A nucleic acid-lipid nanoparticle freeze-dried composition and its preparation method and application

Abstract

The present invention provides a nucleic acid-lipid nanoparticle freeze-dried composition and a preparation method and application thereof. The present invention belongs to the technical field of pharmaceutical preparations. The nucleic acid-lipid nanoparticle freeze-dried composition includes nucleic acid-lipid nanoparticles and a freeze-drying protective agent. The nucleic acid-lipid nanoparticles include nucleic acids and lipid nanoparticles. The lipid nanoparticles include cationic lipids, neutral lipids, auxiliary lipids and polymer conjugated lipids. The cationic lipids include a combination of ionizable cationic lipids and permanently charged cationic lipids. The nucleic acid-lipid nanoparticle freeze-dried composition product of the present invention has high stability and can be stably stored at 4°C for more than 3 months. In addition, the particle uniformity before and after freeze-drying is good, the encapsulation rate and mRNA integrity are excellent, and the nucleic acid before and after freeze-drying can be fully guaranteed not to be degraded; the water content is low, the re-dissolution time is short, and the physical and chemical properties are excellent.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations and relates to a nucleic acid-lipid nanoparticle freeze-dried composition and a preparation method and application thereof. Background Art

[0002] mRNA vaccines introduce mRNA (messenger ribonucleic acid) containing gene sequences encoding antigenic proteins into body cells through a specific delivery system. By expressing the target protein in vivo, the body is stimulated to produce specific immunological responses, which can achieve dual immunity of humoral and cellular immunity with high effectiveness. With the clinical safety and protective efficacy of mRNA vaccines from Moderna and BioNtech verified, mRNA vaccine technology has received widespread attention. The mRNA-LNP (mRNA-lipid nanoparticle) drug carriers that have been marketed are composed of ionizable cationic lipids, neutral lipids, cholesterol and PEG (polyethylene glycol) modified lipids. Ionizable cationic lipids carry positive charges at acidic pH and electrostatically interact with negatively charged nucleic acids to encapsulate nucleic acids. They become neutrally charged at physiological pH and fuse with negatively charged endosomal lipids, thereby releasing nucleic acids into the cytoplasm.

[0003] Permanently charged cationic lipids are a series of amphiphilic compounds consisting of a cationic head group conjugated to one or more hydrophobic fatty acid tail groups with different linkers. The cationic head group usually contains one or more protruding amino groups, such as quaternary ammonium salts, other charged amino groups, guanidine or heterocyclic compounds, which make the whole compound positively charged at physiological pH. The cationic head group of the permanently charged cationic lipid adsorbs nucleic acids through electrostatic adsorption, and its amphiphilic structure helps in the preparation of liposomes or lipid nanoparticles for therapeutic gene delivery. Among them, nanoliposomes of the permanently charged cationic lipid DOTAP ((2,3-dioleoyl-propyl)-trimethylammonium chloride) can deliver mRNA encoding antigens to prevent infectious diseases, and can form polymer hybrid nanoparticles to deliver mRNA molecules to treat cancer and genetic diseases.

[0004] Currently, the mRNA-LNP vaccines on the market have poor long-term stability and need to be stored at ultra-low temperatures (-20°C or -70°C). The transportation and storage stability of vaccines have become the main problems in their application. It is urgent to develop formulations with good long-term stability and long-term stability at low temperatures of 4°C and room temperature, which are convenient for transportation. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a nucleic acid-lipid nanoparticle freeze-dried composition and a preparation method and application thereof.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] On the one hand, the present invention provides a nucleic acid-lipid nanoparticle lyophilized composition, which comprises nucleic acid-lipid nanoparticles and a lyoprotectant, wherein the nucleic acid-lipid nanoparticles comprise nucleic acids and lipid nanoparticles, wherein the lipid nanoparticles comprise cationic lipids, neutral lipids, auxiliary lipids and polymer-conjugated lipids (also referred to as conjugated lipids), and wherein the cationic lipids comprise a combination of ionizable cationic lipids and permanently charged cationic lipids.

[0008] In the present invention, by using a combination of ionizable cationic lipids and permanently charged cationic lipids as cationic lipids, neutral lipids, auxiliary lipids (cholesterol) and polymer conjugated lipids to form nucleic acid-lipid nanoparticles, the freeze-dried composition product can be highly stable and can be stably stored at 4°C for more than 3 months. In addition, the particle uniformity before and after freeze-drying is good, the encapsulation rate and mRNA integrity are excellent, and the nucleic acid before and after freeze-drying can be fully guaranteed not to be degraded; the water content is low, the re-dissolution time is short, and the physical and chemical properties are excellent.

[0009] Preferably, the molar ratio of the ionizable cationic lipid, the permanently charged cationic lipid, the neutral lipid, the auxiliary lipid and the polymer conjugated lipid is (5-65):(5-65):(5-15):(35-45):(1-5), within the molar ratio range, the proportion of the ionizable cationic lipid 5-65 can be 5, 6, 8, 9, 10, 15, 18, 20, 23, 25, 28, 30, 33, 35, 38, 40, 42, 45, 48, 50, 52, 55, 58, 60, 63 or 65, etc., and the proportion of the permanently charged cationic lipid 5-65 can be 5, 6 , 8, 9, 10, 15, 18, 20, 23, 25, 28, 30, 33, 35, 38, 40, 42, 45, 48, 50, 52, 55, 58, 60, 63 or 65, etc., the neutral lipid ratio range 5-15 can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, etc., the auxiliary lipid ratio range 35-45 can be 35, 36, 37, 38, 39, 30, 41, 42, 43, 44 or 45, and the polymer conjugated lipid ratio range 1-5 can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5, etc.

[0010] Preferably, the molar ratio of the ionized cationic lipid to the permanently charged cationic lipid is 1:9 to 9:1, for example 1:9, 1.5:8.5, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 or 9:1, etc., preferably 2:8 to 8:2, further preferably 3:7 to 7:3, and further preferably 5:5, i.e. 1:1.

[0011] Preferably, the ionizable cationic lipid is selected from one or a combination of at least two of YK-009, YK-401, YK305, ALC0315, SM102, and DLIN-MC3-DMA, wherein:

[0012] YK-009 has the structure shown in formula (II):

[0013]

[0014] YK-401 has the structure shown in formula (II-I):

[0015]

[0016] YK-305 has the structure shown in formula (IV-I):

[0017]

[0018] ALC0315 has the structure shown in formula (VI):

[0019]

[0020] SM102 has the structure shown in formula (VI-I):

[0021]

[0022] DLIN-MC3-DMA has the structure shown in formula (VII):

[0023] .

[0024] Preferably, the permanently charged cationic lipid is selected from one or a combination of at least two of (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP); (2,3-dioleoyl-propyl)-trimethylammonium and its salts; 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) or didodecyldimethylammonium bromide (DDAB).

[0025] Preferably, the neutral lipid is selected from one or more of the following substances: dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE) or a mixture thereof; preferably distearoylphosphatidylcholine (DSPC).

[0026] Preferably, the helper lipid is selected from cholesterol, vitamin E, DC-cholesterol or derivatives thereof, preferably cholesterol.

[0027] Preferably, the polymer-conjugated lipid is a lipid modified with polyethylene glycol (PEG).

[0028] Preferably, the polyethylene glycol-modified lipid is selected from any one or a combination of at least two of distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000) or methoxy polyethylene glycol ditetradecanoyl acetamide (ALC-0159).

[0029] Preferably, the lyoprotectant is selected from any one of sucrose, trehalose or mannitol, or a combination of at least two of them.

[0030] Preferably, the nucleic acid is selected from any one or a combination of at least two of small interfering RNA, asymmetric interfering RNA, micro RNA, Dicer-substrate RNA, small hairpin RNA, mRNA, long non-coding RNA, circular RNA, self-amplifying RNA or 5'-pppRNA.

[0031] The sequence of the mRNA used in the present invention is shown as SEQ ID No.1.

[0032] Preferably, the mass ratio of the lipid nanoparticles to the nucleic acid is 10-30:1, for example, 10:1, 13:1, 15:1, 18:1, 20:1, 22:1, 25:1, 28:1 or 30:1.

[0033] In another aspect, the present invention provides a method for preparing the above-mentioned nucleic acid-lipid nanoparticle freeze-dried composition, the preparation method comprising the following steps:

[0034] The freeze-dried protective agent solution is mixed with the nucleic acid-lipid nanoparticle solution to obtain a mixed solution to be freeze-dried, and freeze-dried to obtain the nucleic acid-lipid nanoparticle freeze-dried composition.

[0035] Preferably, the mass percentage of the lyoprotectant in the lyoprotectant solution is 5% to 20%, for example 5%, 6%, 8%, 10%, 12%, 15%, 18% or 20%.

[0036] Preferably, the solvent in the lyoprotectant solution is enzyme-free water or a Tris buffer or DPBS buffer with a pH value of 5-9 (eg, 5, 6, 7, 8 or 9).

[0037] Preferably, the mass percentage content of nucleic acid and lipid nanoparticles in the nucleic acid-lipid nanoparticle solution is 0.1% to 1%, for example, 0.1%, 0.3%, 0.5%, 0.8% or 1%.

[0038] Preferably, the solvent in the nucleic acid-lipid nanoparticle solution is a Tris buffer or a DPBS buffer with a pH value of 5-9 (eg, 5, 6, 7, 8 or 9).

[0039] Preferably, the mixed solution to be lyophilized is filled into a 2 mL sample bottle with a filling volume of 0.5-1.5 mL (eg, 0.5 mL, 1.0 mL or 1.5 mL) for freeze drying.

[0040] In the present invention, the sample bottle is a vial.

[0041] Preferably, the freeze drying comprises the following steps: a pre-freezing stage, a sublimation drying stage, and a desorption drying stage.

[0042] Preferably, liquid nitrogen is used for cooling in the pre-freezing stage.

[0043] Preferably, the liquid nitrogen cooling time is 30 min to 6 h, for example, 30 min, 45 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h or 6 h.

