Preparation method of nucleic acid-lipid nanoparticles

Abstract

The invention discloses a preparation method of nucleic acid-lipid nanoparticles. The preparation method comprises the following steps: (1) mixing a lipid solution of which the solvent is an alcohol solvent with a first buffer solution of which the pH value is 3.6-4.5 by adopting a microfluidic mixing device to obtain a mixture; (2) carrying out liquid exchange on the mixture by adopting a second buffer solution with the pH value of 5.5-6.5 to remove the alcohol solvent in the mixture, and concentrating an obtained liquid exchange product to obtain a concentrated product; and (3) mixing the concentrated product with nucleic acid in the presence of a protective agent according to a nitrogen-phosphorus ratio of 15-35 at 25-37 DEG C, and incubating to obtain the nucleic acid-lipid nanoparticles. The invention provides a high-quality nucleic acid-lipid nanoparticle drug preparation scheme which is high in flexibility, high in material utilization rate, good in product quality and easy for large-scale production for nucleic acid drugs, and has important significance for development and industrial large-scale production of nucleic acid-lipid nanoparticle drugs.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid drug preparation technology, specifically relating to a method for preparing nucleic acid-lipid nanoparticles. Background Technology

[0002] The application of nucleic acid therapy in areas such as mRNA vaccines, personalized cancer treatment, and gene editing for genetic diseases marks a significant advancement in the biomedical field. However, nucleic acid molecules, as negatively charged hydrophilic macromolecules, are difficult to directly penetrate negatively charged cell membranes and are susceptible to degradation by nucleases or self-hydrolysis, limiting their clinical applications. Lipid nanoparticles (LNPs), as an effective nucleic acid delivery system, effectively prevent in vivo degradation by encapsulating nucleic acids internally and achieve endosome escape through ionizable lipids. They possess good biocompatibility and a mature clinical application basis (such as COVID-19 mRNA vaccines), and have become a research hotspot in nucleic acid drug delivery.

[0003] Traditional LNP preparation primarily employs the ethanol dilution method, which involves mixing a lipid ethanol solution with an acidic nucleic acid buffer using a microfluidic mixer to form the LNP. This is then replaced with a neutral buffer via dialysis, ultrafiltration, or tangential flow filtration to obtain a stable formulation. While this method offers high encapsulation efficiency, it suffers from several drawbacks: nucleic acids are exposed to ethanol, acidic environments, and shear stress during the initial LNP assembly phase, making them prone to degradation; the entire preparation process requires nucleic acid participation, which struggles to meet the flexibility and rapid preparation requirements of personalized therapy; furthermore, small-scale preparation results in significant nucleic acid waste, while large-scale production presents challenges such as complex logistics and storage, and difficulties in batch consistency control.

[0004] To address this, we began exploring a post-encapsulation strategy: preparing empty LNPs and then encapsulating nucleic acid drugs within them through subsequent processing. This aims to prevent nucleic acids from participating in the assembly process, reduce their risk of damage, and improve preparation flexibility and material utilization. However, mature empty LNPs possess a complete phospholipid bilayer structure similar to a cell membrane, with a hydrophobic core that effectively isolates hydrophilic molecules. Nucleic acids, being hydrophilic macromolecules, cannot passively diffuse through this hydrophobic barrier. The primary challenge is to enable nucleic acids to temporarily and efficiently pass through the lipid bilayer of the LNP, achieving high encapsulation efficiency. Simultaneously, preventing uncontrolled particle size and uniformity is crucial. Post-encapsulation processing can lead to uncontrolled fusion, aggregation, or rupture of the LNPs, resulting in increased particle size and wider distribution, severely impacting product uniformity, stability, and subsequent efficacy.

