Lipid nanoparticle freeze-drying preparation as well as preparation method and application thereof

Abstract

The invention relates to the technical field of drug delivery, in particular to a lipid nanoparticle freeze-drying preparation as well as a preparation method and application thereof. According to the invention, by screening proper freeze-drying preparation prescriptions including freeze-drying protective agent type and concentration, buffer solution type and concentration and lipid nanoparticle prescriptions, sucrose with the concentration of 10-20% w / v is used as a single-component freeze-drying protective agent, a Tris buffer solution or HEPES buffer solution with the concentration of 1-20 mM is used as a buffer system, and the freeze-drying protective agent and the lipid nanoparticle prescriptions are prepared. The lipid nano-particle freeze-dried preparation has the advantages that the preparation is simple, excellent physicochemical properties and in-vivo transfection efficiency are still kept after redissolution, and the lipid nano-particle freeze-dried preparation has a good prospect in the field of development and application of nucleic acid drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of drug delivery, and in particular to a lipid nanoparticle freeze-dried preparation and a preparation method and application thereof. Background Art

[0002] Compared with traditional small molecule drugs and antibody drugs, nucleic acid drugs have a wider range of application scenarios. Nucleic acids are a type of electronegative macromolecules that are easily degraded, have low cellular uptake and transfection efficiency, and therefore need to develop safe and effective delivery vectors to protect them from degradation and promote cellular uptake. Currently, lipid nanoparticles (LNP) are one of the mainstream delivery vectors for nucleic acid drugs.

[0003] However, the nucleic acid vaccines currently on the market are all liquid products, and LNPs carrying nucleic acids (such as mRNA) have poor stability in aqueous media. The main reasons include two aspects: first, mRNA itself is easily inactivated due to oxidation, hydrolysis, enzymatic hydrolysis, etc.; second, LNPs are usually composed of ionizable lipids, auxiliary lipids, cholesterol, and PEG-lipids in a certain molar ratio. There are complex interactions between the components, which are easily affected by various factors such as solvent salt, pH, temperature, and oxygen, resulting in the release of mRNA or the aggregation of nanoparticles during storage or transportation, which greatly affects the efficacy. Therefore, in order to extend the shelf life of nucleic acid-carrying nanoparticle drugs, they often need to be transported and stored in ultra-low temperature environments. Taking the vaccine BNT162b2 as an example, it needs to be transported and stored at -80 to -60°C, and can only be stable for 2 hours after thawing at room temperature. Such harsh storage conditions greatly limit the accessibility of nucleic acid vaccines.

[0004] In order to overcome the instability of the physical and chemical properties of nucleic acid vaccines during long-term storage, nucleic acid vaccines can be freeze-dried to make freeze-dried preparations, which can be stored at 2-8°C for a long time and rehydrated before administration. However, the development of freeze-dried preparations is also full of huge challenges: mRNA-LNP has a shell-core structure, and the mRNA encapsulated in the core not only has a large molecular weight, but also has extremely high requirements for the integrity of the mRNA molecule. During the high-intensity pressure changes of vacuum freeze-drying, mRNA-LNP needs to keep its phospholipid layer and nanostructure intact, and maintain its original physical and chemical properties after reconstitution, so as to exert its original therapeutic effect.

[0005] In the face of the above challenges, some solutions are being tried in this field, such as using sucrose and mannitol as two components or sucrose, trehalose, and mannitol as three components as freeze-dried protective agents, and mixing them in a specific ratio to prepare freeze-dried preparations. However, the above technical solutions all use more than two freeze-dried protective agents, and it is necessary to explore the appropriate ratio with LNP, and the preparation process is complicated. Studies have also shown that although the addition of conventional freeze-dried protective agents can prevent particle aggregation during freezing, it may still make the freeze-dried preparations non-functional.

[0006] Therefore, there is still a need in the art to develop LNP lyophilized preparations that can be stored for a long time, maintain efficient delivery, and ensure mRNA expression in vivo. Summary of the invention

[0007] In view of the problems existing in the prior art mentioned above, the present invention realizes long-term storage of lyophilized preparations and maintains their efficient delivery and in vivo expression of mRNA by rationally designing and strictly controlling the types and amounts of excipients in lyophilized preparations and lipids in LNPs (especially ionizable lipids).

[0008] To achieve the above-mentioned purpose and other related purposes, the present invention provides a lipid nanoparticle lyophilized preparation and a preparation method and application thereof. By screening a suitable lyophilized preparation formulation, including a protective agent and concentration, a buffer and concentration, and an LNP formulation (especially ionizable lipids), an mRNA-LNP lyophilized preparation with better delivery effect is designed, so that the lyophilized powder still maintains excellent physicochemical properties and in vivo transfection efficiency after reconstitution.

[0009] The first aspect of the present invention provides the lipid nanoparticle lyophilized preparation, comprising lipid nanoparticles, a lyophilization protectant and a buffer substance; the lipid nanoparticles include an ionizable lipid or an isomer thereof or a pharmaceutically acceptable salt thereof, and the ionizable lipid has the following structure:

[0010]

[0011] wherein n1 and n2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0012] G1 and G2 are each independently a C1-C10 alkylene group;

[0013] R1, R2, R3, and R4 are each independently H, a C1-C20 straight-chain or branched alkane group, or a C2-C20 straight-chain or branched alkene group;

[0014] G3 is C1-C10 alkylene; or G3 is (CH2) a -O-(CH2) b, wherein a and b are each independently 1, 2, 3, 4, 5, 6, 7, 8 or 9, and a+b is an integer from 2 to 10;

[0015] L1 is -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, or -O(C=O)O-;

[0016] L2 is -NH(C=O)O- or -O(C=O)NH-;

[0017] The lyoprotectant is sucrose, and its concentration in the liquid preparation before lyophilization is 10%-20% w / v;

[0018] The buffer substance is a Tris buffer or a HEPES buffer, and its concentration in the liquid preparation before lyophilization is 1-20 mM.