[0044] Preferably, the temperature in the sublimation drying stage is -40~0°C (for example, -40°C, -30°C, -20°C, -10°C, -5°C or 0°C), maintained for 50~100h (for example, 50h, 55h, 58h, 60h, 65h, 70h, 75h, 80h, 85h, 90h, 95h or 100h), and the temperature is increased in a gradient manner in 2~4 sections (for example, 2, 3 or 4 sections), each section is increased by 5~20°C (for example, 5°C, 8°C, 10°C, 13°C, 15°C, 18°C ​​or 20°C), and each section is maintained for 6~70h (for example, 6h, 8h, 10h, 15h, 20h, 25h, 30h, 35h, 40h, 45h, 50h, 55h, 60h, 65h or 70h).

[0045] Further preferably, the temperature rising process in the sublimation drying stage is: -40°C for 50h, -35°C for 12h, -15°C for 7h, and 0°C for 6h.

[0046] Preferably, the temperature of the desorption drying stage is 15-30°C (e.g., 15°C, 18°C, 20°C, 23°C, 25°C, 28°C or 30°C), and the operation time is 2-5h (e.g., 2h, 2.5h, 3h, 3.5h, 4h, 4.5h or 5h).

[0047] Preferably, during the freeze-drying process, the vacuum pressure is maintained at ≤0.05 mbar. For example, the vacuum pressure can be controlled at 0.05 mbar, 0.03 mbar, 0.01 mbar, 0.008 mbar, 0.005 mbar, 0.003 mbar, 0.001 mbar, etc.

[0048] In another aspect, the present invention provides use of the above-mentioned lyophilized nucleic acid-lipid nanoparticle composition in the preparation of nucleic acid drugs.

[0049] In another aspect, the present invention provides use of the above-described lyophilized nucleic acid-lipid nanoparticle composition in the preparation of a medicament for treating a disease or disorder in a mammal in need thereof.

[0050] Preferably, the disease or disorder is selected from the group consisting of infectious diseases, proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0051] Preferably, the disease is cancer.

[0052] Preferably, the disease is diabetes.

[0053] Preferably, the infectious disease is selected from: diseases caused by coronavirus, influenza virus or HIV virus, childhood pneumonia, Rift Valley fever, yellow fever, rabies or various herpes.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] The nucleic acid-lipid nanoparticle freeze-dried composition product of the present invention has high stability and can be stably stored at 4°C for more than 3 months. In addition, the particle uniformity before and after freeze-drying is good, the encapsulation rate and mRNA integrity are excellent, and the nucleic acid before and after freeze-drying can be fully guaranteed not to be degraded; the water content is low, the re-dissolution time is short, and the physical and chemical properties are excellent. DETAILED DESCRIPTION

[0056] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0057] Example 1

[0058] 1) Preparation of mRNA stock solution:

[0059] The mRNA transcribed from the DNA encoding the SARS-CoV-2 spike protein (S protein) was used as a template as the model mRNA. The preparation process was as follows:

[0060] (i) Preparation of mRNA

[0061] 1-1) Thawing of reagents

[0062] a) Thaw 10× transcription buffer and four ribonucleotides (ATP, CTP, GTP, TTP) at room temperature, mix thoroughly, and centrifuge briefly; keep 10× transcription buffer at room temperature and the four ribonucleotides on ice for later use.

[0063] b) Mix T7 RNA polymerase, inorganic pyrophosphatase and mouse RNase inhibitor separately, centrifuge briefly, and place on ice for later use.

[0064] 1-2) Transcription

[0065] If co-transcription capping is selected, prepare the co-transcription capping system according to Table 1 below:

[0066] Table 1 Co-transcriptional capping system

[0067]

[0068] Note: DNA template needs to be added later. Since 10× transcription buffer contains spermidine, too high spermidine concentration will cause DNA template precipitation.

[0069] The reagent information used is as follows:

[0070] RNase-free H2O, manufacturer: Solarbio Life Sciences; catalog number: R1600;

[0071] Transcription buffer, manufacturer: Yisheng Biotechnology (Shanghai) Co., Ltd.;

[0072] Cytidine triphosphate solution CTP Solution (specification 100 mM), manufacturer: Yisheng Biotechnology (Shanghai) Co., Ltd.;

[0073] Guanosine triphosphate solution GTP Solttion (specification 100mM), manufacturer: Yisheng Biotechnology (Shanghai) Co., Ltd.;

[0074] Adenosine triphosphate solution ATP Solttion (specification 100mM), manufacturer: Yisheng Biotechnology (Shanghai) Co., Ltd.;

[0075] Uridine triphosphate solution TTP Solttion (specification 100mM), manufacturer: Yisheng Biotechnology (Shanghai) Co., Ltd.;

[0076] Cap1-GAGm7G(5')ppp(5')(2'0MeA)pG (specification 100mM), manufacturer: Yisheng Biotechnology (Shanghai) Co., Ltd.;

[0077] Template DNA: Linearized plasmid DNA of S protein of Omicron variant SARS-CoV-2 strain, manufacturer: GenScript Biotech Co., Ltd.;

[0078] T7 RNA polymerase, manufacturer: Yisheng Biotechnology (Shanghai) Co., Ltd.;

[0079] Inorganic pyrophosphatase, manufacturer: Yisheng Biotechnology (Shanghai) Co., Ltd.;

[0080] Mouse RNase inhibitor; Manufacturer: Yisheng Biotechnology (Shanghai) Co., Ltd.;

[0081] 1-3) Incubation

[0082] The above reaction solutions were mixed evenly, centrifuged briefly, and incubated at 37°C for 2 h.

[0083] 1-4) DNase I treatment

[0084] After the transcription reaction was completed, 1 μL DNase I (RNase-free) was added to each tube, mixed, centrifuged briefly, and incubated at 37°C for 30 min to remove the template DNA.

[0085] 1-5) mRNA purification

[0086] Purification by lithium chloride precipitation

[0087] i) To 20 μL of reaction mixture, add 30 μL of RNase-free H2O and 30 μL of 7.5 M LiCl.

[0088] ii) After mixing, place the mixture at -20°C for at least 30 min, centrifuge at maximum speed at 4°C for 15 min, and collect the precipitate.

[0089] iii) Add 500 μL of ice-cold 70% ethanol to wash the RNA pellet.

[0090] iv) Dissolve the mRNA precipitate in 20 μL RNase-free H2O. The purified mRNA (whose sequence is shown in SEQ ID No. 1) solution was stored at -80°C.

[0091] 2) Organic phase:

[0092] The organic phase is an anhydrous ethanol solution of cationic lipids (two components), neutral lipids, auxiliary lipids and conjugated lipids, and the concentration of the solution is 20 mg / ml.

[0093] a) cationic lipids consist of YK-009 (ionizable cationic lipid) and DOTAP (permanently charged cationic lipid) accounting for 49% of the total lipids, wherein the molar ratio of YK-009 to DOTAP is 1:1;

[0094] b) The molar ratio of neutral lipid DSPC to total lipids is 10%;

[0095] c) The molar ratio of auxiliary lipid cholesterol to total lipids is 39.5%;

[0096] d) The molar ratio of conjugated lipid DMG-PEG2000 to total lipids was 1.5%.

[0097] Among them, the lipid source information is as follows:

[0098] Ionizable cationic lipid; YK-009 (prepared with reference to CN114044741A);

[0099] Permanently charged cationic lipids; (2,3-dioleoyl-propyl)-trimethylammonium chloride DOTAP (purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.);

[0100] Neutral lipids: distearoylphosphatidylcholine DSPC (purchased from Nippon Seika Co., Ltd.);

[0101] Auxiliary lipids: cholesterol (purchased from Nippon Seika Co., Ltd.);

[0102] Conjugated lipid: DMG-PEG2000 (purchased from Xiamen Sinopang Biotechnology Co., Ltd.).

[0103] Among them, the structural formula of YK009 is as follows:

[0104]

[0105] Among them, the DOTAP structure is as follows:

[0106]

[0107] 3) Aqueous phase: Dissolve the mRNA stock solution in pH 4.0 citrate buffer to prepare the aqueous phase;

[0108] 4) Encapsulation:

[0109] The organic phase and the aqueous phase were quickly mixed in a microfluidic device (manufacturer: Precision NanoSystems) for encapsulation. The encapsulation method was that the ratio of the organic phase and the aqueous phase inflow rate was 1:3, the total inflow flow rate was 12mL / min, and the chip model was Ignite NxGen. After encapsulation, the sample was placed in a dialysis bag (source leaf, 50KD) and dialyzed overnight in Tris buffer to obtain an mRNA-LNP sample.

[0110] 5) Preparation of freeze-drying protective agent: Prepare freeze-drying protective agent solution by mixing sucrose solution in nuclease-free water.

[0111] 6) Preparation of mRNA-LNP freeze-dried preparation using freeze-drying protective agent

[0112] The freeze-drying protective agent (i.e., freeze-drying protective agent) solution prepared in step 4) is added to the mRNA-LNP sample in step 3) to obtain a mixed solution to be freeze-dried, so that the mass concentration of sucrose in the mixed solution to be freeze-dried is 10%, and then divided into vials, 0.5 mL per vial, and freeze-dried.

[0113] Freeze drying process

[0114] Pre-freezing stage: liquid nitrogen freezing for 3h

[0115] Sublimation drying stage: -45℃50h, -35℃12h, -15℃7h, 0℃6h

[0116] Analysis and drying stage: 15℃2h, 30℃2h

[0117] The whole process vacuum pressure control is ≤0.05mbar.

[0118] Example 2

[0119] In this example, the molar ratio of the ionizable cationic lipid to the permanently charged cationic lipid was varied (as shown in Table 2).

[0120] On the basis of Example 1, the molar ratio of cationic lipids (two components) to total lipids remained unchanged at 49%, the molar ratio of YK009 to DOTAP was adjusted to 1:1 as shown in the following table, and the other steps were consistent with Example 1.