[0005] Therefore, developing a post-encapsulation process that can efficiently encapsulate nucleic acids and achieve precise control over LNP particle size and uniformity is of great significance for the development and industrial-scale production of nucleic acid-lipid nanoparticle drugs. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing nucleic acid-lipid nanoparticles that can achieve robust and controllable control over the particle size, distribution, and uniformity of nucleic acid-lipid nanoparticles while ensuring high nucleic acid encapsulation efficiency, so as to meet the needs of industrialization.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing nucleic acid-lipid nanoparticles, comprising the following steps: (i) A lipid solution in alcohol solvent is mixed with a first buffer solution with a pH of 3.6-4.5 using a microfluidic mixing device at a flow rate ratio of (2-4):1 and a total flow rate of 15-25 mL / min to obtain a mixture. The lipid solution contains cationic lipids, cofactor phospholipids, cholesterol and PEGylated lipids in a molar ratio of (40-60):(5-15):(35-40):(1-2). The total lipid concentration of the lipid solution is 5-25 mmol / L. (ii) The mixture is replaced with a second buffer solution with a pH of 5.5 to 6.5 to remove the alcohol solvent from the mixture, and the resulting product is concentrated to obtain a concentrated product; (iii) The concentrated product and nucleic acid are mixed and incubated at 25-37°C in the presence of a protective agent at a nitrogen-to-phosphorus ratio of 15-35 to obtain the nucleic acid-lipid nanoparticles.

[0008] In some embodiments, the pH value of the first buffer solution is 3.8 to 4.2, specifically 3.8, 3.9, 4.0, 4.1 or 4.2.

[0009] In some embodiments, the pH value of the second buffer solution is 5.8 to 6.2, specifically 5.8, 5.9, 6.0, 6.1 or 6.2.

[0010] In some preferred embodiments, the first buffer solution is an acetate buffer or a citrate buffer.

[0011] In some preferred embodiments, the second buffer is a Tris-HCl buffer.

[0012] In some specific and preferred embodiments, when the nucleic acid is RNA, the cationic lipid is Dlin-MC3-DMA, and the first buffer is acetate buffer.

[0013] In other specific and preferred embodiments, when the nucleic acid is DNA, the cationic lipid is DOTAP-Cl, the second buffer is citrate buffer, and the higher the total flow rate, the lower the nitrogen-to-phosphorus ratio.

[0014] In some embodiments, the buffer salt concentrations of the first buffer solution and the second buffer solution are independently 10-50 mM, specifically 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, or 50 mM. More preferably, the buffer salt concentrations of the first buffer solution and the second buffer solution are independently 10~30mM, and even more preferably 15~25mM.

[0015] In some embodiments, the lipid solution contains cationic lipids, cofactor phospholipids, cholesterol, and PEGylated lipids.

[0016] In some embodiments, the alcohol solvent is ethanol and / or tert-butanol. The tert-butanol comprises a 90% to 100% (v / v) tert-butanol solution.

[0017] In some embodiments, the total lipid concentration of the lipid solution is specifically 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L, 21 mmol / L, 22 mmol / L, 23 mmol / L, 24 mmol / L, or 25 mmol / L. More preferably, the total lipid concentration of the lipid solution is 5-20 mmol / L, even more preferably 5-15 mmol / L, and particularly preferably 6-10 mmol / L.

[0018] In some embodiments, the protective agent is one or more of sucrose, sodium chloride, and mannitol.

[0019] More preferably, the protective agent is a sucrose solution with a concentration of 160-400 mg / mL, specifically a sucrose solution with concentrations of 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, 210 mg / mL, 220 mg / mL, 230 mg / mL, 240 mg / mL, 250 mg / mL, 260 mg / mL, 270 mg / mL, 280 mg / mL, 290 mg / mL, 300 mg / mL, 310 mg / mL, 320 mg / mL, 330 mg / mL, 340 mg / mL, 350 mg / mL, 360 mg / mL, 370 mg / mL, 380 mg / mL, 390 mg / mL, or 400 mg / mL.

[0020] In some embodiments, the nitrogen-phosphorus ratio is 20 to 30, specifically 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

[0021] In some embodiments, in step (a), the flow rate ratio of the lipid solution to the first mixed solution is specifically 2:1, 2.5:1, 3:1, 3.5:1, or 4:1, and the total flow rate is specifically 15 mL / min, 16 mL / min, 17 mL / min, 18 mL / min, 19 mL / min, 20 mL / min, 21 mL / min, 22 mL / min, 23 mL / min, 24 mL / min, or 25 mL / min.

[0022] More preferably, in step (a), the lipid solution and the first mixed solution are mixed at a flow rate ratio of (2.5~3.5):1, with a total flow rate of 15~20 mL / min.