[0019] The second aspect of the present invention provides a method for preparing the lipid nanoparticle lyophilized preparation, comprising the following steps:

[0020] 1) using the ionizable lipid or its isomer or its pharmaceutically acceptable salt as a component raw material, preparing a lipid nanoparticle solution encapsulating mRNA, and replacing the buffer in the solution with a Tris buffer or a HEPES buffer with a concentration of 1-20 mM to obtain a replaced solution;

[0021] 2) adding sucrose to the replaced solution to make the sucrose concentration be 10%-20% w / v to obtain a liquid preparation;

[0022] 3) Freeze-drying the liquid preparation to obtain a freeze-dried preparation.

[0023] The third aspect of the present invention provides the use of the above-mentioned lipid nanoparticle lyophilized preparation in the preparation of nucleic acid drugs.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention designs a lipid nanoparticle lyophilized preparation that can maintain the physicochemical properties of the original lipid nanoparticles after reconstitution and ensure the mRNA delivery effect by screening a suitable lyophilized preparation formulation, including a protective agent and its concentration, a buffer and its concentration, and an LNP formulation (especially an ionizable lipid).

[0026] 2. The lipid nanoparticle freeze-dried preparation provided by the present invention has good storage stability. After being placed at 2-8°C for 3 months, the average particle size, polydispersity index and encapsulation rate are not significantly changed after reconstitution, and the stability is good; it is suitable for delivering different types of mRNA. Compared with the liquid preparation before freeze-drying, the in vivo transfection biological activity of the freeze-dried preparation is not reduced after reconstitution. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the appearance of the mRNA-LNP lyophilized preparation prepared in Example 2 of the present invention after reconstitution.

[0028] Figure 2 These are the experimental fluorescence imaging results of the mRNA-LNP solution prepared in Example 7 of the present invention before and after freeze-drying, wherein a is after freeze-drying and reconstitution; b is before freeze-drying.

[0029] Figure 3 It is a quantitative display and statistics of the experimental fluorescence imaging results of the mRNA-LNP solution E5 group prepared in Example 7 of the present invention before and after freeze-drying, wherein the horizontal axis 1 represents after freeze-drying and reconstitution; 2 represents before freeze-drying. DETAILED DESCRIPTION

[0030] In order to solve the deficiencies of the prior art, the object of the present invention is to provide a lipid nanoparticle freeze-dried preparation and a preparation method and application thereof.

[0031] In order to achieve the above object, the present invention adopts the following technical solution:

[0032] The first aspect of the present invention provides a lipid nanoparticle lyophilized preparation, comprising lipid nanoparticles, a lyophilization protectant and a buffer substance; the lipid nanoparticles include an ionizable lipid or an isomer thereof or a pharmaceutically acceptable salt thereof, and the ionizable lipid has the following structure:

[0033]

[0034] wherein n1 and n2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0035] G1 and G2 are each independently a C1-C10 alkylene group;

[0036] R1, R2, R3, and R4 are each independently H, a C1-C20 straight-chain or branched alkane group, or a C2-C20 straight-chain or branched alkene group;

[0037] G3 is C1-C10 alkylene; or G3 is (CH2) a -O-(CH2) b , wherein a and b are each independently 1, 2, 3, 4, 5, 6, 7, 8 or 9, and a+b is an integer from 2 to 10;

[0038] L1 is -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, or -O(C=O)O-;

[0039] L2 is -NH(C=O)O- or -O(C=O)NH-;

[0040] The lyoprotectant is sucrose, and its concentration in the liquid preparation before lyophilization is 10%-20% w / v;

[0041] The buffer substance is a Tris buffer or a HEPES buffer, and its concentration in the liquid preparation before lyophilization is 1-20 mM.

[0042] The present invention selects ionizable lipids with specific structures, and cooperates with preferred lyoprotectants and buffer substances to form a lyophilized preparation, which improves the stability of lipid nanoparticles before and after lyophilization, and ensures that the delivery effect of nucleic acids after lyophilization is not affected. It can be seen from Example 1 of the present invention that the lyophilized preparation of the present invention is in the form of a powder cake, which can be quickly re-dissolved, and the re-dissolved lipid nanoparticles maintain a good average particle size distribution, encapsulation rate, mRNA integrity and in vivo transfection effect.

[0043] The main component of the "Tris buffer" described in the present invention is tris(hydroxymethyl)aminomethane, usually referred to as Tris. Preferably, when the buffer substance described in the present invention is Tris buffer, its concentration in the liquid preparation before lyophilization is not greater than 10 mM.

[0044] The main component of the "HEPES buffer" described in the present invention is N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), usually referred to as HEPES. Preferably, when the buffer substance described in the present invention is HEPES buffer, its concentration in the liquid preparation before lyophilization is 20mM.

[0045] In some specific embodiments of the present invention, the buffer is a Tris buffer with a pH of 6.0-7.5, and its concentration in the liquid preparation before lyophilization is 1-10mM. In some preferred embodiments of the present invention, the buffer is a Tris buffer with a pH of 7.5, and its concentration in the liquid preparation before lyophilization is 10mM, and the concentration of sucrose in the liquid preparation before lyophilization is 10% w / v. In other preferred embodiments of the present invention, the buffer is a Tris buffer with a pH of 6.0, and its concentration in the liquid preparation before lyophilization is 1mM, and the concentration of sucrose in the liquid preparation before lyophilization is 15% w / v.

[0046] In some specific embodiments of the present invention, the buffer is a HEPES buffer with a pH of 6.0-7.5, and its concentration in the liquid preparation before lyophilization is 20mM. In some preferred embodiments of the present invention, the buffer is a HEPES buffer with a pH of 6.0, and its concentration in the liquid preparation before lyophilization is 20mM, and the concentration of sucrose in the liquid preparation before lyophilization is 10% w / v.

[0047] The "C1-C20 straight-chain or branched alkane group", "C2-C20 straight-chain or branched olefin group" and "C1-C10 alkylene group" mentioned in the present invention are as described in

[0047] ,

[0048] and

[0049] of the specification of Chinese patent application CN115947671A, respectively.

