPE, DOPC, DOPS and DMPC, preferably DSPC and / or DOPE; The PEGylated lipid is selected from one or more of 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, 2-[(polyethylene glycol)-2000]-N,N-tetracosyl acetamide, 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DSG-PEG2000), DMG-PEG2000, ALC-0159 or n-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine sodium salt, preferably DMG-PEG2000 and / or ALC-0159; The structured lipid is selected from cholesterol and cholesterol derivatives, preferably cholesterol or plant sterols.

7. The preparation method according to any one of claims 1 to 6, wherein the nucleic acid drug comprises any one of an RNA drug, a DNA drug, and a plasmid, preferably an RNA drug, and more preferably siRNA and mRNA.

8. The lipid nanoparticles obtained according to the preparation method according to any one of claims 1 to 7; Preferably, the average particle size of the lipid nanoparticles is 50-150 nm.

9. A pharmaceutical composition comprising the aforementioned lipid nanoparticles and a pharmaceutically acceptable carrier.

10. Use of the lipid nanoparticles according to claim 8 and the pharmaceutical composition according to claim 9 in the preparation of a medicament for preventing or treating tumors, infectious diseases, skin and musculoskeletal diseases, digestive system diseases, respiratory system diseases, immune system diseases, genitourinary system diseases, endocrine and metabolic diseases, and nervous system diseases.

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