[0023] In some embodiments, in step (ii), both the liquid exchange and the concentration are performed using an ultrafiltration centrifugation tube with a molecular weight cutoff of 50-150 kDa. More preferably, an ultrafiltration tube with a molecular weight cutoff of 80-120 kDa is used; even more preferably, an ultrafiltration tube with a molecular weight cutoff of 90-110 kDa is used.

[0024] In some implementations, the centrifugal force used in step (ii) is 1500 ×g to 3000 ×g, specifically such as 1500 ×g, 1600 ×g, 1700 ×g, 1800 ×g, 1900 ×g, 2000 ×g, 2100 ×g, 2200 ×g, 2300 ×g, 2400 ×g, 2500 ×g, 2600 ×g, 2700 ×g, 2800 ×g, 2900 ×g, or 3000 ×g.

[0025] In some embodiments, in step (ii), the mixture is diluted 2 to 5 times with the second buffer solution, and then the mixture is exchanged 80 to 120 times with the second buffer solution to obtain the exchanged product.

[0026] More preferably, in step (ii), the mixture is diluted 2 to 4 times with the second buffer solution, and then the mixture is exchanged 90 to 110 times with the second buffer solution to obtain the exchanged product.

[0027] In some embodiments, in step (ii), the total lipid concentration of the concentrated product is 5-25 mmol / L, specifically 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L, 21 mmol / L, 22 mmol / L, 23 mmol / L, 24 mmol / L, or 25 mmol / L. More preferably, the total lipid concentration of the concentrated product is 5-20 mmol / L, even more preferably 5-15 mmol / L, and particularly preferably 6-10 mmol / L.

[0028] In some embodiments, in step (iii), the concentrated product is mixed with a sucrose solution with a concentration of 160~400 mg / mL at a volume ratio of (0.8~1.2):1, and then mixed with a nucleic acid solution with a concentration of 0.5~1 mg / mL and incubated.

[0029] More preferably, in step (iii), the concentrated product is mixed with a sucrose solution with a concentration of 250~400 mg / mL at a volume ratio of (0.8~1.2):1.

[0030] More preferably, in step (iii), the concentrated product is mixed with a sucrose solution with a concentration of 300~350mg / mL at a volume ratio of (0.8~1.2):1.

[0031] In some embodiments, in step (iii), the incubation solution obtained from the incubation is mixed with a third buffer solution with a pH of 7 to 7.5 and then stored.

[0032] More preferably, the third buffer solution is a Tris-HCl buffer solution.

[0033] More preferably, the buffer salt concentration of the third buffer solution is 10~50mM, specifically 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, or 50 mM. More preferably, the concentration of the buffer salt in the third buffer solution is 10-30 mM, and even more preferably 15-25 mM.

[0034] In some specific and preferred embodiments, the preparation method includes the following steps: (1) Provide a lipid solution with a total lipid concentration of 5-25 mmol / L and an alcohol solvent, and provide a first buffer solution with a pH of 3.6-4.5; (2) The lipid solution and the first buffer solution are mixed at a flow rate ratio of (1~5):1 and a total flow rate of 15~25 mL / min to obtain a mixture; (3) Dilute the mixture 2 to 5 times with a second buffer solution to obtain a diluted product; (4) First, the diluted product is replaced with the second buffer solution at a volume of 80 to 120 times to obtain the replacement product. Then, the replacement product is concentrated to a total lipid concentration of 5 to 25 mmol / L to obtain the concentrated product. Both the replacement and the ultrafiltration are performed by ultrafiltration centrifugation using an ultrafiltration tube with a molecular weight cutoff of 50 to 150 kDa. (5) The concentrated product is mixed with a sucrose solution with a concentration of 160~400 mg / mL at a volume ratio of (0.8~1.2):1, and then mixed with a nucleic acid solution with a concentration of 0.5~1 mg / mL at 25~37℃ and incubated. The resulting incubation solution is mixed with a third buffer solution with a pH of 7~7.5 and stored.