[0048] In some specific embodiments of the present invention, -CH(R1)R2 and -CH(R3)R4 in the ionizable lipid are as described in

[0019] ,

[0020] and

[0021] of the specification of Chinese patent application CN115947671A.

[0049] In some specific embodiments of the present invention, the ionizable lipid is as described in

[0022] ,

[0023] ,

[0024] and

[0025] of the specification of Chinese patent application CN115947671A.

[0050] In some specific embodiments of the present invention, the ionizable lipid is selected from the group consisting of:

[0051]

[0052] The "isomers" include stereoisomers and tautomers.

[0053] The term "stereoisomer" refers to isomers that have the same sequence of atoms connected but different arrangements of atoms in space.

[0054] The term "tautomer" refers to a phenomenon in which the structure of a compound undergoes equilibrium interconversion between two functional group isomers, and the corresponding isomers are called tautomers.

[0055] The "pharmaceutically acceptable salt" refers to an acid addition salt or a base addition salt. All compounds of the present invention that exist in the form of free base or free acid can be converted into their pharmaceutically acceptable salts by treatment with appropriate inorganic or organic bases or acids according to methods known to those skilled in the art. Salts of compounds of the present invention can be converted into their free bases or acids by standard techniques.

[0056] Pharmaceutically acceptable salts of the compounds of the invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts with amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods such as ion exchange used in the art. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, citrate, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconate heptanoate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts. Representative alkali metal salts or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, etc. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates and aryl sulfonates. Other pharmaceutically acceptable salts include salts formed by quaternization of amines, which are carried out using suitable electrophilic reagents (e.g., alkyl halides) to form quaternized alkylated amino salts.

[0057] In some specific embodiments of the present invention, the lipid nanoparticles further comprise nucleic acids, structural lipids, auxiliary lipids and PEG-lipids.

[0058] The "nucleic acid" of the present invention can be a nucleotide polymer of any length. Including but not limited to single-stranded DNA, double-stranded DNA, plasmid DNA, short isomers, mRNA, tRNA, rRNA, long non-coding RNA (lncRNA), miRNA, siRNA, telomerase RNA (Telomerase RNA), small RNA (snRNA and scRNA), circular RNA (circRNA), synthetic miRNA (miRNAmimics, miRNA agomir, miRNA antagomir), antisense oligonucleotide (ASO), ribozyme, asymmetric interfering RNA (aiRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), guide RNA (gRNA), small guide RNA (sgRNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholino antisense oligonucleotide, morpholino oligonucleotide or biological custom oligonucleotide one or more combinations.

[0059] In some specific embodiments of the present invention, the nucleic acid is mRNA. The mRNA is a type of single-stranded ribonucleic acid that is transcribed from a strand of DNA as a template and carries genetic information and can guide protein synthesis. The mRNA can encode one protein or multiple proteins at the same time. Preferably, the mRNA is synthesized by in vitro transcription.

[0060] The "structured lipids" mentioned above refer to lipids containing structures that can stabilize the composition, including but not limited to a combination of one or more of sterols and their derivatives and non-sterols and their derivatives.

[0061] In some specific embodiments, the structural lipids include, but are not limited to, a combination of one or more of sterols and their derivatives, non-sterols, sitosterols, ergosterols, cholestanones, cholestenones, campesterols, stigmasterols, brassicasterols, tomatine, ursolic acid, coprosterols, α-tocopherols, or corticosteroids. Sterols are preferably cholesterol and its derivatives; non-limiting examples of cholesterol derivatives include: polar analogs such as 5α-cholestanol, 5α-coprosterol, cholesteryl-(2'-hydroxy)ethyl ether, cholesteryl-(4'-hydroxy)butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestane, cholesterenone, 5α-cholestenone, and decanoic acid cholesterol ester; and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog, such as cholesteryl-(4'-hydroxy)butyl ether. This is not exhaustive, the selection of structural lipids is not limited, and any structural lipid can be applied to the present invention.

[0062] In some embodiments, the structured lipid is a combination of one or more of cholesterol, sitosterol, ergosterol, corticosteroids and their derivatives.

[0063] In some embodiments, the structured lipid is cholesterol.

[0064] The type of the "auxiliary lipid" is not limited, and phospholipids are preferred, including but not limited to: a combination of one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, and dimyristoylphosphatidylglycerol.

[0065] In some specific embodiments, the helper lipid can be selected from: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC ), 1,2-heneicosanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), dipalmitoylphosphatidylethanolamine (DPPE), 1- Oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-O-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-docosahexanoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl A combination of one or more of acyl-sn-glycero-3-phosphoethanolamine, 1,2-dialinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docosahexanoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), diacetyl-phosphatidylethanolamine (DEPE), stearoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, and sphingomyelin.

[0066] In some specific embodiments, the phosphatidylcholine is a combination of one or more of DSPC, DPPC, DMPC, DOPC, and POPC.

[0067] In some specific embodiments, the helper lipid is phosphatidylcholine, specifically DSPC.

[0068] In some specific embodiments, the helper lipid is phosphatidylcholine, specifically DPPC.

[0069] In some specific embodiments, the helper lipid is phosphatidylcholine, specifically a combination of DSPC and DPPC.

[0070] In some specific embodiments, the helper lipid is phosphatidylethanolamine, specifically DOPE.

[0071] The "PEG-lipid" described in the present invention generally refers to a conjugate formed by linking PEG (polyethylene glycol) and lipid molecules through chemical bonds, including but not limited to PEG-modified phospholipids and derived lipids, such as PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, methoxypolyethylene glycol ditetradecyl acetamide, or a combination of one or more thereof.

