A lipid nanoparticle freeze-dried preparation and its preparation method and application

Abstract

The present invention relates to the field of drug delivery technology, and in particular to a lipid nanoparticle lyophilized preparation and its preparation method and application. The present invention screens a suitable lyophilized preparation prescription, including the type and concentration of a lyoprotectant, the type and concentration of a buffer, and a lipid nanoparticle prescription, to obtain a lipid nanoparticle lyophilized preparation having a concentration of 10%-20% w / v sucrose as a single-component lyoprotectant, a concentration of 1-20mM Tris buffer or HEPES buffer as a buffer system, and a specific ionizable lipid. The lyophilized preparation is simple to prepare, maintains excellent physicochemical properties and in vivo transfection efficiency after reconstitution, and has good prospects in the 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] Nucleic acid drugs offer a wider range of applications than traditional small molecule drugs and antibody drugs. Nucleic acids are electronegative macromolecules that are susceptible to degradation and have low cellular uptake and transfection efficiency. Therefore, the development of safe and effective delivery vehicles is crucial to protect them from degradation and promote cellular uptake. Currently, lipid nanoparticles (LNPs) are one of the leading delivery vehicles for nucleic acid drugs.

[0003] However, all currently marketed nucleic acid vaccines are liquid products. LNPs loaded with nucleic acids (such as mRNA) exhibit poor stability in aqueous media. This is primarily due to two factors: first, mRNA itself is easily inactivated by oxidation, hydrolysis, and enzymatic degradation; second, LNPs are typically composed of ionizable lipids, helper lipids, cholesterol, and PEG-lipids in specific molar ratios. These components interact with each other in complex ways and are easily affected by factors such as solvent salinity, pH, temperature, and oxygen. This can lead to mRNA release or nanoparticle aggregation during storage or transportation, significantly impacting drug efficacy. Therefore, to extend the shelf life of nucleic acid-loaded nanoparticles, they often require ultra-low temperature transportation and storage. For example, the vaccine BNT162b2 must be transported and stored at -80 to -60°C and is stable for only two hours after thawing at room temperature. These demanding storage conditions significantly limit the accessibility of nucleic acid vaccines.

[0004] To overcome the physical and chemical instability of nucleic acid vaccines during long-term storage, nucleic acid vaccines can be freeze-dried to produce lyophilized preparations. Lyophilized preparations can be stored at 2-8°C for a long time and can be rehydrated and reconstituted before administration. However, the development of lyophilized 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 requires extremely high integrity of the mRNA molecule. mRNA-LNP needs to maintain its phospholipid layer and nanostructure without being destroyed during the high-intensity pressure changes of vacuum freeze-drying, and maintain its original physical and chemical properties after reconstitution, so as to exert its original therapeutic effect.

[0005] In response to these challenges, several solutions are being explored in the field, such as using sucrose and mannitol or sucrose, trehalose, and mannitol as lyoprotectants, and mixing them in specific proportions to prepare lyophilized preparations. However, these solutions all utilize two or more lyoprotectants, requiring the exploration of a suitable ratio for LNPs and resulting in complex preparation processes. Studies have also shown that while the addition of conventional lyoprotectants can prevent particle aggregation during freezing, it can still render the lyophylized preparation nonfunctional.

[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 described above, the present invention achieves 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 and other related purposes, the present invention provides a lipid nanoparticle lyophilized formulation, a preparation method and application thereof, and designs an mRNA-LNP lyophilized formulation with better delivery effect by screening a suitable lyophilized formulation formulation, including a protective agent and concentration, a buffer and concentration, and an LNP formulation (especially ionizable lipids), 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 lyoprotectant, and a buffer substance; the lipid nanoparticles comprise an ionizable lipid or an isomer thereof or a pharmaceutically acceptable salt thereof, wherein 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 a C1-C10 alkylene group; 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 freeze-drying protective agent is sucrose, and its concentration in the liquid preparation before freeze-drying is 10%-20% w / v;

[0018] The buffer substance is Tris buffer or 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 lyophilized lipid nanoparticle preparation, comprising the following steps:

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

[0021] 2) adding sucrose to the replaced solution to obtain a sucrose concentration of 10% to 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 formulation that can maintain the original physicochemical properties of the lipid nanoparticles after reconstitution and ensure the mRNA delivery effect by screening a suitable lyophilized formulation formula, including a protective agent and concentration, a buffer and concentration, and a 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 efficiency were not significantly changed after reconstitution, indicating good stability. It is suitable for delivering different types of mRNA. Compared with the liquid preparation before lyophilization, the in vivo transfection biological activity of the freeze-dried preparation was 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, where a is after freeze-drying and reconstitution; b is before freeze-drying.