[0121] Table 2 Molar ratio of cationic lipid YK009 and DOTAP

[0122]

[0123] Example 3

[0124] In this example, the types of ionizable cationic lipids were changed (as shown in Table 3).

[0125] On the basis of Example 1, the molar ratio of ionizable cationic lipid to permanently charged cationic lipid remained unchanged at 1:1, YK009 was replaced with the ionizable cationic lipid in the following table, and the other steps were consistent with Example 1.

[0126] Table 3 Types of ionizable cationic lipids

[0127]

[0128] Example 4

[0129] In this example, the types of permanently charged cationic lipids were varied (as shown in Table 4).

[0130] On the basis of Example 1, the molar ratio of ionizable cationic lipid to permanently charged cationic lipid remained unchanged at 1:1, DOTAP was replaced with the permanently charged cationic lipid in the following table, and the other steps were consistent with Example 1.

[0131] Table 4 Types of permanently charged cationic lipids

[0132]

[0133] Example 5

[0134] In this embodiment, the sublimation drying procedure was changed.

[0135] 1. Influence of sublimation drying method

[0136] On the basis of Example 1, the sublimation drying in Part 3) "Freeze Drying Procedure" "-45°C 50h, -35°C 12h, -15°C 7h, 0°C 6h" is replaced by:

[0137] Example 5-1: -40°C for 50h, -30°C for 12h, -15°C for 7h, 0°C for 6h;

[0138] Example 5-2: -35°C for 50h, -25°C for 12h, -10°C for 7h, 0°C for 6h;

[0139] Example 5-3: -35°C for 63h, -15°C for 12h;

[0140] Example 5-4: -35°C for 75h;

[0141] Example 5-5: -25°C for 75h;

[0142] The other ingredients and freeze-drying steps were the same as those in Example 1.

[0143] 2. Influence of sublimation drying time

[0144] On the basis of Example 1, the sublimation drying in Part 3) "Freeze Drying Procedure" "-45°C 50h, -35°C 12h, -15°C 7h, 0°C 6h" is replaced by:

[0145] Example 5-6: -45°C for 20h, -35°C for 12h, -15°C for 7h, 0°C for 6h;

[0146] Example 5-7: -45°C for 2h, -35°C for 2h, 0°C for 2h;

[0147] The other ingredients and freeze-drying steps were the same as those in Example 1.

[0148] Example 6

[0149] In this embodiment, the pre-freezing process is changed (as shown in Table 5).

[0150] The freeze-dried sample preparation method is the same as in Example 1, except that the liquid nitrogen freezing in the pre-freezing process is replaced by the process listed in the table below, and the other components and freeze-drying steps remain the same as in Example 1.

[0151] Table 5 Pre-freezing process

[0152]

[0153] Example 7

[0154] In this example, the filling volume was changed (as shown in Table 6).

[0155] The freeze-dried sample preparation method is the same as in Example 1. 1.0 ml and 1.5 ml are filled in the vials respectively. The total freeze-drying time of 0.5 ml is 82 h. Other filling preparation methods, pre-freezing processes and analytical drying processes are consistent with those in Example 1. Only the sublimation drying time is extended on the basis of Example 1. Specific information is shown in the table below;

[0156] Table 6 Filling volume

[0157]

[0158] Comparative Example 1

[0159] The sample (mRNA-LNP (YK009)) was prepared according to the single-component cationic lipid composition YK009: DSPC: cholesterol: DMG-PEG2000 with a molar ratio of 49:10:39.5:1.5, and freeze-dried according to the freeze-drying process of Example 1 of the present invention.

[0160] Comparative Example 2

[0161] According to the single-component cationic lipid composition ALC0315: DSPC: cholesterol: DMG-PEG2000, the molar ratio is 49:10:39.5:1.5, and freeze-drying is performed according to the freeze-drying process of Example 1 of the present invention.

[0162] Experimental Example 1

[0163] In this experiment, the physical and chemical properties of

[0164] 1-1 The mRNA-LNP samples and their lyophilized preparations were tested as follows:

[0165] 1) Particle size and particle size dispersion index (PDI): Particle size and PDI were measured using dynamic light scattering (DLS) in a nano-laser particle size analyzer (manufacturer: MALVERN; model: ZST3305) at 25±1°C. 60 μL of sample was taken and placed in a quartz micro-sample cell for testing. The test parameters were set as shown in Table 7 below:

[0166] Table 7 Detection parameters

[0167]

[0168] Particle size and PDI are important features to characterize liposomes. There is a liposome fusion process during the freeze-thaw process. Small-sized liposomes fuse into liposomes with slightly larger particle sizes. As the number of freeze-thaw cycles increases, the liposome particle size will increase. Within a certain range, the smaller the particle size, the smaller the change in particle size after freeze-drying, indicating that the product quality and process are better. The dispersion coefficient is the distribution of particle size. The smaller the dispersion coefficient, the more uniform the liposome particle size.

[0169] 2) Encapsulation efficiency: Dilute the sample to 2.8μg / mL, mix a portion with an equal volume (50μL) of TritonX-100, fully break the emulsion, and use it to determine the total mRNA concentration. Mix the other portion with an equal volume (50μL) of TE (Tris-EDTA) to determine the concentration of free, unencapsulated mRNA. After incubating the samples with TE (Tris-EDTA) and TritonX-100 at 37°C for a certain period of time, add 100μL of Ribogreen reagent diluted 200 times, centrifuge to remove bubbles, and use a multifunctional microplate reader to measure the fluorescence value at an excitation wavelength of 485nm and an emission wavelength of 528nm to read the plate.

[0170] 3) Integrity: Extract mRNA from mRNA-LNP using a kit, denature at 65°C after dilution, and place on ice after the test sample is prepared. In addition, prepare gel buffer, add the prepared test sample solution, gel buffer and RNase-free water to the sample plate in sequence, and determine the sample mRNA integrity spectrum using the PA800Plts gel electrophoresis principle.

[0171] 4) Water content: Take about 300 mg of the sample to be tested, weigh it accurately, and measure it with a Karl Fischer moisture meter V30s according to the water content determination method (General Rules 0832 Method 1 of Part 4 of the 2020 edition of the Chinese Pharmacopoeia). Perform parallel measurements on two samples and calculate based on the average water content.

[0172] Moisture content is a key indicator for evaluating the quality of freeze-dried products and also a key indicator for evaluating the freeze-drying process. High moisture content indicates that the freeze-drying process has failed to remove moisture from the product, and high moisture content will lead to reduced product stability.

[0173] 5) Reconstitution time: Add 0.5 mL of nuclease-free water to the freeze-dried sample for reconstitution. Observe and record the time required for reconstitution.

[0174] Reconstitution time is also an indicator for evaluating product quality and freeze-drying process. The shorter the reconstitution time, the more conducive it is to clinical use.

[0175] 1-2 Stability test

[0176] The freeze-dried preparations obtained in the above embodiment were placed at 2-8°C, room temperature and 40°C for 3 months respectively, and the particle size, PDI, encapsulation efficiency and integrity of the preparation samples at 0 days, 14 days, 1 month, 2 months and 3 months were tested to measure their stability.

[0177] Experimental Example 2

[0178] In this experimental example, biological characteristics were detected

[0179] Transfection efficiency experiment:

[0180] First, 293T cells were plated in a 24-well plate one day in advance. The next day, 200 ng of the test sample was added to the culture wells where the cells were plated the day before, and the culture was continued for 18 to 24 hours. The cells were then collected to extract total protein, and the protein samples were separated by SDS-PAGE (polyacrylamide gel electrophoresis) and electrotransferred to a solid phase carrier (nitrocellulose membrane).

[0181] The protein on the solid phase carrier is used as the antigen, and an immune reaction is carried out with the specific antibody (primary antibody) against the new coronavirus S protein, and then reacted with the secondary antibody labeled with horseradish peroxidase. The substrate is added for color development and detected by a multifunctional imager. The intensity of the band can reflect the expression level of the specific S protein expressed by the test preparation.

[0182] Conclusion Analysis 1: Molar Ratio of Ionizable Cationic Lipids to Permanently Charged Cationic Lipids

[0183] In Example 1 and Example 2, lipid compositions of different molar ratios of ionizable cationic lipid YK009 and permanently charged cationic lipid DOTAP were used for encapsulation, and 10% sucrose was used as a freeze-drying protective agent for freeze drying to investigate the effect on the freeze-drying effect of the samples. The lipid molar ratios of each example in the following table are: cationic lipid: DSPC: Chol (cholesterol): DMG-PEG2000 = 49: 10: 39.5: 1.5, wherein the cationic lipids are YK009 and DOTAP, and the specific molar ratios are as follows:

[0184] Table 8 Molar ratio of cationic lipid YK009 and DOTAP

[0185]

[0186] The results of measuring the particle size, PDI, encapsulation efficiency, integrity, moisture content, and reconstitution time of the mRNA-LNP sample and its lyophilized preparation (at room temperature of 25°C, 0 days after lyophilization) are shown in the following table:

[0187] Table 9 Comparison of various detection indexes of cationic lipid composition preparations

[0188]

[0189] Conclusion:

[0190] 1) Particle size and particle size distribution coefficient PDI:

[0191] In Examples 2-1 to 2-3, the molar ratio of YK009 > DOTAP, which are 1:9, 2:8, and 3:7 respectively. As the proportion of YK009 decreases, the particle size after lyophilization changes from 25.9 nm to 14.3 nm, and the change in PDI after lyophilization is relatively small. In Examples 2-4 to 2-6, the molar ratio of YK009 < DOTAP, which are 7:3, 8:2, and 9:1 respectively. The particle size after lyophilization increases from 13.6 nm to 26.4 nm, and the change in PDI after lyophilization is relatively small. Compared with Example 1, in Example 1, the molar ratio of YK009:DOTAP is 1:1, the particle size before lyophilization is the smallest at 70.6 nm, the change in particle size after lyophilization is the smallest at 9.6 nm < 10 nm, the change in PDI is also relatively small, and the particle size after lyophilization has good uniformity.