[0072] In some embodiments, the PEG-lipids include but are not limited to PEG-C-DMG, PEG-C-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, PEG-DPPC, PEG-distearoylphosphatidylethanolamine (PEG-DSPE), PEG-DS, Chol (cholesterol)-PEG, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (PEG-DMG), PEG-S-DMG, polyethylene glycol phosphatidylethanolamine, polyethylene glycol ceramide, polyethylene glycol A combination of one or more of PEG-DMA, PEG-distearylglycerol, PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide, PEG-dipalmitoylphosphatidylethanolamine, PEG-phosphatidylethanol, PEG-phosphatidylethylmyristyloxypropyl-3-amine, PEG-oxypropyl alcoholamine, 1,2-distearoyloxypropyl-3-amine-N[methoxy(polyethylene glycol)](PEG-DSA), methoxypolyethylene glycol lauric acid, and methoxypolyethylene glycol ditetradecylacetamide (ALC0159).

[0073] In some embodiments, the PEG-lipid is PEG-DMG.

[0074] In some specific embodiments of the present invention, the weight average molecular weight of PEG in the PEG-lipid is 1000-10000, for example, 1000-2000, 2000-4000, 4000-6000, 6000-8000, 8000-10000, preferably 2000.

[0075] In some specific embodiments, the average particle size of the lipid nanoparticles ranges from 60-300 nm, and can be 60-70 nm, 70-80 nm, 80-90 nm, 90-100 nm, 100-150 nm, 150-200 nm, 200-250 nm, or 250-300 nm.

[0076] In the present invention, there is no special restriction on the concentration of the lipid nanoparticles. It can be seen from the experimental results that the parameters of the lyophilized preparations of lipid nanoparticles with different concentrations change little before and after lyophilization, and the lipid nanoparticles have good stability.

[0077] In some specific embodiments, the mass ratio of the total mass of lipid molecules in the lipid nanoparticles to the mass of mRNA is 10-30:1.

[0078] In some specific embodiments, the molar percentage of the ionizable lipid or its isomer or its pharmaceutically acceptable salt, the auxiliary lipid, the structural lipid and the PEG-lipid in the lipid nanoparticle is 30-65: 5-40: 15-45: 1-5. In some preferred embodiments of the present invention, the molar percentage of the ionizable lipid or its isomer or its pharmaceutically acceptable salt, the auxiliary lipid, the structural lipid and the PEG-lipid in the lipid nanoparticle is 46.3: 9.4: 42.7: 1.6.

[0079] The second aspect of the present invention provides a method for preparing the above-mentioned lipid nanoparticle lyophilized preparation, comprising the following steps:

[0080] 1) using the ionizable lipid or its isomer or its pharmaceutically acceptable salt as a component raw material, preparing a lipid nanoparticle solution encapsulating mRNA, and replacing the buffer in the solution with a Tris buffer or a HEPES buffer with a concentration of 1-20 mM to obtain a replaced solution;

[0081] 2) adding sucrose to the replaced solution to make the sucrose concentration be 10%-20% w / v to obtain a liquid preparation;

[0082] 3) Freeze-drying the liquid preparation to obtain a freeze-dried preparation.

[0083] In some specific embodiments, in step 1), a lipid nanoparticle solution containing mRNA is prepared by microfluidic mixing. Specifically, the preparation of the lipid nanoparticle solution containing mRNA by microfluidic mixing includes: dissolving an ionizable lipid or its isomer or a pharmaceutically acceptable salt thereof, an auxiliary lipid, a structural lipid and a PEG-lipid in ethanol to obtain an organic phase, then dissolving the mRNA in an acidic buffer to obtain an aqueous phase, mixing the organic phase and the aqueous phase by microfluidics in a certain volume ratio to prepare lipid nanoparticles, immediately diluting with a buffer, purifying and removing ethanol, and obtaining a lipid nanoparticle solution containing mRNA. Preferably, the acidic buffer is a sodium acetate buffer or a citric acid buffer with a pH of 4.0-5.0; the volume ratio of the organic phase to the aqueous phase is 1:2-4. Preferably, the buffer in the replacement solution while purifying and removing ethanol is a Tris buffer or a HEPES buffer with a concentration of 1-20mM. Preferably, the purification includes ultrafiltration, dialysis and tangential flow filtration (TFF).

[0084] In some specific embodiments, in step 3), the freeze-drying includes pre-freezing, primary drying and desorption drying. Preferably, the pre-freezing temperature is less than -40°C, the pressure is atmospheric pressure, and the time is 160-200 min; the primary drying temperature is -40 to -30°C, the pressure is 0.01-0.15 mbar, and the time is 1600-2000 min; the desorption drying temperature is 20-40°C, the pressure is 0.01-0.3 mbar, and the time is 300-800 min.

[0085] The third aspect of the present invention provides the use of the above-mentioned lipid nanoparticle lyophilized preparation in the preparation of nucleic acid drugs.

[0086] The lipid nanoparticle lyophilized preparation of the present invention is reconstituted before use. In some specific embodiments, the lipid nanoparticle lyophilized preparation of the present invention is reconstituted with water for injection, saline or acidic buffer solution of the same volume as the liquid preparation before lyophilization. It can be seen from the in vivo efficacy verification of Example 7 of the present invention that the in vivo transfection biological activity of the lyophilized preparation provided by the present invention does not decrease after reconstitution.

[0087] The nucleic acid drug of the present invention may contain one or more adjuvants or may be combined with one or more adjuvants. The adjuvant may refer to any substance that prolongs, enhances or accelerates the immune response. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligonucleotides, growth factors and cytokines, such as monokines, lymphokines, interleukins, and chemokines.

[0088] In some specific embodiments, the nucleic acid drug of the present invention is administered intravenously, intramuscularly, intradermally, subcutaneously, or intraspinal.

[0089] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0090] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials in the embodiments of the present invention may be used to implement the present invention, based on the prior art mastery of those skilled in the art and the description of the present invention.

[0091] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related conventional techniques in the art. Unless otherwise specified, the materials and equipment used in the present invention are all commercially available.