[0035] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: This invention optimizes the overall process steps and parameters of the preparation method, achieving high encapsulation efficiency of nucleic acid drugs while successfully obtaining lipid nanoparticle formulations with moderate particle size, uniform distribution, and excellent batch-to-batch consistency. The post-encapsulation strategy effectively overcomes the problems of easy loss of nucleic acid efficacy and difficulty in controlling batch-to-batch consistency in the traditional lipid nanoparticle preparation process, further enhancing the stability of the formulation and the flexibility of the process.

[0036] Furthermore, the preparation method of this invention requires no hazardous chemical reagents throughout the entire process, ensuring high process safety and ease of operation. The post-encapsulation technology, combined with an efficient concentration and liquid exchange strategy, significantly reduces the difficulty of process scale-up and energy consumption, facilitating large-scale production, effectively improving production efficiency, and reducing overall costs. Attached Figure Description

[0037] Figure 1 The particle size distribution of nucleic acid-lipid nanoparticles in three batches of nucleic acid-lipid nanoparticle solutions in Example 1 is shown. Figure 2 The particle size distribution of nucleic acid-lipid nanoparticles in three batches of nucleic acid-lipid nanoparticle solutions in Example 3 is shown. Figure 3 The particle size distribution of nucleic acid-lipid nanoparticles in three batches of nucleic acid-lipid nanoparticle solutions in Comparative Example 1 is shown. Figure 4 The particle size distribution of nucleic acid-lipid nanoparticles in the three batches of nucleic acid-lipid nanoparticle solutions in Comparative Example 3 is shown. Detailed Implementation

[0038] To address the problems of easy loss of nucleic acid molecules, difficulty in controlling batch-to-batch consistency, and low flexibility in traditional methods for preparing nucleic acid-lipid nanoparticle drugs, the applicant has developed a post-encapsulation strategy. Through extensive research on optimizing the overall process steps and parameters, and after numerous experimental verifications, the applicant has arrived at the technical solution proposed in this invention: a method for preparing nucleic acid-lipid nanoparticles, comprising the following steps: (i) A mixture is obtained by mixing a lipid solution with an alcohol solvent and a first buffer solution with a pH of 3.6 to 4.5 using a microfluidic mixing device; (ii) The mixture is replaced with a second buffer solution with a pH of 5.5 to 6.5 to remove the alcohol solvent from the mixture, and the resulting product is concentrated to obtain a concentrated product; (iii) The concentrated product and nucleic acid are mixed and incubated at 25-37°C in the presence of a protective agent at a nitrogen-to-phosphorus ratio of 15-35 to obtain the nucleic acid-lipid nanoparticles.

[0039] Furthermore, based on the optimization of each process step, the more detailed preparation method is as follows: (1) Provide a lipid solution with a total lipid concentration of 5-25 mmol / L and an alcohol solvent, and provide a first buffer solution with a pH of 3.6-4.5; (2) The lipid solution and the first buffer solution are mixed at a flow rate ratio of (1~5):1 and a total flow rate of 15~25 mL / min to obtain a mixture; (3) Dilute the mixture 2 to 5 times with a second buffer solution to obtain a diluted product; (4) First, the diluted product is replaced with the second buffer solution at a volume of 80 to 120 times to obtain the replacement product. Then, the replacement product is concentrated to a total lipid concentration of 5 to 25 mmol / L to obtain the concentrated product. Both the replacement and the ultrafiltration are performed by ultrafiltration centrifugation using an ultrafiltration tube with a molecular weight cutoff of 50 to 150 kDa. (5) The concentrated product is mixed with a sucrose solution with a concentration of 160~400 mg / mL at a volume ratio of (0.8~1.2):1, and then mixed with a nucleic acid solution with a concentration of 0.5~1 mg / mL at 25~37℃ and incubated. The resulting incubation solution is mixed with a third buffer solution with a pH of 7~7.5 and stored.

[0040] Existing experiments have confirmed that the nucleic acid-lipid nanoparticle preparation method of this invention can efficiently encapsulate nucleic acids, achieving advantages such as moderate particle size and uniform distribution. The post-encapsulation strategy improves the effective utilization rate of nucleic acid molecules, ensures drug efficacy, and enhances the flexibility of nucleic acid drug preparation. This invention provides a high-quality nucleic acid-lipid nanoparticle drug preparation scheme with high flexibility, high material utilization, excellent product quality, and ease of scalability, which is of great significance to the development and industrial-scale production of nucleic acid-lipid nanoparticle drugs.