[0192] The experimental results show that for different ratios of YK009 and DOTAP in the organic phase in Examples 2-1 to 2-6, as the ratio approaches a single organic phase, the particle size after lyophilization shows an increasing trend. When the ratio is 1:1, the change in particle size after lyophilization is the smallest, with a change < 10 nm, and the particle size after lyophilization is only 80.2 nm, and the changes in particle size and PDI after lyophilization are the smallest.

[0193] 2) Encapsulation efficiency:

[0194] In Example 2-6, the molar ratio of YK009:DOTAP is 9:1, and DOTAP in the organic phase is the main cationic lipid, which plays a major role in mRNA encapsulation. At this time, the encapsulation efficiency before lyophilization is significantly lower than that of other batches at 95.0%, and the encapsulation efficiency of the mRNA-LNP after lyophilization decreases significantly, with a change of 8.9%. This indicates that the proportion of unencapsulated and unprotected mRNA is too high. Since mRNA is extremely unstable, the unencapsulated mRNA is easily degraded.

[0195] In Example 1 and Example 2-1 to Example 2-5, the molar ratio of YK009 to DOTAP is 1~8:9~2, and the encapsulation rates before lyophilization are all >95%, and the encapsulation rates after lyophilization are all >89%, and the encapsulation rates are all high. Among them, the encapsulation rate before and after lyophilization decreased the least when the ratio was 1:1, with a change of 5.3%. The encapsulation rate after lyophilization was the highest at 93.0%. The higher the encapsulation rate, the greater the proportion of mRNA encapsulated by liposomes, the smaller the proportion of naked mRNA, and the less mRNA degradation occurs.

[0196] The experimental results show that the encapsulation rate decreases the least before and after freeze-drying when the ratio is 1:1, the encapsulation rate is the highest before and after freeze-drying, and the mRNA is the most stable and not easily degraded.

[0197] 3) Completeness:

[0198] The integrity of the samples prepared in each batch of examples decreased slightly after lyophilization. The molar ratios of the mixed lipids YK009 and DOTAP in Example 2-1 and Example 2-6 were 1:9 and 9:1, respectively. YK009 or DOTAP was the main cationic lipid in the organic phase, which played a major role in mRNA encapsulation. At this time, the integrity before lyophilization was significantly lower than that of other batches, and the mRNA integrity decreased significantly after lyophilization.

[0199] The integrity of Example 1, Example 2-2 to Example 2-5 changed slightly before and after freeze-drying, only decreasing by 0.2% to 0.9%, among which Example 1 had the best integrity, decreasing by only 0.2%.

[0200] The experimental results show that the mRNA integrity of the nanolipid particle freeze-dried preparation prepared with cationic lipids at a molar ratio of YK009:DOTAP of 1:1 has the smallest change.

[0201] 4) Water content and reconstitution time:

[0202] The moisture content of the freeze-dried samples prepared in each batch of the examples did not exceed 3%, and the reconstitution time was short, <10s.

[0203] Example 1 A nanolipid particle freeze-dried preparation was prepared using a cationic lipid with a molar ratio of YK009:DOTAP of 1:1, which had the lowest moisture content of 0.878% and a short reconstitution time of <10s.

[0204] From the above results, it can be seen that the closer the molar ratio of Example 2-1 and Example 2-6 is to the single cationic lipid YK009 or DOTAP, the larger the particle size, the lowest encapsulation efficiency and integrity. The freeze-dried preparations of Example 1, Example 2-2 to Example 2-5 have the smallest change in particle size, the highest encapsulation efficiency and integrity, the lowest water content, the shortest reconstitution time, and the best freeze-drying effect.

[0205] summary:

[0206] Compared with the mRNA-LNP freeze-dried preparations prepared by adding permanently charged cationic lipids at a ratio of 1:9 and 9:1, the addition of permanently charged cationic lipids at a ratio of 2:8 to 8:2 can significantly improve the particle size and uniformity, encapsulation efficiency, integrity, moisture content, and reconstitution effect of the freeze-dried preparations;

[0207] Among them, DOTAP with a cationic lipid addition ratio of 1:1 in the nanoparticle lipid was used as the membrane material of mRNA-LNP, and the sample solution was freeze-dried, which had the best effect. Specifically:

[0208] The mRNA-LNP sample was a mixture of YK009 and DOTAP in a ratio of 1:1 as the lipid material for encapsulating nucleic acid, and freeze-dried to prepare a lyophilized preparation (Example 1). The particle size increased by 9.6 nm, the PDI did not change significantly, the encapsulation efficiency decreased by 5.3%, the integrity decreased by 0.2%, and the water content was minimal, about 0.9%, and the reconstitution time was <10 s;

[0209] The mRNA-LNP lyophilized preparations were prepared with the molar ratio of YK009:DOTAP of 9:1 and 1:9 in the mRNA-LNP samples. The particle size changed by about 26 nm after lyophilization, the PDI did not change significantly, the encapsulation efficiency changed slightly by 6.4%~8.9% compared with other batches, the integrity changed slightly by about 1.4% compared with other batches, the moisture content was not much different from other batches, all <2%, and the reconstitution time was <10s.

[0210] The mRNA-LNP samples were added with permanent charged cationic lipids in a ratio of 2:8~8:2 to prepare mRNA-LNP freeze-dried preparations. After freeze-drying, the particle size changed from 9.6nm to 19.3nm, the particle size changed by <20nm, the PDI did not change significantly, the encapsulation rate changed by 5.3%~6.6%, the encapsulation rate decreased slightly after freeze-drying, the integrity changed by 0.2%~0.9%, and the mRNA-LNP integrity was good after freeze-drying, indicating that the lipids in this prescription ratio had a good protective effect on mRNA during the freeze-drying process. The water content was not much different from other batches, all <2%, and the reconstitution time was all <10s.

[0211] Conclusion Analysis 2: Types of Ionizable Cationic Lipids

[0212] Example 3 Different types of ionizable cationic lipids and permanently charged cationic lipid DOTAP were used to encapsulate mRNA in a 1:1 molar ratio of cations, and freeze-dried with 10% sucrose as a lyophilization protectant. The lipid ratios of each example in the following table are: cationic lipid: DSPC: Chol: DMG-PEG2000 = 49: 10: 39.5: 1.5, wherein the cationic lipids are the lipids listed in the following table and the molar ratio of DOTAP is 1: 1, and the types of ionizable cationic lipids are shown in the following table:

[0213] Table 10 Ionizable cationic lipid types

[0214]

[0215] The results of testing the particle size, PDI, encapsulation efficiency, integrity, moisture content and reconstitution time of the mRNA-LNP samples and their lyophilized preparations (room temperature 25°C, 0 days after lyophilization) are shown in the following table:

[0216] Table 11 Comparison of various test indicators of ionizable lipid types and preparations

[0217]

[0218] in conclusion:

[0219] 1) Particle size and particle size distribution coefficient PDI:

[0220] In Examples 3-1 to 3-5, different ionizable cationic lipids were used to replace YK009 to prepare nucleic acid-lipid nanoparticles, and freeze-dried. The ionizable cationic lipids were SM102, ALC0315, DLin-MC3-DMA, YK-401 and YK-305. The particle sizes of the samples in each example before freeze-drying were not much different, all <80nm. The particle sizes of the freeze-dried samples changed to varying degrees after re-dissolution. The particle sizes of Examples 3-2 and 3-3 changed greatly, 20.6nm and 22.1nm, respectively. The particle sizes after freeze-drying were 95.0nm and 98.1nm, and the PDI changes were 0.0161 and 0.0275, respectively. The particle size changes of Example 3-1, Example 3-4 and Example 3-5 are all <20nm, the particle size after freeze-drying is <90nm, and the PDI changes slightly from 0.0046 to 0.0156. Compared with Example 1, the particle size change of Example 1 after freeze-drying is smaller, and the PDI change after freeze-drying is the smallest.

[0221] The experimental results show that nucleic acid-lipid nanoparticles prepared by different ionizable cationic lipids were freeze-dried. After freeze-drying and re-dissolution, YK009 had the best particle size and particle size distribution effect, and the order of the effects of other lipids was YK009>YK-401>SM102≈YK-305>ALC0315>DLin-MC3-DMA.

[0222] 2) Encapsulation efficiency:

[0223] In Examples 3-1 to 3-5, SM102, ALC0315, DLin-MC3-DMA, YK-401 and YK-305 were used to replace YK009, respectively. The encapsulation efficiency before lyophilization was >95%, and the encapsulation efficiency after lyophilization decreased insignificantly, ranging from 5.7% to 8.6%. The encapsulation efficiency of Examples 3-4 and 3-5 was higher at 92.1% and 90.8%, respectively. The encapsulation efficiency of Example 1 after freeze drying changed to 5.3% and the encapsulation efficiency was 93.0%, indicating that the method of Example 1 can be used to replace with other ionizable cationic lipids to improve the protection of the freeze-dried mRNA-LNP preparation. Reduce the leakage of mRNA caused by the damage to LNP caused by the stress generated by water freezing.

[0224] The experimental results show that the use of YK009 combined with DOTAP as cationic lipids has the highest encapsulation rate before and after freeze-drying, and the mRNA is the most stable and not easily degraded. Replacing YK009 with SM102, YK-401, and YK-305 can also achieve certain effects.

[0225] 3) Completeness:

[0226] For Example 3-1 to Example 3-5, the integrity changes after freeze-drying are between 0.6% and 1.9%, for Example 3-2 and Example 3-3, the integrity changes after freeze-drying are >1%, for Example 3-1, Example 3-4, and Example 3-5, the integrity changes after freeze-drying are <1%, and are not much different from those in Example 1. The corresponding ionizable cationic lipids are SM102, YK-401, and YK-305, respectively.