[0092] The E series ionizable lipids described in the following examples have the following structures:

[0093]

[0094] wherein n1 and n2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0095] G1 and G2 are each independently a C1-C10 alkylene group;

[0096] R1, R2, R3, and R4 are each independently H, a C1-C20 straight-chain or branched alkane group, or a C2-C20 straight-chain or branched alkene group;

[0097] G3 is C1-C10 alkylene; or G3 is (CH2) a -O-(CH2) b , wherein a and b are each independently 1, 2, 3, 4, 5, 6, 7, 8 or 9, and a+b is an integer from 2 to 10;

[0098] L1 is -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, or -O(C=O)O-;

[0099] L2 is -NH(C=O)O- or -O(C=O)NH-.

[0100] Embodiment 1:

[0101] 1. Preparation of mRNA-LNP

[0102] The mRNA-LNP preparation method comprises the following steps:

[0103] Step 1: Ionizable lipids E1, E5, E11, DSPC (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG in Table 1 were respectively added. 2000 It was dissolved in ethanol according to the molar percentage of 46.3:9.4:42.7:1.6 to prepare a lipid ethanol solution, which was the organic phase.

[0104] Step 2: Dissolve the mRNA in sodium acetate buffer (pH=4.7) to obtain an aqueous phase.

[0105] Step 3: The organic phase obtained in step 1 and the aqueous phase obtained in step 2 were prepared by a microfluidic device in a volume ratio of 1:3. The prepared LNP was immediately diluted with sodium acetate buffer (pH = 4.7), purified (ultrafiltration / dialysis / TFF) to remove ethanol, and the buffer was replaced with 10mM Tris (pH = 7.5) to obtain an mRNA-LNP (lipid nanoparticles encapsulating mRNA) solution.

[0106] 2. Preparation of lyophilized preparations

[0107] The lyophilization protective agent sucrose was added to the mRNA-LNP solution prepared above to obtain a pre-lyophilized liquid preparation, wherein the concentration of sucrose in the pre-lyophilized liquid preparation was 10% w / v; the pre-lyophilized liquid preparation was added to a borosilicate controlled injection bottle at a filling volume of 0.5 mL / bottle, and the lyophilization program was set according to the conditions and parameters shown in Table 2 below to perform lyophilization.

[0108] Table 1 Structural formula of ionizable lipids

[0109]

[0110]

[0111] Table 2 Freeze-drying conditions and parameters

[0112] step describe Temperature(℃) Pressure(mbar) Duration (min) 1 Pre-freeze <-40 Atmospheric pressure 180 2 One-time drying -38 0.1 1800 3 Analytical drying 30 0.2 600

[0113] The finished product obtained after freeze-drying is in the form of powder cake. After adding 0.5 mL of water for injection / normal saline, the freeze-dried powder is quickly reconstituted within 10 seconds. After testing and experiments, the mRNA-LNP after the freeze-dried powder is reconstituted maintains a good average particle size distribution, encapsulation efficiency, mRNA integrity and in vivo transfection effect.

[0114] Example 2: Screening of lyophilization protectants

[0115] 1. Selection and comparison of single-component lyophilization protective agents

[0116] Using E1 as an ionizable lipid and the substances in Table 3 below as lyoprotectants, an mRNA-LNP solution was first prepared according to the prescription and preparation method in Example 1, and then the corresponding lyoprotectants were added to prepare a pre-lyophilized liquid preparation and observe the appearance, and then lyophilization was performed, and the obtained lyophilized preparation product was reconstituted, and the appearance after reconstitution was observed. The results are as follows: Figure 1 shown.

[0117] Table 3 Screening of lyophilization protective agents

[0118]

[0119]

[0120] Depend on Figure 1 The experimental results show that compared with trehalose and mannitol at the same concentration, the freeze-dried preparations prepared with sucrose as a single prescription protective agent have no obvious changes in appearance before and after freeze-drying, and all are light blue liquid, which shows the Tyndall effect of nanoparticles. The other groups are light blue liquid before freeze-drying, but become turbid after freeze-drying, indicating that the nanoparticles have aggregated, flocculated or settled.

[0121] 2. Comparative experiment before and after freeze-drying

[0122] Using E5 as the ionizable lipid, sucrose (the concentration of sucrose in the preparation is 15% w / v, denoted as 15% Suc) and maltose (the concentration of maltose in the preparation is 15% w / v, denoted as 15% Mal) were added as lyoprotectants, and the mRNA-LNP solution was first prepared according to the prescription and preparation method in Example 1, and the average particle size (Z-Ave), polydispersity index (PDI) and encapsulation efficiency (EE) of the LNP without lyoprotectant (i.e. before lyophilization, denoted as CL) and after lyophilization and reconstitution (denoted as DG) of the sample with the above-mentioned lyoprotectant were measured. The results are shown in Table 4.

[0123] Table 4 Z-Ave, PDI and EE of mRNA-LNP before and after lyophilization

[0124]

[0125] As shown in Table 4, compared with the preparation with maltose as the lyoprotectant, the Z-Ave, PDI and EE of LNP in the preparation with sucrose as the lyoprotectant changed less before and after lyophilization.

[0126] 3. Screening of single-prescription and multi-prescription lyoprotectants

[0127] E5 was used as the ionizable lipid, and sucrose (the concentration of sucrose in the preparation was 10% w / v, recorded as 10% Suc), sucrose and trehalose complex (the concentration of sucrose in the preparation was 10% w / v, the concentration of trehalose was 5% w / v, recorded as 10% Suc+5% Tre), sucrose and maltose complex (the concentration of sucrose in the preparation was 10% w / v, the concentration of maltose was 5% w / v, recorded as 10% Suc+5% Mal), sucrose and mannitol complex (the concentration of sucrose in the preparation was 10% w / v, the concentration of mannitol was 5% w / v, recorded as 10% Suc+5% Mal), and sucrose and mannitol complex (the concentration of sucrose in the preparation was 10% w / v, the concentration of mannitol was 5% w / v, recorded as 10% Suc+5% Mal). / v, recorded as 10% Suc+5% Man), sucrose and poloxamer 188 complex (the concentration of sucrose in the preparation is 10% w / v, the concentration of poloxamer 188 is 5% w / v, recorded as 10% Suc+5% P188) lyoprotectant, the mRNA-LNP solution was prepared according to the prescription and preparation method in Example 1, and the Z-Ave, PDI and EE of the LNP without adding lyoprotectant (i.e. before lyophilization, recorded as CL) and after lyophilization and reconstitution of the sample with the above lyoprotectant (recorded as DG) were measured, and the results are shown in Table 5.