[0029] Figure 3 This is a quantitative statistical display of the 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] To address the deficiencies of the prior art, the present invention aims to provide a lyophilized lipid nanoparticle preparation and a preparation method and application thereof.

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

[0032] The first aspect of the present invention provides a lyophilized lipid nanoparticle preparation comprising lipid nanoparticles, a lyoprotectant, and a buffering substance; the lipid nanoparticles comprise an ionizable lipid or an isomer thereof or a pharmaceutically acceptable salt thereof, wherein 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 a C1-C10 alkylene group; 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 freeze-drying protective agent is sucrose, and its concentration in the liquid preparation before freeze-drying is 10%-20% w / v;

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

[0042] The present invention utilizes ionizable lipids with specific structures, which are synergistically combined with preferred lyoprotectants and buffers to form a lyophilized formulation. This improves the stability of the lipid nanoparticles before and after lyophilization, while ensuring that the nucleic acid delivery effect remains unaffected after lyophilization. As shown in Example 1, the lyophilized formulation of the present invention is in the form of a powder cake that can be rapidly reconstituted. After reconstitution, the lipid nanoparticles maintain a good average particle size distribution, encapsulation efficiency, mRNA integrity, and in vivo transfection efficacy.

[0043] The main component of the "Tris buffer" of the present invention is tris (hydroxymethyl)aminomethane, usually referred to as Tris. Preferably, when the buffer substance of the present invention is Tris buffer, its concentration in the liquid preparation before lyophilization is no more 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), commonly 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 20 mM.

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

[0046] In some specific embodiments of the present invention, the buffer is a HEPES buffer having a pH of 6.0-7.5, and its concentration in the liquid preparation before lyophilization is 20 mM. In some preferred embodiments of the present invention, the buffer is a HEPES buffer having a pH of 6.0, and its concentration in the liquid preparation before lyophilization is 20 mM, 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" described 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 term "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 a free base or free acid can be converted into their pharmaceutically acceptable salts by treating with an appropriate inorganic or organic base or acid according to methods known to those skilled in the art. Salts of the compounds of the present invention can be formed by converting them into their free base or acid form using standard techniques.

[0056] Pharmaceutically acceptable salts of the compounds of the present 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 used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, citrate, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconoheptate, 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. The salt derived from suitable base comprises alkali metal salt, alkaline earth metal salt, ammonium salt.Representational alkali metal salt or alkaline earth metal salt comprises sodium salt, lithium salt, potassium salt, calcium salt, magnesium salt etc.In appropriate cases, other pharmaceutically acceptable salt comprises the non-toxic ammonium, quaternary ammonium and amine cation formed using the counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate.Other pharmaceutically acceptable salt comprises the salt formed by the quaternization of amine, and this quaternization is carried out using suitable electrophilic reagent (for example, alkyl halide), to form quaternized alkylated amino salt.

[0057] In some embodiments of the present invention, the lipid nanoparticles further comprise nucleic acids, structural lipids, helper 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 a combination of one or more of a custom biological oligonucleotide.

[0059] In some specific embodiments of the present invention, the nucleic acid is mRNA. mRNA is a type of single-stranded RNA transcribed from a single strand of DNA as a template, carrying genetic information and guiding protein synthesis. mRNA can encode a single protein or multiple proteins simultaneously. 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 one or more combinations of sterols and their derivatives and non-sterols and their derivatives.