[0227] The experimental results show that the mRNA integrity of the nanolipid particle freeze-dried preparation prepared with cationic lipids at a molar ratio of 1:1 among YK009, SM102, YK-401, YK-305 and DOTAP has little change.

[0228] 4) Water content and reconstitution time:

[0229] The moisture content of the freeze-dried samples prepared in each batch of the examples was relatively low at about 1%, and the reconstitution time was short, <10s.

[0230] Example 1: A nucleic acid nanoparticle freeze-dried preparation was prepared using SM102, YK-401, YK-305 and DOTAP (molar ratio of 1:1) as cationic lipids. The preparation had low water content and a short reconstitution time of <10 s.

[0231] From the above results, it can be seen that the lyophilized preparations of Example 3-1, Example 3-4 and Example 3-5 respectively replaced YK009 with SM102, YK-401 and YK-305, and compared with the lyophilized preparations of Example 3-2 and Example 3-3 respectively replaced YK009 with ALC0315 and DLin-MC3-DMA, the particle size change was smaller, the encapsulation rate and integrity were higher, the water content was low, the reconstitution time was short, and the lyophilization effect was better. This shows that replacing YK009 with SM102, YK-401 and YK-305 can also achieve a certain effect of Example 1.

[0232] summary:

[0233] Compared with the mRNA-LNP lyophilized preparation prepared by the organic phase composition of YK009 as an ionizable cationic lipid in Example 1, YK009 is replaced by other ionizable cationic lipids and the DOTAP organic phase in a 1:1 ratio to prepare nanoparticle lyophilized preparations, which can also significantly improve the particle size and uniformity, encapsulation efficiency, integrity, and re-dissolution effect of the lyophilized preparations; among them, the ionizable cationic lipids with better effects are SM102, YK-401, and YK-305, and the ionizable cationic lipids with poorer effects are ALC0315 and DLin-MC3-DMA.

[0234] Conclusion Analysis 3: Permanently Charged Cationic Lipid Species

[0235] Example 4 Different types of permanently charged cationic lipids were mixed with the permanently charged cationic lipid YK009 in a molar ratio of 1:1 as organic phases for mRNA encapsulation, and freeze-dried with 10% sucrose as a lyophilization protectant. The lipid ratios of the examples in the following table were: cationic lipid: DSPC: Chol: DMG-PEG2000 = 49:10:39.5:1.5, wherein the cationic lipid was YK009 and the permanently charged cationic lipids listed in the table below in a molar ratio of 1:1, and the types of permanently charged cationic lipids are shown in the table below.

[0236] surface 2 Types of permanent cationic lipids

[0237]

[0238] The results of testing the particle size, PDI, encapsulation efficiency, integrity, moisture content and reconstitution time of the mRNA-LNP samples and their lyophilized preparations (room temperature 25°C, 0 days after lyophilization) are shown in the following table:

[0239] Table 13 Comparison of various test indicators of permanently charged cationic lipid preparations

[0240]

[0241] in conclusion:

[0242] 1) Particle size and particle size distribution coefficient PDI:

[0243] In Example 4-1 and Example 4-2, different types of permanently charged cationic lipids were used to replace DOTAP to prepare nucleic acid-lipid nanoparticles, and freeze-dried. The permanently charged cationic lipids were DOTMA and DDAB, respectively. The particle size of the samples of Example 4-1 and Example 4-2 before freeze-drying was slightly larger than that of Example 1, which was 80 nm. After freeze-drying, the particle size increased by 15.7 nm to 19.8 nm. The particle size change after freeze-drying was <20 nm. The PDI changes were 0.0142 and 0.0080, respectively, which were slightly larger than those of Example 1.

[0244] The experimental results show that different types of permanently charged cationic lipids were used to prepare nucleic acid-mixed lipid nanoparticles for freeze-drying. The particle size and particle size distribution before and after freeze-drying can also achieve certain effects by replacing DOTAP with DOTMA and DDAB, which are slightly worse than DOTAP.

[0245] 2) Encapsulation efficiency:

[0246] In Example 4-1 and Example 4-2, different types of permanently charged cationic lipids were used to replace DOTAP to prepare nucleic acid-lipid nanoparticles, and freeze-dried. The permanently charged cationic lipids were DOTMA and DDAB, respectively. The encapsulation efficiency before freeze-drying was >96%, and the encapsulation efficiency after freeze-drying decreased to a higher level than that of DOTAP, ranging from 8.8% to 9.7%.

[0247] The experimental results show that after replacing DOTAP with DOTMA and DDAB, the freeze-drying effect is significantly reduced.

[0248] 3) Completeness:

[0249] From Example 4-1 to Example 4-2, the integrity after freeze-drying changed from 0.8% to 1.1%, indicating that replacing DOTAP with DOTMA and DDAB did not produce as good integrity effect after freeze-drying as Example 1 using permanently charged cationic lipids.

[0250] The experimental results showed that the freeze-drying effect decreased after DOTAP was replaced by DOTMA and DDAB.

[0251] 4) Water content and reconstitution time:

[0252] In Example 4-1 and Example 4-2, the moisture content of the freeze-dried preparations was 1.234% and 1.413%, respectively, slightly higher than that in Example 1, and the reconstitution time was relatively short, <10s.

[0253] From the above results, it can be seen that the freeze-dried preparations of Example 4-1 and Example 4-2 replaced DOTAP with DOTMA and DDAB respectively, and the particle size changed greatly compared with Example 1, especially the particle size change of DDAB before and after freeze-drying was the largest at 19.8.

[0254] summary:

[0255] Compared with the mRNA-LNP lyophilized preparation prepared with DOTAP as the permanently charged cationic lipid in the organic phase in Example 1, after DOTAP was replaced with other permanently charged cationic lipids and prepared with the YK009 organic phase in a 1:1 ratio for the nanoparticle lyophilized preparation, the particle size and uniformity, encapsulation efficiency, integrity, and re-dissolution effect of the lyophilized preparation were significantly reduced.

[0256] Conclusion Analysis 4: Sublimation Drying;

[0257] In Example 5, different heating methods or times are used in the sublimation drying procedure to freeze-dry the sample mRNA-LNP, and the obtained freeze-dried preparations are analyzed.

[0258] 1. Influence of heating method

[0259] The total time of the sublimation drying method used is equal, and the specific heating method is shown in the following table:

[0260] Table 14 Difference in sublimation drying temperature

[0261]

[0262] The results of testing the particle size, PDI, encapsulation efficiency, integrity, moisture content and reconstitution time of the mRNA-LNP samples and their lyophilized preparations (room temperature 25°C, 0 days after lyophilization) are shown in the following table:

[0263] Table 15 Comparison of various test indicators of sublimation drying temperature preparations

[0264]

[0265] in conclusion:

[0266] 1) Particle size and particle size distribution coefficient PDI:

[0267] In Example 1, Example 5-1 and Example 5-2, sublimation drying was performed by gradient heating, and the particle size changed slightly, which were 9.6 nm, 10.1 nm and 10.4 nm, respectively, and PDI did not increase significantly. The particle size changed slightly before and after freeze-drying, indicating that the product quality and process were better. PDI did not increase significantly, indicating that the size uniformity of mRNA-LNP was good. Among them, the gradient heating mode of Example 1 was adopted, and the particle size changed the least and the uniformity was optimal.

[0268] On the contrary, in Example 5-3 to Example 5-5, the ordinary heating method was adopted without gradient temperature regulation, and the particle size increased significantly by about 13nm~20nm, indicating that the size uniformity of mRNA-LNP was significantly deteriorated.

[0269] 2) Encapsulation rate: Example 1, Example 5-1 and Example 5-2, the sublimation drying was carried out by gradient heating, and the encapsulation rate was 91.1%~93.0%, and the degree of reduction was 5.3%~6.7%. The encapsulation rate was high, and the reduction after freeze-drying was not obvious, indicating that the proportion of mRNA encapsulated by liposomes was large, and a large amount of mRNA was prevented from being degraded. On the contrary, Example 5-3 to Example 5-5, the ordinary heating method was adopted, and the gradient temperature was not regulated, and the encapsulation rate was low, with a decrease of 12.9%~13.2%. The encapsulation rate decreased significantly, indicating that the proportion of mRNA not encapsulated increased significantly, and the degraded mRNA increased significantly.

[0270] 3) Integrity: The integrity of Example 1, Example 5-1 and Example 5-2, which were subjected to sublimation drying by gradient heating, remained basically unchanged, at 83.4%, 82% and 82.3%, respectively, which was only about 0.2% to 0.3% less than that of the mRNA-LNP sample. In Example 5-3 to Example 5-5, the integrity was reduced by using the ordinary heating method, down to 79.6% to 80.6%, with a change of 1.5% to 2%.

[0271] 4) Water content and re-dissolution time: Although the re-dissolution time of the above examples is within 10 seconds, the water content of Example 1, Example 5-1 and Example 5-2 using the gradient heating method is less than 1%. The water content of Examples 5-3 to 5-5 using the ordinary heating method exceeds 1%, which may affect the long-term stable storage of the samples.

[0272] summary:

[0273] During the freeze-drying process, different heating methods are used for sublimation drying, which affects the particle size uniformity, encapsulation rate, integrity and moisture content of the preparation. Among them, when the temperature control method is gradient heating (for example, Example 1, Example 5-1 and Example 5-2), the freeze-drying effect is good.

[0274] Sublimation drying was performed by gradient temperature increase (for example, Example 1, Example 5-1, Example 5-2), and the physicochemical properties, mRNA encapsulation ratio and integrity of the freeze-dried preparation did not change significantly. The particle size increased by 9.6nm~10.4nm, the PDI did not change significantly, the encapsulation rate decreased by 5.3%~6.7%, the integrity decreased by about 0.2%~0.3%, the water content was less than 1%, and the reconstitution time was <10s.

[0275] Among them, the freeze-dried preparation (Example 1) obtained by adopting a lower temperature gradient heating sublimation drying method has a particle size increase of only 9.6nm, an encapsulation efficiency decrease of only 5.3%, a integrity decrease of 0.2%, a moisture content of only 0.878%, and a reconstitution time of <10s.