[0128] Table 5 Z-Ave, PDI and EE of mRNA-LNP before and after freeze-drying

[0129]

[0130] As shown in Table 5, compared with the addition of trehalose, maltose, mannitol or the composite lyoprotectant of poloxamer 188 and sucrose, the key data such as Z-Ave, PDI, and EE of LNP after lyophilization changed less when sucrose was used as a single lyoprotectant.

[0131] Example 3: Screening of sucrose concentration

[0132] Using E5 as the ionizable lipid, 10% sucrose (the concentration of sucrose in the preparation is 10% w / v, recorded as 10% Suc), 12% sucrose (the concentration of sucrose in the preparation is 12% w / v, recorded as 12% Suc), 14% sucrose (the concentration of sucrose in the preparation is 14% w / v, recorded as 14% Suc), 16% sucrose (the concentration of sucrose in the preparation is 16% w / v, recorded as 16% Suc), and 20% sucrose (the concentration of sucrose in the preparation is 20% w / v, recorded as 20% Suc) of lyoprotectants were added, and mRNA-LNP solutions were prepared according to the prescription and preparation method in Example 1. The Z-Ave, PDI and EE of LNP before and after lyophilization of the lyophilized preparation samples to which the above lyoprotectants were added were measured. The results are shown in Table 6.

[0133] Table 6 Z-Ave, PDI and EE of LNP before and after freeze-drying at different sucrose concentrations

[0134]

[0135] Note: 0.1 means the mRNA concentration is 0.1 mg / ml.

[0136] As shown in Table 6, when 10%-20% w / v sucrose is used as a protective agent, the key data of mRNA-LNP before and after freeze-drying change little. Among them, the ideal effect can be achieved with a lower dose of 10% w / v, which can reduce costs.

[0137] Example 4: Screening of buffer concentration

[0138] Using E5 as the ionizable lipid, adding 10% sucrose (the concentration of sucrose in the preparation is 10% w / v, denoted as 10% Suc) as a lyoprotectant, and using 5mM Tris (pH 7.5), 10mM Tris (pH 7.5), 20mM Tris (pH7.5), and 50mM Tris (pH 7.5) as buffers (i.e., buffer substances in the lyophilized preparation), respectively, the mRNA-LNP solution was prepared according to the prescription and preparation method in Example 1, and the Z-Ave, PDI and EE of the LNP before and after lyophilization of the above-prepared lyophilized preparation samples were measured. The results are shown in Table 7.

[0139] Table 7 Z-Ave, PDI and EE of LNP before and after lyophilization at different buffer concentrations

[0140]

[0141] As shown in Table 7, when the concentration of Tris buffer increases from 5mM to 20mM, the key data of mRNA-LNP before and after freeze-drying change little, and the freeze-dried preparation with 10mM Tris buffer has the best effect. As the concentration of Tris buffer increases from 20mM to 50mM, the key data of mRNA-LNP before and after freeze-drying change greatly.

[0142] Using Tris buffer at pH 6.0 as the buffer system, the concentration of Tris buffer was screened. The results showed that when the concentration of Tris buffer was no more than 10mM, the key data of mRNA-LNP before and after freeze-drying changed little, and the freeze-dried preparation using 1mM Tris buffer as the buffer system had the best effect. Under this premise, the sucrose concentration was further screened, and the freeze-dried preparation had the best effect when the sucrose concentration was 15% w / v.

[0143] The concentration of HEPES buffer was screened with a pH of 6.0 as the buffer system. The results showed that the freeze-dried preparation with a concentration of 20 mM HEPES buffer as the buffer system had the best effect. Under this premise, the sucrose concentration was further screened. When the sucrose concentration was 10% w / v, the freeze-dried preparation had the best effect.

[0144] Example 5: Screening of LNP concentration

[0145] Using E5 as the ionizable lipid, adding 10% sucrose (the concentration of sucrose in the preparation is 10% w / v, denoted as 10% Suc) as a lyoprotectant, and using 10 mM Tris (pH 7.5) as a buffer (i.e., the buffer substance in the lyophilized preparation), mRNA-LNP solutions with different LNP concentrations were prepared according to the prescription and preparation method in Example 1, and the Z-Ave, PDI and EE of the LNP before and after lyophilization of the lyophilized preparation samples prepared above were measured. The results are shown in Table 8.

[0146] Table 8 Z-Ave, PDI and EE of LNP before and after freeze-drying at different LNP concentrations

[0147]

[0148] Note: 0.1, 0.2, 0.5 means the mRNA concentration is 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL.

[0149] As shown in Table 8, different LNP concentrations have almost no effect on the Z-Ave, PDI and EE of LNP before and after freeze-drying.

[0150] Example 6: Screening of ionizable lipids

[0151] Different ionizable lipid formulations were screened, and E5, BNT:ALC-0315 in Table 9, and H1 were used as ionizable lipids, respectively. According to the formulation in Table 10, 10% sucrose (the concentration of sucrose in the preparation was 10% w / v, recorded as 10% Suc) was added as a lyophilization protectant, 10 mM Tris (pH 7.5) was used as a buffer (i.e., the buffer substance in the lyophilized preparation), and the mRNA concentration was controlled to be 0.1 mg / mL. According to the formulation and preparation method in Example 1, mRNA-LNP solutions with different ionizable lipids were prepared, and the Z-Ave, PDI and EE of the LNP before and after lyophilization of the above-prepared lyophilized preparation samples were measured. The results are shown in Table 11.