[0061] In some specific embodiments, the structured lipids include, but are not limited to, a combination of one or more of sterols and their derivatives, non-sterols, sitosterol, ergosterol, cholestanone, cholestenone, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, coprosterol, α-tocopherol, 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-ketocholesterol; non-polar analogs such as 5α-cholestane, cholesterenone, 5α-cholestenone, and cholesterol decanoate; 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, and the selection of structured lipids is not limited, and any structured 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] There is no limitation on the type of the “helper lipid”, 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 embodiments, the helper lipid can be selected from the group consisting of: 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 (DSPPC), 1,2-di ...oleoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-s ), 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-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diachidonoyl-sn-glycero-3-phosphocholine, 1,2-docohexanoyl-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-docohexanoyl-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 embodiments, the phosphatidylcholine is a combination of one or more of DSPC, DPPC, DMPC, DOPC, and POPC.

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

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

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

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

[0071] The term "PEG-lipid" as used herein generally refers to a conjugate formed by chemically linking PEG (polyethylene glycol) to a lipid molecule. This includes, but is not limited to, PEG-modified phospholipids and derived lipids, exemplified by combinations of one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and methoxypolyethylene glycol ditetradecylacetamide.

[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-oxypropylolamine, 1,2-distearoyloxypropyl-3-amine-N[methoxy(polyethylene glycol)] (PEG-DSA), methoxypolyethylene glycol laurate, and methoxypolyethylene glycol ditetradecyl acetamide (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. Experimental results show that the parameters of the lyophilized preparations of lipid nanoparticles with different concentrations change little before and after lyophilization, and 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 helper lipid, the structural lipid, and the PEG-lipid in the lipid nanoparticles 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 helper lipid, the structural lipid, and the PEG-lipid in the lipid nanoparticles is 46.3: 9.4: 42.7: 1.6.

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

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

[0081] 2) adding sucrose to the replaced solution to obtain a sucrose concentration of 10% to 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 microfluidic mixing method is used to prepare a lipid nanoparticle solution encapsulating mRNA. Specifically, the microfluidic mixing method comprises: dissolving an ionizable lipid or its isomer or pharmaceutically acceptable salt thereof, a helper 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; microfluidically mixing the organic phase and the aqueous phase at a specific volume ratio to form lipid nanoparticles; immediately diluting the solution with a buffer solution; and purifying the solution to remove ethanol to obtain the mRNA-encapsulated lipid nanoparticle solution. Preferably, the acidic buffer solution is a sodium acetate buffer or a citric acid buffer solution having a pH of 4.0-5.0; and the volume ratio of the organic phase to the aqueous phase is 1:2-4. Preferably, the ethanol is purified and replaced with a Tris buffer or HEPES buffer solution having a concentration of 1-20 mM. Preferably, the purification comprises 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 minutes; the primary drying temperature is -40 to -30°C, the pressure is 0.01-0.15 mbar, and the time is 1600-2000 minutes; and the desorption drying temperature is 20-40°C, the pressure is 0.01-0.3 mbar, and the time is 300-800 minutes.

[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 lyophilized lipid nanoparticle formulations of the present invention are reconstituted prior to use. In some embodiments, the lyophilized lipid nanoparticle formulations of the present invention are reconstituted with the same volume of water for injection, physiological saline, or acidic buffer solution as the pre-lyophilized liquid formulation. As demonstrated by the in vivo efficacy validation of Example 7, the in vivo transfection bioactivity of the lyophilized formulations of the present invention was not diminished after reconstitution.

[0087] The nucleic acid drugs of the present invention may contain one or more adjuvants or may be combined with one or more adjuvants. The adjuvant may be 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 herein have the same meanings as commonly understood by those skilled in the art. In addition to the specific methods, devices, and materials used in the examples, any prior art methods, devices, and materials similar or equivalent to those in the examples may be used to implement the present invention, based on the knowledge of the prior art by those skilled in the art and the disclosure of this invention.

[0091] Unless otherwise noted, the experimental methods, detection methods, and preparation methods disclosed herein utilize conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Unless otherwise noted, the materials and equipment used herein are 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 a C1-C10 alkylene group; 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] Example 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 added. 2000 The lipid ethanol solution was prepared by dissolving the lipids in ethanol according to the molar percentage of 46.3:9.4:42.7:1.6, which served as 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 mixed in a volume ratio of 1:3 using a microfluidic device to prepare LNPs. The prepared LNPs were immediately diluted with sodium acetate buffer (pH = 4.7), purified (ultrafiltration / dialysis / TFF) to remove ethanol, and the buffer was replaced with 10 mM Tris (pH = 7.5) to obtain an mRNA-LNP (lipid nanoparticles encapsulating mRNA) solution.