[0276] The particle size uniformity, encapsulation rate, integrity and moisture content of the freeze-dried preparation obtained by sublimation drying using the common heating method (e.g., Examples 5-3 to 5-5) are significantly lower than those of the gradient heating method, and the freeze-drying effect is worse than that of the sublimation drying method using the gradient heating method. The particle size increases by 13.4 nm to 20.1 nm (3.8 to 10.5 nm more than the particle size increase in Example 1), the encapsulation rate decreases by about 12.9% to 13.2% (about 7.6 to 7.9% higher than the encapsulation rate decrease in Example 1), the integrity decreases by 1.5% to 2% (1.3 to 1.8% higher than the integrity decrease in Example 1), and the moisture content is 1.025% to 1.116%.

[0277] 2. Influence of sublimation drying time

[0278] All of them adopt the gradient heating method for sublimation drying, and the time taken is different, as shown in the following table:

[0279] Table 16 Difference in sublimation drying time

[0280]

[0281] The particle size, PDI, encapsulation efficiency and integrity, moisture content and reconstitution time of mRNA-LNP samples and their lyophilized preparations (room temperature 25°C, 0 days after lyophilization) were tested. The results are shown in the following table:

[0282] Table 17 Comparison of various test indicators of sublimation drying time preparations

[0283]

[0284] in conclusion:

[0285] 1) Particle size and PDI: In Example 1, the sublimation drying time was controlled to 75 hours, and the particle size changed the least, increasing by 9.6 nm; in Example 5-6, the sublimation drying time was controlled to 45 hours, and the particle size increased by 14.9 nm. When the sublimation drying time of Example 5-7 was only 6 hours, the particle size increased significantly, increasing by 33.7 nm, and the particle uniformity was poor.

[0286] 2) Encapsulation efficiency: When the sublimation drying time in Example 1 and Example 5-6 was controlled at 45h~75h, the encapsulation efficiency changed the least, decreasing by about 5.3%~9.7%, which was a small change; while when the sublimation drying time in Example 5-7 was only 6h, the change was large, decreasing by 15.1%, and the proportion of unencapsulated mRNA increased, resulting in a significant increase in degraded mRNA.

[0287] 3) Integrity: When the sublimation drying time of Example 1 and Example 5-6 was controlled at 45h~75h, the integrity remained basically unchanged, only decreasing by 0.2% and 0.6%, which was a small decrease; when the sublimation drying time of Example 5-7 was only 6h, there was a significant change, decreasing by 4.5%, and the integrity decreased significantly.

[0288] 4) Water content and reconstitution time: When the sublimation drying time of Example 1 was 75 hours, the water content was the lowest, which was 0.878%; followed by Example 5-6 with a sublimation drying time of 45 hours, with a water content of 1.346%; Example 5-7 had a sublimation drying time of only 6 hours and the highest water content, reaching 14.625%, which may be due to the fact that the freeze-drying time was not reached, and therefore it could not be considered a qualified freeze-dried product.

[0289] From the above results, it can be seen that the physicochemical properties, mRNA packaging ratio and integrity of Example 1 changed the least, Examples 5-6 were similar to Example 1, and Examples 5-7 changed greatly. All three methods adopted a gradient heating method, but the durations used were different, namely 75h, 45h and 6h. It can be seen that the freeze-drying effect is better when the total sublimation drying time reaches more than 45h.

[0290] Summary: The mRNA-LNP samples were freeze-dried, and a gradient temperature was used in the sublimation drying stage, and the duration was controlled at 45~75h. The freeze-dried preparation obtained had a good freeze-drying effect.

[0291] The sublimation drying method was adopted with a gradient temperature increase of 45-75h (Example 1, Example 5-1, Example 5-2 and Example 5-6). The particle size increased by about 10nm, the PDI had no obvious change, the encapsulation efficiency decreased by no more than 10%, the integrity decreased by about 0.2%-0.6%, the moisture content was within 1%, and the re-dissolution time was <10s.

[0292] The sublimation drying stage adopts ordinary heating, and the total process time is 75h. The mRNA-LNP samples are freeze-dried (Example 5-3 to Example 5-5). The physicochemical properties, mRNA encapsulation ratio and integrity change greatly, and the freeze-drying effect is worse than that of the step-by-step heating method. The particle size of the freeze-dried preparation obtained by ordinary heating increases by about 13nm~20nm, the encapsulation rate decreases by >10%, the integrity decreases by about 2%, and the water content increases to about 1.2%.

[0293] On the contrary, if the total duration of the sublimation drying process is too short, such as 6 hours, the freeze-drying effect is poor and cannot be used as a qualified freeze-dried product.

[0294] Even if a gradient temperature sublimation drying method is used, but the total time is too short (6 h in Example 6-7), the mRNA-LNP sample is freeze-dried, and the freeze-dried preparation obtained has a poor freeze-drying effect; the particle size increases by 43.7 nm, the encapsulation efficiency decreases by 15.1%, the integrity decreases by 4.5%, and the water content is 14.625%.

[0295] Conclusion Analysis 5: Pre-freezing Method

[0296] Example 6 The sample mRNA-LNP was freeze-dried and pre-frozen at different temperatures or times (as shown in Table 17), and the obtained freeze-dried preparations were analyzed.

[0297] Table 18 Pre-freezing method

[0298]

[0299] The particle size, PDI, encapsulation efficiency and integrity, moisture content and reconstitution time of mRNA-LNP samples and their lyophilized preparations (room temperature 25°C, 0 days after lyophilization) were tested. The results are shown in the following table:

[0300] Table 19 Comparison of various test indicators of pre-freezing preparations

[0301]

[0302] in conclusion:

[0303] 1) Particle size and PDI: Example 1 uses liquid nitrogen for pre-freezing, and the particle size and PDI change the least, which are 9.6nm and 0.0022. Examples 6-1 to 6-3 use direct cooling for pre-freezing, and the particle size of the lyophilized preparations obtained changes from 16.2nm to 20.3nm and the PDI changes from 0.0126 to 0.0244, and the particle size uniformity deteriorates. It can be seen that the pre-freezing effect of liquid nitrogen is better, and the effect of ordinary cooling is worse.

[0304] 2) Encapsulation rate: Example 1 used liquid nitrogen pre-freezing, and the encapsulation rate changed the least, controlled at 5.3%; Example 6-1 to Example 6-3 used ordinary cooling methods, and the encapsulation rate changed greatly, decreasing by about 9%.

[0305] 3) Integrity: The integrity of Example 1 changed the least, only 0.2%, which can be ignored; the integrity of Example 6-1 to Example 6-4 changed greatly, and the change increased significantly to about 2.3%~3.2%. It can be seen that the freeze-drying effect of liquid nitrogen is better than ordinary cooling.

[0306] 4) Water content and reconstitution time: The water content of Example 1 is the lowest (<1%), and the water content of Examples 6-1 to 6-4 is >1%. Increased water content may lead to difficulty in storage. It can be seen that the freeze-drying effect of liquid nitrogen and gradient cooling is better than that of ordinary cooling.

[0307] Summary: The mRNA-LNP samples were freeze-dried, and the pre-freezing procedure used liquid nitrogen. The freeze-dried preparations obtained had excellent particle uniformity, encapsulation efficiency, integrity, and moisture content.

[0308] The mRNA-LNP sample was pre-frozen for 3 hours and freeze-dried to prepare a freeze-dried preparation (Example 1). The particle size increased by only 9.6 nm, with a small change in particle size. The encapsulation efficiency decreased by 5.3%, and the integrity decreased by 0.2%. The mRNA encapsulation efficiency and integrity did not change significantly, indicating that the proportion of mRNA exposure and damage caused by freeze-drying was small and the impact was not obvious. The moisture content of the freeze-dried sample was <1%, and the reconstitution time was <10s.

[0309] On the contrary, the particle uniformity, encapsulation efficiency, integrity and moisture content of the freeze-dried preparation obtained by ordinary cooling are significantly worse than those by gradient cooling and liquid nitrogen pre-freezing.

[0310] The pre-freezing adopts the common cooling method (Example 6-1 to Example 6-3), and the mRNA-LNP samples are freeze-dried, and the particle size, encapsulation efficiency and integrity change greatly, and the freeze-drying effect is poor. The particle size of the obtained freeze-dried preparation increases by about 20nm, the encapsulation efficiency decreases by up to 9%, the integrity decreases by more than 2.3%, and the moisture content is >1.0%.

[0311] Conclusion Analysis 6: Filling Volume

[0312] Example 7 freeze-dried RNA-LNP using different filling volumes, and analyzed the obtained freeze-dried preparations. The freeze-dried sample preparation method was the same as in Example 1, and 1.0 ml and 1.5 ml were filled in the vials respectively. The total freeze-drying time for 0.5 ml was 82 h. Other filling preparation methods, pre-freezing processes and analytical drying processes were consistent with those in Example 1. Only the sublimation drying time was extended based on that in Example 1. See the table below for specific information;

[0313] Table 20 Filling volume

[0314]

[0315] The results of testing the particle size, PDI, encapsulation efficiency, integrity, moisture content and reconstitution time of the mRNA-LNP samples and their lyophilized preparations (room temperature 25°C, 0 days after lyophilization) are shown in the following table:

[0316] Table 21 Comparison of various test indicators of filling volume preparations

[0317]

[0318] in conclusion:

[0319] 1) Particle size and particle size distribution coefficient PDI:

[0320] In Example 1, Example 7-1 and Example 7-2, the filling volume of 2 ml vials was 0.5 ml, 1 ml, and 1.5 ml, respectively, and the particle size changes were small, 9.6 nm, 10.0 nm, and 14.4 nm, respectively, and PDI did not increase significantly. It shows that the filling volume of 2 ml vials is within the range of 0.5 ml to 1.5 ml, and the particle size changes before and after lyophilization are small, indicating that the product quality and process are better. PDI did not increase significantly, indicating that the size uniformity of mRNA-LNP is good.