[0041] The present invention will be further described below with reference to embodiments and comparative examples. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0042] The sources of some of the raw materials and instruments used in the following examples and comparative examples are as follows: Dlin-MC3-DMA: DLin-MC3-dimethylamine, purchased from Sinobond Biotechnology, catalog number 06040008900; DOTAP-Cl: 1,2-dioleoyl-3-trimethylammonium-propane in chloride form, purchased from Sinobo Bio, catalog number 06040009900; mPEG-DMG-2K is named methoxy-polyethylene glycol-dilauroyl glycerol (molecular weight 2000), purchased from Sinobond Biotechnology, catalog number 06020112402; Cholesterol, purchased from Sinobond Biotechnology, product number 06040010300; DSPC: Distearylphosphatidylcholine, purchased from Lipoid GmbH, Germany, product number 556500; Anhydrous ethanol was purchased from Nanjing Chemical Reagent Co., Ltd., product number C0691555010; Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) was purchased from Jiangsu Hanstone Pharmaceutical Co., Ltd., product number A03002; Tris(hydroxymethyl)aminomethane (free base, Tris) was purchased from Merck, Germany, product number 1.08386.1000; Sodium citrate dihydrate was purchased from Gedian Renfu, product number F323C220301; Citric acid monohydrate was purchased from Renfu in Gedian, product number F301C220701; Sodium acetate trihydrate was purchased from Nanjing Chemical Reagent Co., Ltd., product number C0130665023; Glacial acetic acid was purchased from Nanjing Chemical Reagent Co., Ltd., product number C0680675223; Hydroxyethylpiperazine ethanesulfonic acid (HEPES) was purchased from Aivito Pharmaceutical Technology Co., Ltd., product number F20220000151; The DNA and RNA were prepared in the laboratory. The DNA length ranged from 4000 to 5000 bp, and the RNA length ranged from 1000 to 4000 nt.

[0043] The purified water was prepared in the laboratory. The MaxMix mixer is a mixing device independently developed by Kaituo Biotechnology. Specifically, it is the lipid nanoparticle preparation mixer disclosed in patent CN 220968881 U, with a mixing channel inner diameter of 0.5 mm. The ultrafiltration tubes were purchased from Hangzhou Kebaite Filter Material Co., Ltd., with a molecular weight cutoff of 100 kDa. The nanoparticle size analyzer used was a Malvern Panalytical Zetasizer Pro.

[0044] The multi-functional microplate reader is the SpectraMax Id5 from Meigu Molecular Instruments Co., Ltd.

[0045] In the following examples and comparative examples, when preparing the buffer solution, 1M NaOH solution and 1M HCl solution were used to adjust the pH value of the buffer solution to the required pH value.

[0046] Example 1 This embodiment provides a nucleic acid-lipid nanoparticle product, the preparation method of which is as follows: Organic phase preparation: Dlin-MC3-DMA, DSPC, Cholesterol and mPEG-DMG-2K were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 and incubated in a water bath at 40°C for 30 min to obtain a four-component lipid solution with a concentration of 8 mmol / L.

[0047] Preparation of aqueous phase: Based on the content of 1.8 g sodium acetate trihydrate and 0.4 g acetic acid per liter of purified water, weigh out sodium acetate trihydrate and acetic acid respectively, add them to an appropriate amount of purified water, dissolve them and adjust the pH to 4.0 to obtain 20 mM acetate buffer.

[0048] Mixing: Using a MaxMix mixer, the organic phase (MC3 lipid solution) and the aqueous phase (20 mM acetate buffer) were mixed at a flow rate ratio of 3:1, with a total flow rate of 16 mL / min to obtain the mixed product.

[0049] Dilution: The mixture was diluted 3-fold with 20 mM Tris-HCl buffer at pH 6.0 to obtain the diluted product.

[0050] Ultrafiltration centrifugation and liquid exchange: Transfer the diluted product to an ultrafiltration tube (molecular weight cutoff of 100 kDa), and exchange the diluted product 100 times with 20 mM Tris-HCl buffer at pH 6.0 under a centrifugal force of 2000 × g to obtain the liquid exchanged product.