[0152] Table 9 Structural formulas of different ionizable lipids

[0153]

[0154] Note: ALC-0315 is commercially available, H1 is a compound disclosed in Chinese patent 202210344395.1, and was prepared by referring to the method in the patent.

[0155] Table 10 mRNA-LNP prescription

[0156]

[0157]

[0158] Table 11 Z-Ave, PDI and EE of LNP before and after freeze-drying with different ionizable lipid formulations

[0159]

[0160] As shown in Table 11, the ionizable lipids ALC0315 and H1 and E5 were used to prepare the LNP freeze-dried preparations with the same buffer composition and the same type and concentration of lyoprotectants. Compared with ALC0315 and H1, the E5 formulation had smaller changes in key parameters during the freeze-drying process and better stability, indicating that the selection of ionizable lipids has an important influence on the stability of the LNP freeze-dried preparations.

[0161] Example 7: In vivo efficacy verification experiment

[0162] Male ICR mice (6-8 weeks, Shanghai Jiesijie Experimental Animal Co., Ltd.) were housed under experimental conditions of 22±2℃ and relative humidity of 45–75%, with a light / dark cycle of 12h. Luciferase mRNA was used as a reporter gene. Luciferase catalyzes luciferin to produce bioluminescence, and the transfection efficiency of LNP is reflected by detecting the intensity of bioluminescence per unit time. Taking luciferase mRNA (purchased from ApexBio Technology) as an example, the mRNA-LNP freeze-dried preparation samples E1 group, E11 group and E5 group obtained in Example 1 were prepared; the reconstituted samples of each group were injected intramuscularly into the lateral thigh muscle of mice at a dose of 150μg / kg mRNA, with two mice and two legs in each group of samples. At a specific time point, 200uL of luciferin potassium substrate (20μg / mL) was injected intraperitoneally into the mouse. After 5 minutes, the mouse was placed in a small animal in vivo imager to measure the fluorescence intensity. The experimental results are shown as follows Figure 2 shown.

[0163] Depend on Figure 2It can be seen that the lyophilized powder E1, E11 and E5 groups encapsulating luciferase mRNA are compared with the corresponding non-lyophilized liquid preparations. The quantitative results of in vivo imaging by intramuscular injection in mice show that there is no significant difference between the two. The in vivo transfection biological activity of the lyophilized powder reconstituted is not reduced compared with the non-lyophilized liquid preparation. The in vivo efficacy of the lyophilized preparations prepared with other ionizable lipids in the E series was simultaneously verified. The formula of the lyophilized preparation is the same as that of the E1, E11 and E5 groups in the embodiment. The quantitative results of intramuscular injection in vivo imaging in mice show that the in vivo transfection biological activity does not decrease after the lyophilized powder is reconstituted.

[0164] Figure 3 Quantitative display and statistics of fluorescence imaging results of E5 group before and after freeze-drying ( Figure 3 1 means after freeze-drying and reconstitution; 2 means before freeze-drying). Figure 3 It can be seen that there is no statistical difference between the before and after comparison. According to statistics, the E1 group and the E11 group also have similar results.

[0165] Example 8: Stability test

[0166] 1. Long-term stability test

[0167] E5 was used as an ionizable lipid, 10% sucrose (the concentration of sucrose in the preparation was 10% w / v, recorded as 10% Suc) was added as a lyophilization protective agent, the buffer was a Tris buffer with a pH of 7.5 and a concentration of 10 mM, and the reconstitution solution was water for injection. First, mRNA-LNP solutions with different mRNA concentrations were prepared according to the prescription and preparation method in Example 1, and then a lyophilization protective agent was added to prepare a pre-lyophilized liquid preparation, which was then lyophilized, and the resulting lyophilized preparation product was stored at 2-8°C, and the Z-Ave, PDI, EE and mRNA integrity of the LNP of the lyophilized preparation product were measured at the beginning of storage (0h), 1 month (1M), 2 months (2M) and 3 months (3M), respectively. The results are shown in Table 12.

[0168] Table 12 Results of long-term stability test of lyophilized preparations

[0169]

[0170] From the results in Table 12, it can be seen that the lyophilized preparation samples with different mRNA concentrations are stored at 2-8°C for 3 months, and the physical and chemical properties are basically stable, with good long-term stability.

[0171] 2. Stability test of different buffer systems

[0172] 2.1 Preparation of lyophilized preparations with different buffer systems

[0173] As shown in Table 13 below, lyophilized preparations were prepared using E11 as an ionizable lipid, 10% sucrose (the concentration of sucrose in the preparation was 10% w / v, recorded as 10% Suc) as a lyophilization protectant, a HEPES buffer with a pH of 6.0 and a concentration of 20 mM as a buffer system, and 15% sucrose (the concentration of sucrose in the preparation was 15% w / v, recorded as 15% Suc) as a lyophilization protectant, a Tris buffer with a pH of 6.0 and a concentration of 1 mM as a buffer system. First, an mRNA-LNP solution was prepared according to the prescription and preparation method in Example 1, and the Z-Ave, PDI, EE and mRNA integrity of the LNP were measured, and then a lyophilization protectant was added to prepare a pre-lyophilized liquid preparation, which was then lyophilized, and the Z-Ave, PDI, EE and mRNA integrity of the lyophilized LNP were measured. The results are shown in Table 14.

[0174] Table 13 Lyophilized preparations with different buffer systems

[0175]

[0176] Table 14 Z-Ave, PDI and EE of LNP before and after freeze-drying

[0177]

[0178] From the results in Table 14, it can be seen that the lyophilized preparations prepared by adding 10% sucrose as a lyophilization protectant and using HEPES buffer with a pH of 6.0 and a concentration of 20 mM as a buffer system and by adding 15% sucrose as a lyophilization protectant and Tris buffer with a pH of 6.0 and a concentration of 1 mM as a buffer system have little change in Z-Ave, PDI, and EE of mRNA-LNP before and after lyophilization, and have good stability.