[0321] 2) Encapsulation efficiency: In Example 1, Example 7-1 and Example 7-2, different volumes were filled in 2 ml vials, and the encapsulation efficiency was between 91.5% and 93.0%, with a reduction degree of 5.3% to 6.7%. As the filling volume increased, the change in particle size before and after lyophilization increased, and the encapsulation efficiency decreased.

[0322] 3) Integrity: Example 1, Example 7-1 and Example 7-2 used 2 ml vials to fill different volumes, and the integrity was 82.4% to 83.4%, which was only about 0.2% to 0.8% less than that of the mRNA-LNP sample.

[0323] 4) Water content and reconstitution time: In Example 1, Example 7-1 and Example 7-2, different volumes were filled in 2 ml vials. After freeze-drying, the water content of the preparations was less than 1%, and the reconstitution time was <10 s.

[0324] summary:

[0325] During the freeze-drying process, different filling volumes directly affect the thickness of the freeze-dried cake layer. Different sublimation resistances are generated during the sample sublimation drying stage. Different filling volumes are investigated, and freeze drying is performed by extending the sublimation drying time as the filling volume increases. The results show that the freeze-drying effect of different filling volumes is good, and the particle size uniformity, encapsulation rate, integrity and moisture can all reach the effect of Example 1.

[0326] Conclusion Analysis 7: Transfection Efficiency

[0327] After re-dissolving the freeze-dried samples of Example 1 and Example 3-1 to Example 3-5, Example 4-1 and Example 4-2, Comparative Example 1 and Comparative Example 2, the transfection efficiency was investigated. The results are shown in the following table:

[0328] Table 22 Comparison of transfection efficiency of lyophilized preparations

[0329]

[0330] in conclusion:

[0331] After freeze-drying, clear bands were visible in each example, among which the bands of Example 3-2, Example 3-3, and Example 4-2 were slightly lighter than those of Example 1. This shows that the transfection effect of Example 1 is similar to that of Example 3-1, Example 3-4, Example 3-5, and Example 4-1, and slightly better than that of Example 3-2, 3-3, and 4-2. This shows that the hybrid lipid nanoparticles disclosed herein were used to successfully transfect cells.

[0332] When mixed nanoparticles were prepared using different ionizable cations and DOTAP, and when mixed nanoparticles were prepared using different permanently charged cationic lipids and YK009, clear bands were visible, and cells could be successfully transfected; there was a slight difference in the expression level of S protein, but the difference was not obvious, and there was no obvious change in the cell transfection efficiency; this proved that different types of mixed lipid nanoparticle mRNA-LNP samples disclosed in the present invention could successfully transfect cells.

[0333] Summary: The freeze-dried preparations prepared by freeze-drying the different types of mixed lipid nanoparticle mRNA-LNP samples disclosed in the present invention can successfully transfect cells. The transfection effect of the mixed lipid nanoparticles is not significantly different from that of the control group.

[0334] Conclusion Analysis 8: Stability Investigation

[0335] The mRNA-LNP lyophilized preparation prepared by the present invention was placed at low temperature of 2-8°C, room temperature of 25°C and high temperature of 40°C for 3 months respectively to detect the particle size, encapsulation efficiency and integrity, and to investigate its stability; since the change in moisture during the stability period is not related to the lyophilization process but only to the sealing of the packaging material, the moisture and reconstitution time are not tested during the stability experiment stage.

[0336] The cationic lipid composition of the formulation is shown in Table 23, and the stability of the preparation is shown in Table 24-Table 26.

[0337] Table 23 Prescription cationic lipid composition

[0338]

[0339] Table 24 Stability of formulation (room temperature)

[0340]

[0341] Table 25 Stability of preparations (2~8℃)

[0342]

[0343] Table 26 Stability of preparations (40°C)

[0344]

[0345] in conclusion:

[0346] 1) Particle size and PDI: In Example 1, Example 2-2 and Example 2-5 of the present disclosure, under different temperature conditions of room temperature, 2-8°C and 40°C, the particle size increases within 3nm from 0 days to 3 months at room temperature and 2-8°C; under the condition of high temperature 40°C, the increase is also controlled within 7.0nm, indicating that the stability of the lyophilized preparation under different temperature conditions is good. Compared with Comparative Example 1, the particle size increases within 7nm at room temperature and 2-8°C, and increases by 13.7nm at high temperature 40°C, and the stability effect at different temperatures is better.

[0347] 2) Encapsulation rate: In the disclosed Examples 1, 2-2 and 2-5, the encapsulation rate changes were controlled within 0.5% to 1.2% from 0 days to 3 months at room temperature and 2 to 8°C, respectively, and the changes were not significant. At 40°C, after being placed for 3 months, the changes were 1.6% to 2.1%. Compared with Comparative Example 1, the encapsulation rate changes were 1.8% to 2.4% at room temperature and 2 to 8°C, and the encapsulation rate changes were 4.2% at high temperature of 40°C. The stability effect at different temperatures is better.

[0348] 3) Integrity: In Example 1, Example 2-2 and Example 2-5 of the present disclosure, at different temperature conditions of room temperature, 2~8°C and 40°C, from 0 days to 3 months, the integrity decreased by 0.5%~1.3% at room temperature and 2~8°C, and the change was not significant; the integrity decreased the most at 40°C, but it was also maintained at 1.9%~2.4%, with less change, high integrity and high stability. Compared with Comparative Example 1, the package integrity changed by 1.2%~1.7% at room temperature and 2~8°C, and the encapsulation rate changed by 2.8% at high temperature 40°C, and the stability effect at different temperatures was better.

[0349] The above results show that the physicochemical properties, encapsulated mRNA ratio and integrity of Example 1, Example 2-2 and Example 2-5 did not change significantly or changed slightly under different temperature conditions, and the stability was better than the freeze-dried preparation of mRNA-LNP containing only ionizable cationic lipid nanoparticles.

[0350] Summary: The mRNA-LNP freeze-dried preparation disclosed in the present invention was placed at 2-40°C for three months, and its particle size, encapsulation efficiency and integrity did not change significantly, and had good stability.

[0351] It can be seen from the above embodiments that the present invention has achieved at least the following technical effects:

[0352] 1. Physical and chemical properties

[0353] 1. Molar ratio of ionizable cationic lipid (YK009) to permanently charged cationic lipid (DOTAP):

[0354] The sample with a molar ratio of ionizable cationic lipid (YK009) to permanently charged cationic lipid (DOTAP) of 1:1 had the best particle size and uniformity (PDI), encapsulation efficiency, integrity, moisture, re-dissolution effect and stability; the freeze-dried preparations prepared with a ratio of 8:2 to 2:8 could achieve good preparation effects.

[0355] i. The molar ratio of ionizable cationic lipid (YK009) to permanently charged cationic lipid (DOTAP) was 1:1, and its particle size increased by 9.6nm, PDI did not change significantly, encapsulation efficiency decreased by 5.3%, integrity decreased by 0.2%, water content was about 0.9%, and re-dissolution time was <10s;

[0356] ii. The freeze-dried preparations were prepared with a ratio of 8:2 to 2:8. After freeze-drying, the particle size changed from 9.6nm to 19.3nm, the PDI did not change significantly, the encapsulation efficiency changed from 5.3% to 6.6%, the encapsulation efficiency decreased slightly after freeze-drying, the integrity changed from 0.2% to 0.9%, the moisture content was <2%, and the reconstitution time was <10s.

[0357] iii. The freeze-dried preparations were prepared with a ratio of 9:1 and 1:9. The particle size changed by about 26 nm after freeze-drying, the PDI did not change significantly, the encapsulation efficiency changed by 6.4%~8.9% slightly more than other batches, the integrity changed by about 1.4% slightly more than other batches, the moisture content was <2%, and the reconstitution time was <10s.

[0358] 2. Types of ionizable cationic lipids

[0359] Using different types of ionizable cationic lipids to prepare mixed lipid nanoparticles after freeze-drying can also significantly improve the particle size and uniformity (PDI), encapsulation efficiency, integrity, moisture and re-dissolution effect of the freeze-dried preparation; the order of effect is YK009>YK-401>SM102≈YK-305>ALC0315>DLin-MC3-DMA.

[0360] i. After freeze-drying, the particle size changed from 15.3 to 22.1 nm, and the PDI did not change significantly. The order of lipid effect was YK009>YK-401>SM102≈YK-305>ALC0315>DLin-MC3-DMA.

[0361] ii. The encapsulation rate before freeze-drying was >95%, and the encapsulation rate after freeze-drying decreased slightly, ranging from 5.7% to 8.6%. The integrity changed from 0.6% to 1.9%. The moisture content was relatively low, around 1%, and the reconstitution time was short, <10s.

[0362] iii. Among them, YK-401 and YK-305 were used, with a higher encapsulation rate ranging from 5.7% to 6.6%, and a higher integrity ranging from 0.6% to 0.7%. The encapsulation rate and integrity were good, and the reconstitution time was short, <10s.

[0363] 3. Types of Permanently Charged Cationic Lipids

[0364] Using different types of permanently charged cationic lipids to prepare mixed lipid nanoparticles after freeze-drying can also significantly improve the particle size and uniformity (PDI), encapsulation efficiency, integrity, moisture and re-dissolution effect. The order of effect is DOTAP>DOTMA>DDAB.

[0365] i. After freeze-drying, the particle size increased from 15.7 to 19.8 nm, with a change of <20 nm. PDI changed from 0.0142 to 0.0080, the encapsulation rate decreased from 8.8% to 9.7%, the integrity changed from 0.8% to 1.1%, the moisture content was <2%, and the reconstitution time was short <10s.

[0366] 4. Effect of freeze drying

[0367] 4-1 Heating method of sublimation drying

[0368] When the sublimation drying adopts a gradient temperature increase, the particle size and uniformity, encapsulation rate, integrity, moisture content and re-dissolution effect after freeze-drying are good. On the contrary, if the gradient temperature is not regulated, the freeze-drying effect is slightly worse.