[0051] Ultrafiltration centrifugation concentration: Continue to concentrate the liquid exchange product at a centrifugal force of 2000 ×g to obtain a concentrated product with a lipid concentration of 8 mmol / L.

[0052] Encapsulating RNA: Add an equal volume of sucrose solution with a concentration of 320 mg / mL to the concentrated product, and add RNA solution under vortex conditions at a nitrogen-to-phosphorus ratio of 30. After incubation at 37°C for 5 min, add an equal volume of 20 mM Tris-HCl buffer with a pH of 7.4 to obtain a nucleic acid-lipid nanoparticle solution.

[0053] The three batches of nucleic acid-lipid nanoparticle solutions prepared according to the above preparation method were mixed to obtain nucleic acid-lipid nanoparticle products. Nanoparticle size analysis was performed on them, and the target nucleic acid encapsulation rate was detected by a multifunctional microplate reader.

[0054] Example 2 This embodiment provides a nucleic acid-lipid nanoparticle product, the preparation method of which is as follows: Organic phase preparation: Dlin-MC3-DMA, DSPC, Cholesterol and mPEG-DMG-2K were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 and incubated in a water bath at 40°C for 30 min to obtain a four-component lipid solution with a concentration of 8 mmol / L.

[0055] Preparation of aqueous phase: Based on the content of 1.8 g sodium acetate trihydrate and 0.4 g acetic acid per liter of purified water, weigh out sodium acetate trihydrate and acetic acid respectively, add them to an appropriate amount of purified water, dissolve them and adjust the pH to 4.0 to obtain 20 mM acetate buffer.

[0056] Mixing: Using a MaxMix mixer, the organic phase (MC3 lipid solution) and the aqueous phase (20 mM acetate buffer) were mixed at a flow rate ratio of 3:1, with a total flow rate of 16 mL / min to obtain the mixed product.

[0057] Dilution: The mixture was diluted 3-fold with 20 mM Tris-HCl buffer at pH 6.0 to obtain the diluted product.

[0058] Ultrafiltration centrifugation and liquid exchange: Transfer the diluted product to an ultrafiltration tube (molecular weight cutoff of 100 kDa), and exchange the diluted product 100 times with 20 mM Tris-HCl buffer at pH 6.0 under a centrifugal force of 2000 × g to obtain the liquid exchanged product.

[0059] Ultrafiltration centrifugation concentration: Continue to concentrate the liquid exchange product at a centrifugal force of 2000 ×g to obtain a concentrated product with a lipid concentration of 8 mmol / L.

[0060] Encapsulating RNA: Add an equal volume of sucrose solution with a concentration of 320 mg / mL to the concentrated product, and add RNA solution under vortex conditions at a nitrogen-to-phosphorus ratio of 30. After incubation at 25°C for 30 min, add an equal volume of 20 mM Tris-HCl buffer with a pH of 7.4 to obtain a nucleic acid-lipid nanoparticle solution.

[0061] The three batches of nucleic acid-lipid nanoparticle solutions prepared according to the above preparation method were mixed to obtain nucleic acid-lipid nanoparticle products. Nanoparticle size analysis was performed on them, and the target nucleic acid encapsulation rate was detected by a multifunctional microplate reader.

[0062] Example 3 This embodiment provides a nucleic acid-lipid nanoparticle product, the preparation method of which is as follows: Organic phase preparation: DOTAP-Cl, DSPC, Cholesterol and mPEG-DMG-2K were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 and incubated in a water bath at 40°C for 30 min to obtain a four-component lipid solution with a concentration of 8 mmol / L.

[0063] Preparation of aqueous phase: Based on the content of 2.4 g sodium citrate dihydrate and 2.5 g sodium citrate monohydrate per liter of purified water, weigh out sodium citrate dihydrate and sodium citrate monohydrate respectively, add them to an appropriate amount of purified water, dissolve them and adjust the pH to 4.0 to obtain 20 mM citrate buffer.

[0064] Mixing: Using a MaxMix mixer, the organic phase (DOTAP lipid solution) and the aqueous phase (20 mM citrate buffer) were mixed at a flow rate ratio of 3:1, with a total flow rate of 32 mL / min to obtain the mixed product.