[0106] 2. Preparation of lyophilized preparations

[0107] The lyoprotectant sucrose was added to the mRNA-LNP solution prepared above to obtain a pre-lyophilized liquid preparation. 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 freeze-drying program was set according to the conditions and parameters shown in Table 2 below for freeze-drying.

[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 lyophilization is in the form of a powder cake. When 0.5 mL of water for injection / normal saline is added, the lyophilized powder is quickly reconstituted within 10 seconds. After testing and experiments, the mRNA-LNP after reconstitution of the lyophilized powder maintains a good average particle size distribution, encapsulation efficiency, mRNA integrity and in vivo transfection effect.

[0114] Example 2: Screening of lyoprotectants

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

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

[0117] Table 3 Screening of lyophilization protectants

[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 the sole protective agent showed no significant change in appearance before and after freeze-drying, with all formulations exhibiting a light blue liquid state, demonstrating the Tyndall effect of nanoparticles. The other groups exhibited a light blue liquid state before freeze-drying but became turbid after freeze-drying, indicating aggregation, flocculation, or sedimentation of the nanoparticles.

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

[0122] Using E5 as the ionizable lipid, sucrose (the concentration of sucrose in the preparation was 15% w / v, denoted as 15% Suc) and maltose (the concentration of maltose in the preparation was 15% w / v, denoted as 15% Mal) were added as lyoprotectants. First, mRNA-LNP solutions were prepared according to the prescription and preparation method in Example 1. The average particle size (Z-Ave), polydispersity index (PDI) and encapsulation efficiency (EE) of the LNPs without the addition of the lyoprotectant (i.e., before lyophilization, denoted as CL) and after lyophilization and reconstitution (denoted as DG) of the samples 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-formulation and multi-formulation freeze-dried protective agents

[0127] E5 was used as the ionizable lipid, and the 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), a lyoprotectant of sucrose and poloxamer 188 (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), and an mRNA-LNP solution was prepared according to the prescription and preparation method in Example 1. The Z-Ave, PDI and EE of the LNP without the addition of the lyoprotectant (i.e., before lyophilization, recorded as CL) and after lyophilization and reconstitution (recorded as DG) of the sample with the addition of the above-mentioned lyoprotectant were measured. The results are shown in Table 5.

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

[0129]

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

[0131] Example 3: Screening of sucrose concentration

[0132] Using E5 as the ionizable lipid, mRNA-LNP solutions were prepared according to the prescription and preparation method in Example 1, and the Z-Ave, PDI and EE of the lyophilized preparation samples with the addition of the above lyophilized protective agents were measured before and after lyophilization of the LNPs. 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 sucrose was used as a protective agent at 10%-20% w / v, the key data of mRNA-LNP changed little before and after freeze-drying. A lower dose of 10% w / v was sufficient to achieve the desired effect, thus reducing costs.

[0137] Example 4: Screening of buffer concentration

[0138] Using E5 as the ionizable lipid, 10% sucrose (the concentration of sucrose in the preparation was 10% w / v, denoted as 10% Suc) was added as a lyoprotectant, and 5 mM Tris (pH 7.5), 10 mM Tris (pH 7.5), 20 mM Tris (pH 7.5), and 50 mM Tris (pH 7.5) were used as buffers (i.e., the buffer substance in the lyophilized preparation), respectively. mRNA-LNP solutions were prepared according to the formulation and preparation method in Example 1. The Z-Ave, PDI, and EE of the LNPs of the lyophilized preparation samples prepared above were measured before and after lyophilization. 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 Tris buffer concentration increased from 5mM to 20mM, the key data of mRNA-LNP before and after lyophilization changed little, and the lyophilized preparation with 10mM Tris buffer had the best effect. However, as the Tris buffer concentration increased from 20mM to 50mM, the key data of mRNA-LNP before and after lyophilization changed significantly.