[0179] 2.2 Long-term stability and accelerated stability test of freeze-dried preparations with different buffer systems

[0180] The freeze-dried preparation samples obtained in 2.1 were divided into two groups, one group was stored at 2-8°C (normal condition stability test), and the other group was stored at 25°C (accelerated condition stability test), and the Z-Ave, PDI, EE and mRNA integrity of LNP of the two groups of freeze-dried preparation products were measured at the beginning of storage (0h), storage for 20 days (20d), storage for one month (1M), storage for two months (2M) and storage for 3 months (3M), respectively. The results are shown in Table 15. Among them, the reconstitution solution is water for injection.

[0181] Table 15 Long-term stability and accelerated stability test results

[0182]

[0183]

[0184] As shown in Table 15, the physical and chemical properties of the two batches of samples of LNP-1mM Tris-15%Suc ​​and LNP-20mM HEPES-10%Suc were basically stable when stored at 2-8°C for 3 months. The two batches of samples of LNP-1mM Tris-15%Suc ​​and LNP-20mM HEPES-10%Suc were stored at 25°C for 3 months, and Z-Ave and EE were basically stable. The integrity of mRNA decreased with the storage time, but was still greater than 80% at 3 months.

[0185] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A lipid nanoparticle freeze-dried preparation, characterized in that: The invention comprises lipid nanoparticles, a freeze-drying protective agent and a buffer substance; the lipid nanoparticles comprise an ionizable lipid or an isomer thereof or a pharmaceutically acceptable salt thereof, and the ionizable lipid has the following structure: wherein n1 and n2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; G1 and G2 are each independently a C1-C10 alkylene group; R1, R2, R3, and R4 are each independently H, a C1-C20 straight-chain or branched alkane group, or a C2-C20 straight-chain or branched alkene group; G3 is C1-C10 alkylene; or G3 is (CH2) a -O-(CH2) b , wherein a and b are each independently 1, 2, 3, 4, 5, 6, 7, 8 or 9, and a+b is an integer from 2 to 10; L1 is -(C=O)O-, -O(C=O)-, -NH(C=O)O-, -O(C=O)NH-, or -O(C=O)O-; L2 is -NH(C=O)O- or -O(C=O)NH-; The lyoprotectant is sucrose, and its concentration in the liquid preparation before lyophilization is 10%-20% w / v; The buffer substance is a Tris buffer or a HEPES buffer, and its concentration in the liquid preparation before lyophilization is 1-20 mM.

2. The lipid nanoparticle lyophilized preparation according to claim 1, characterized in that The buffer substance is a Tris buffer with a pH of 6.0-7.5, and its concentration in the liquid preparation before lyophilization is 1-10 mM; or, the buffer substance is a HEPES buffer with a pH of 6.0-7.5, and its concentration in the liquid preparation before lyophilization is 20 mM.

3. The lipid nanoparticle lyophilized preparation according to claim 2, characterized in that The buffer substance is a Tris buffer with a pH of 7.5, and its concentration in the liquid preparation before freeze-drying is 10 mM, and the concentration of sucrose in the liquid preparation before freeze-drying is 10% w / v; or, the buffer substance is a Tris buffer with a pH of 6.0, and its concentration in the liquid preparation before freeze-drying is 1 mM, and the concentration of sucrose in the liquid preparation before freeze-drying is 15% w / v; or, the buffer substance is a HEPES buffer with a pH of 6.0, and its concentration in the liquid preparation before freeze-drying is 20 mM, and the concentration of sucrose in the liquid preparation before freeze-drying is 10% w / v.

4. The lipid nanoparticle lyophilized preparation according to claim 1, characterized in that The ionizable lipid is selected from the group consisting of:

5. The lipid nanoparticle lyophilized preparation according to claim 1, characterized in that The lipid nanoparticles also include nucleic acids, structural lipids, helper lipids and PEG-lipids.

6. The lipid nanoparticle lyophilized preparation according to claim 5, characterized in that Includes one or more of the following characteristics: 1) The nucleic acid is mRNA; 2) The structural lipid is cholesterol; 3) The helper lipid is DSPC, DOPE, DPPC or a combination of DSPC and DPPC; 4) the PEG-lipid is PEG-DMG; 5) The average particle size of the lipid nanoparticles is in the range of 60-300 nm; 6) The mass ratio of the total mass of lipid molecules in the lipid nanoparticles to the mass of mRNA is 10-30:1; 7) The molar percentage of the ionizable lipid or its isomer or its pharmaceutically acceptable salt, the auxiliary lipid, the structural lipid and the PEG-lipid in the lipid nanoparticles is 30-65:5-40:15-45:1-5.

7. The method for preparing the lipid nanoparticle preparation according to any one of claims 1 to 6, characterized in that: The steps include: 1) using the ionizable lipid or its isomer or its pharmaceutically acceptable salt as a component raw material, preparing a lipid nanoparticle solution encapsulating mRNA, and replacing the buffer in the solution with a Tris buffer or a HEPES buffer with a concentration of 1-20 mM to obtain a replaced solution; 2) adding sucrose to the replaced solution to make the sucrose concentration be 10%-20% w / v to obtain a liquid preparation; 3) Freeze-drying the liquid preparation to obtain a freeze-dried preparation.

8. The method for preparing the lyophilized lipid nanoparticle preparation according to claim 7, characterized in that: In step 1), a lipid nanoparticle solution encapsulating mRNA is prepared by microfluidic mixing; and / or, in step 3), the freeze-drying includes pre-freezing, primary drying and analytical drying.

9. The method for preparing the lyophilized lipid nanoparticle preparation according to claim 8, characterized in that: In step 1), while ethanol is purified and removed, the buffer in the replacement solution is a Tris buffer or a HEPES buffer with a concentration of 1-20 mM.

10. Use of the lyophilized lipid nanoparticle preparation according to any one of claims 1 to 6 or the lyophilized lipid nanoparticle preparation obtained by the preparation method according to any one of claims 7 to 9 in the preparation of nucleic acid drugs.

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