[0369] i. The sublimation drying was carried out by gradient heating. The particle size changed slightly between 9.6 and 10.4 nm, the PDI did not increase significantly, the encapsulation efficiency was between 91.1% and 93.0%, the reduction degree was between 5.3% and 6.7%, the integrity decreased by about 0.2% to 0.3%, the moisture content was <1%, and the re-dissolution time was <10 s.

[0370] ii. Without gradient temperature regulation, the particle size increased significantly, by about 13~20nm, the encapsulation efficiency decreased by 12.9%~13.2%, the integrity changed by 1.5%~2%, the moisture content was >1%, and the re-dissolution time was <10s.

[0371] 4-2 Effect of sublimation drying time

[0372] The sublimation drying stage adopts gradient temperature increase and controls the duration at 45h~75h. The freeze-drying effect of the freeze-dried preparation obtained is good. On the contrary, if the total duration of the sublimation drying process is too short, such as 6h, the freeze-drying effect is poor and cannot be used as a qualified freeze-dried product.

[0373] i. When the sublimation drying time is 45h~75h, the particle size changes the least, increasing by 9.6~14.9nm, the encapsulation rate changes the least, decreasing by about 5.3%~9.7%, the integrity remains basically unchanged, only decreasing by 0.2%~0.6%, and the moisture content is 0.878%~1.346%.

[0374] ii. When the sublimation drying time was only 6h, the particle size increased significantly, by 33.7nm, the encapsulation efficiency changed greatly, decreasing by 15.1%, the integrity decreased significantly, by 4.5%, and the moisture content was 14.625%.

[0375] 4-3 Impact of pre-freezing program

[0376] The pre-freezing procedure uses liquid nitrogen pre-freezing, and the resulting freeze-dried preparation has excellent particle size and uniformity (PDI), encapsulation efficiency, integrity, moisture and reconstitution effect.

[0377] i. Pre-freezing by liquid nitrogen resulted in the smallest changes in particle size and PDI, which were 9.6 nm and 0.0022, and the smallest change in encapsulation efficiency, which was controlled at 5.3%. The integrity changed by only 0.2%, and the moisture content was <1%.

[0378] ii. Direct cooling for pre-freezing resulted in freeze-dried preparations with a particle size variation of 16.2-20.3 nm, a PDI variation of 0.0126-0.0244, a large variation in encapsulation efficiency, a decrease of about 9%, a significant increase in integrity variation to about 2.3-3.2%, and a moisture content of >1%.

[0379] 5. Influence of filling volume

[0380] The freeze-drying effect of the filling volume of 0.5ml to 1.5ml is good, and the particle size and uniformity (PDI), encapsulation efficiency, integrity, moisture and reconstitution effect can all reach the effect of the present disclosure.

[0381] The filling volumes of 2ml vials were 0.5ml, 1ml and 1.5ml respectively. The particle size varied by 9.6~14.4nm, PDI did not increase significantly, the encapsulation efficiency was 91.5~93.0%, a decrease of 5.3~6.7%, the integrity was 82.4%~83.4%, a decrease of about 0.2~0.8%, the water content was less than 1%, and the reconstitution time was <10s.

[0382] 2. Biological Characteristics

[0383] 1. Transfection effect

[0384] The freeze-dried preparations of mixed lipid nanoparticles prepared by adding different types of permanently charged cationic lipids and the freeze-dried preparations of mixed lipid nanoparticles prepared by adding different types of ionizable cationic lipids and DOTAP can both be successfully transfected into cells; the S protein is well expressed in the cells.

[0385] 3. Stability

[0386] 1. The mRNA-LNP freeze-dried preparation disclosed in the present invention is placed at 2-40°C for three months, and its particle size, encapsulation efficiency and integrity do not change significantly, and the stability is good. Specifically:

[0387] i. After storage for 3 months at room temperature and 2-8°C, the particle size increased within 3 nm, the PDI did not change significantly, and the encapsulation efficiency and integrity decreased by only about 1%, with no significant changes;

[0388] Under high temperature conditions of 40°C, the particle size change was controlled within 7.0nm, the encapsulation efficiency decreased by 1.6~4.2%, and the integrity decreased by 1.9~2.8%, with little change.

[0389] The applicant declares that the present invention illustrates the nucleic acid-lipid nanoparticle freeze-dried composition and its preparation method and application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0390] The sequence of the mRNA used in the present invention is shown as SEQ ID No.1.

[0391] SEQ ID No.1:

[0392]

Claims

1. A nucleic acid-lipid nanoparticle freeze-dried composition, characterized in that: The nucleic acid-lipid nanoparticle freeze-dried composition comprises nucleic acid-lipid nanoparticles and a freeze-drying protective agent, wherein the nucleic acid-lipid nanoparticles comprise nucleic acids and lipid nanoparticles, wherein the lipid nanoparticles comprise cationic lipids, neutral lipids, auxiliary lipids and polymer-conjugated lipids, and wherein the cationic lipids are a combination of ionizable cationic lipids and permanently charged cationic lipids; The ionizable cationic lipid is selected from one of YK-009, YK-401, YK305 or SM102, wherein: YK-009 has the structure shown in formula (II): ； YK-401 has the structure shown in formula (II-I): ； YK-305 has the structure shown in formula (IV-I): ； SM102 has the structure shown in formula (VI-I): ； The permanently charged cationic lipid is selected from (2,3-dioleoyl-propyl)-trimethylammonium chloride; The polymer conjugated lipid is a lipid modified with polyethylene glycol; the lipid modified with polyethylene glycol is selected from any one of distearoylphosphatidylethanolamine polyethylene glycol 2000, dimyristoylglycerol-3-methoxy polyethylene glycol 2000 or methoxy polyethylene glycol ditetradecyl acetamide or a combination of at least two thereof; The neutral lipid is selected from any one of dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine or a combination of at least two thereof; The auxiliary lipid is selected from any one or a combination of at least two of cholesterol, vitamin E, DC-cholesterol or their derivatives; The molar ratio of the ionizable cationic lipid to the permanently charged cationic lipid is 1:1; The lyoprotectant is selected from any one of sucrose, trehalose or mannitol.

2. The nucleic acid-lipid nanoparticle freeze-dried composition according to claim 1, characterized in that: The nucleic acid is selected from any one or a combination of at least two of small interfering RNA, asymmetric interfering RNA, micro RNA, Dicer-substrate RNA, small hairpin RNA, mRNA, long non-coding RNA, circular RNA, self-amplifying RNA or 5'-pppRNA.

3. The nucleic acid-lipid nanoparticle freeze-dried composition according to claim 2, characterized in that: The sequence of the mRNA is shown as SEQ ID No.

1.

4. The nucleic acid-lipid nanoparticle freeze-dried composition according to claim 1, characterized in that: The mass ratio of the lipid nanoparticles to the nucleic acid is 10-30:

1.

5. A method for preparing the freeze-dried nucleic acid-lipid nanoparticle composition according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: The freeze-dried protective agent solution is mixed with the nucleic acid-lipid nanoparticle solution to obtain a mixed solution to be freeze-dried, and freeze-dried to obtain the nucleic acid-lipid nanoparticle freeze-dried composition.

6. The preparation method according to claim 5, characterized in that: The mass percentage of the lyophilization protectant in the lyophilization protectant solution is 5-20%, and the solvent in the lyophilization protectant solution is enzyme-free water or Tris buffer or DPBS buffer with a pH value of 5-9.

7. The preparation method according to claim 5, characterized in that: The total mass percentage content of nucleic acid and lipid nanoparticles in the nucleic acid-lipid nanoparticle solution is 0.1%-1%, and the solvent in the nucleic acid-lipid nanoparticle solution is Tris buffer or DPBS buffer with a pH value of 5-9.

8. The preparation method according to claim 5, characterized in that: The mixed solution to be lyophilized is filled into a 2 mL sample bottle with a filling volume of 0.5-1.5 mL, and freeze-dried.

9. The preparation method according to claim 5, characterized in that: The freeze drying process comprises the following steps: a pre-freezing stage, a sublimation drying stage, and a desorption drying stage.

10. The preparation method according to claim 9, characterized in that: The pre-freezing stage uses liquid nitrogen for cooling.

11. The preparation method according to claim 10, characterized in that: The liquid nitrogen cooling time is 30min~6h.

12. The preparation method according to claim 9, characterized in that: The temperature of the sublimation drying stage is -40~0°C, maintained for 50~100 hours, and the temperature is increased in a gradient manner in 2~4 stages, with each stage increasing by 5~20°C and each stage maintained for 6~70 hours.

13. The preparation method according to claim 9, characterized in that: The temperature rise process of the sublimation drying stage is: -40°C for 50h, -35°C for 12h, -15°C for 7h, and 0°C for 6h.

14. The preparation method according to claim 9, characterized in that: The temperature of the desorption drying stage is 15-30° C. and the operation time is 2-5 hours.

15. The preparation method according to claim 5, characterized in that: During the freeze-drying process, the vacuum pressure was maintained at ≤0.05 mbar.

16. Use of the freeze-dried nucleic acid-lipid nanoparticle composition according to any one of claims 1 to 4 in the preparation of nucleic acid drugs.

17. Use of the lyophilized nucleic acid-lipid nanoparticle composition according to any one of claims 1 to 4 in the preparation of a medicament for treating a disease or condition in a mammal in need thereof.

18. The use according to claim 17, characterized in that The disease or condition is selected from the group consisting of infectious diseases, proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

19. The use according to claim 17, characterized in that The disease is cancer.

20. The use according to claim 17, characterized in that The disease is diabetes.

21. The use according to claim 18, characterized in that The infectious disease is selected from the group consisting of: diseases caused by coronavirus, influenza virus or HIV virus, childhood pneumonia, Rift Valley fever, yellow fever, rabies or herpes.

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