[0142] Using Tris buffer at pH 6.0 as the buffer system, a screening of Tris buffer concentrations revealed that when the Tris buffer concentration was no greater than 10 mM, key mRNA-LNP parameters showed minimal changes before and after lyophilization. The lyophilized formulation using 1 mM Tris buffer achieved the best results. Furthermore, sucrose concentration screening was conducted under this premise, and the lyophilized formulation achieved the best results at a sucrose concentration of 15% w / v.

[0143] Using HEPES buffer at pH 6.0 as the buffer system, screening of HEPES buffer concentrations revealed that 20 mM HEPES buffer provided the best results for the lyophilized formulation. Furthermore, screening of sucrose concentrations under this condition revealed that 10% w / v sucrose provided the best results for the lyophilized formulation.

[0144] Example 5: Screening of LNP concentration

[0145] Using E5 as the ionizable lipid, 10% sucrose (the sucrose concentration in the preparation was 10% w / v, denoted as 10% Suc) as a lyoprotectant, and 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 formulation and preparation method in Example 1. The Z-Ave, PDI, and EE of the LNPs of the lyophilized preparation samples prepared above were measured before and after lyophilization. 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 had 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, with E5, BNT:ALC-0315 in Table 9, and H1 as ionizable lipids, respectively. According to the formulation in Table 10, 10% sucrose (the concentration of sucrose in the preparation is 10% w / v, denoted as 10% Suc) was added as a lyoprotectant, 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 at 0.1 mg / mL. mRNA-LNP solutions with different ionizable lipids were prepared according to the formulation and preparation method in Example 1, and the Z-Ave, PDI, and EE of the LNPs before and after lyophilization of the lyophilized preparation samples prepared above 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, and H1 is the compound disclosed in Chinese patent 202210344395.1 and was prepared by referring to the method in the patent.

[0155] Table 10 mRNA-LNP formulation

[0156]

[0157]

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

[0159]

[0160] As shown in Table 11, the ionizable lipids ALC0315 and H1 formulations and the E5 formulation were prepared with the same buffer composition and the same type and concentration of lyoprotectants. Compared with the two formulations ALC0315 and H1, the E5 formulation had smaller changes in key parameters during the lyophilization process and better stability, indicating that the selection of ionizable lipids has an important influence on the stability of the LNP lyophilized formulation.

[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°C and a 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, E11 and E5 obtained in Example 1 were prepared; the reconstituted samples of each group were injected intramuscularly into the lateral thigh muscle of the mouse at a dose of 150μg / kg mRNA, with two mice and two legs in each group. At a specific time point, 200uL of luciferin potassium substrate (20μg / mL) was injected into the abdominal cavity of the mouse. After 5 minutes, the mouse was placed in a small animal in vivo imaging instrument to measure the fluorescence intensity. The experimental results are shown as follows. Figure 2 shown.

[0163] Depend on Figure 2Quantitative in vivo imaging of the luciferase mRNA-encapsulated lyophilized powders E1, E11, and E5 compared to the corresponding non-lyophilized liquid formulations via intramuscular injection in mice revealed no significant differences. Reconstitution of the lyophilized powders demonstrated no decrease in in vivo transfection bioactivity compared to the non-lyophilized liquid formulations. Simultaneously, the in vivo efficacy of lyophilized formulations prepared with other ionizable lipids in the E-series was validated. These formulations were identical to those in the examples for Groups E1, E11, and E5. Quantitative in vivo imaging of the lyophilized powders via intramuscular injection in mice demonstrated no decrease in in vivo transfection bioactivity following reconstitution.

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

[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, denoted as 10% Suc) was added as a lyoprotectant, 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 lyoprotectant was added to prepare a pre-lyophilized liquid preparation, which was then lyophilized. The resulting lyophilized preparation was stored at 2-8°C, and the Z-Ave, PDI, EE, and mRNA integrity of the LNP were measured at the beginning of storage (0 h), 1 month (1 M), 2 months (2 M), and 3 months (3 M). The results are shown in Table 12.

[0168] Table 12 Long-term stability test results 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 basically stable in physical and chemical properties when stored at 2-8°C for 3 months, and have 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 formulations were prepared using E11 as an ionizable lipid, 10% sucrose (the concentration of sucrose in the formulation was 10% w / v, denoted as 10% Suc) as a lyoprotectant, a HEPES buffer at pH 6.0 and a concentration of 20 mM as a buffer system, and 15% sucrose (the concentration of sucrose in the formulation was 15% w / v, denoted as 15% Suc) as a lyoprotectant, a Tris buffer at pH 6.0 and a concentration of 1 mM as a buffer system. First, an mRNA-LNP solution was prepared according to the formulation and preparation method of Example 1. The Z-Ave, PDI, EE, and mRNA integrity of the LNPs were measured. Then, a lyoprotectant was added to prepare a pre-lyophilized liquid formulation, which was then lyophilized. The Z-Ave, PDI, EE, and mRNA integrity of the lyophilized LNPs 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 lyophilization

[0177]

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

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

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

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

[0182]

[0183]

[0184] As shown in Table 15, the physicochemical properties of the LNP-1mM Tris-15% Suc and LNP-20mM HEPES-10% Suc batches remained largely stable after storage at 2-8°C for 3 months. Z-Ave and EE remained largely stable after storage at 25°C for 3 months for the LNP-1mM Tris-15% Suc and LNP-20mM HEPES-10% Suc batches. mRNA integrity decreased over time but remained above 80% after 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 skilled in 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 one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A lyophilized lipid nanoparticle 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 is selected from the following group: ； The lyoprotectant is sucrose, the buffer substance is a Tris buffer with a pH of 7.5, the concentration of which in the liquid preparation before lyophilization is 10 mM, and the concentration of sucrose in the liquid preparation before lyophilization is 10% w / v; or, the buffer substance is a Tris buffer with a pH of 6.0, the concentration of which in the liquid preparation before lyophilization is 1 mM, and the concentration of sucrose in the liquid preparation before lyophilization is 15% w / v; or, the buffer substance is a HEPES buffer with a pH of 6.0, the concentration of which in the liquid preparation before lyophilization is 20 mM, and the concentration of sucrose in the liquid preparation before lyophilization is 10% w / v; The lipid nanoparticles further include nucleic acid, structural lipid, auxiliary lipid and PEG-lipid; the nucleic acid is mRNA; the structural lipid is cholesterol; the auxiliary lipid is DSPC; the PEG-lipid is PEG 2000 -DMG; the mass ratio of the total mass of lipid molecules to mRNA in the lipid nanoparticles is 10-30:1; the molar percentage of ionizable lipids or their isomers or pharmaceutically acceptable salts, auxiliary lipids, structural lipids and PEG-lipids in the lipid nanoparticles is 46.3-65:9.4-40:15-42.7:1-1.

6.

2. The lyophilized lipid nanoparticle preparation according to claim 1, wherein The average particle size of the lipid nanoparticles ranges from 60 to 300 nm.

3. The method for preparing the lyophilized lipid nanoparticle preparation according to claim 1 or 2, wherein: The steps include: 1) preparing a lipid nanoparticle solution encapsulating mRNA using the ionizable lipid or its isomer or pharmaceutically acceptable salt as a component raw material, and replacing the buffer in the solution with a Tris buffer having a concentration of 1 mM or 10 mM, or a HEPES buffer having a concentration of 20 mM, to obtain a replaced solution; 2) adding sucrose to the replaced solution to obtain a sucrose concentration of 10% w / v or 15% w / v to obtain a liquid preparation; 3) Freeze-drying the liquid preparation to obtain a freeze-dried preparation.

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

5. The method for preparing the lyophilized lipid nanoparticle preparation according to claim 4, wherein: In step 1), while ethanol is purified and removed, the buffer in the replacement solution is a Tris buffer with a concentration of 1 mM or 10 mM, or a HEPES buffer with a concentration of 20 mM.

6. Use of the lyophilized lipid nanoparticle preparation according to claim 1 or 2 or the lyophilized lipid nanoparticle preparation prepared by the preparation method according to any one of claims 3 to 5 in the preparation of nucleic acid drugs.

Images