[0065] Dilution: The mixture was diluted 3-fold with 20 mM Tris-HCl buffer at pH 6.0 to obtain the diluted product.

[0066] Ultrafiltration centrifugation and liquid exchange: Transfer the diluted product to an ultrafiltration tube (molecular weight cutoff of 100 kDa), and exchange the diluted product 100 times with 20 mM Tris-HCl buffer at pH 6.0 under a centrifugal force of 2000 × g to obtain the liquid exchanged product.

[0067] Ultrafiltration centrifugation concentration: Continue to concentrate the liquid exchange product at a centrifugal force of 2000 ×g to obtain a concentrated product with a lipid concentration of 8 mmol / L.

[0068] DNA encapsulation: Add an equal volume of sucrose solution with a concentration of 320 mg / mL to the concentrated product, and add DNA solution under vortex conditions at a nitrogen-to-phosphorus ratio of 20. After incubation at 25°C for 30 min, add an equal volume of 20 mM Tris-HCl buffer with a pH of 7.4 to obtain a nucleic acid-lipid nanoparticle solution.

[0069] The three batches of nucleic acid-lipid nanoparticle solutions prepared according to the above preparation method were mixed to obtain nucleic acid-lipid nanoparticle products. Nanoparticle size analysis was performed on them, and the target nucleic acid encapsulation rate was detected by a multifunctional microplate reader.

[0070] Example 4 This embodiment provides a nucleic acid-lipid nanoparticle product, the preparation method of which is as follows: Organic phase preparation: DOTAP-Cl, DSPC, Cholesterol and mPEG-DMG-2K were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 and incubated in a water bath at 40°C for 30 min to obtain a four-component lipid solution with a concentration of 8 mmol / L.

[0071] Preparation of aqueous phase: Weigh out sodium citrate dihydrate and sodium citrate monohydrate according to the content of 2.4 g sodium citrate dihydrate and 2.5 g sodium citrate monohydrate per liter of purified water, add them to an appropriate amount of purified water, dissolve them and adjust the pH to 4.0 to obtain 20 mM citrate buffer solution.

[0072] Mixing: Using a MaxMix mixer, the organic phase (DOTAP lipid solution) and the aqueous phase (20 mM citrate buffer) were mixed at a flow rate ratio of 3:1, with a total flow rate of 16 mL / min to obtain the mixed product.

[0073] Dilution: The mixture was diluted 3-fold with 20 mM Tris-HCl buffer at pH 6.0 to obtain the diluted product.

[0074] Ultrafiltration centrifugation and liquid exchange: Transfer the diluted product to an ultrafiltration tube (molecular weight cutoff of 100 kDa), and exchange the diluted product 100 times with 20 mM Tris-HCl buffer at pH 6.0 under a centrifugal force of 2000 × g to obtain the liquid exchanged product.

[0075] Ultrafiltration centrifugation concentration: Continue to concentrate the liquid exchange product at a centrifugal force of 2000 ×g to obtain a concentrated product with a lipid concentration of 8 mmol / L.

[0076] DNA encapsulation: Add an equal volume of 320 mg / mL sucrose solution to the concentrated product, and add DNA solution under vortex conditions at a nitrogen-to-phosphorus ratio of 30. After incubation at 25°C for 30 min, add an equal volume of 20 mM Tris-HCl buffer with a pH of 7.4 to obtain a nucleic acid-lipid nanoparticle solution.

[0077] The three batches of nucleic acid-lipid nanoparticle solutions prepared according to the above preparation method were mixed to obtain nucleic acid-lipid nanoparticle products. Nanoparticle size analysis was performed on them, and the target nucleic acid encapsulation rate was detected by a multifunctional microplate reader.

[0078] Comparative Example 1 This comparative example provides a nucleic acid-lipid nanoparticle product, the preparation method of which is as follows: Organic phase preparation: Dlin-MC3-DMA, DSPC, Cholesterol and mPEG-DMG-2K were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 and incubated in a water bath at 40°C for 30 min to obtain a four-component lipid solution with a concentration of 8 mmol / L.

[0079] Preparation of aqueous phase: Based on the content of 1.8 g sodium acetate trihydrate and 0.4 g acetic acid per liter of purified water, weigh out sodium acetate trihydrate and acetic acid respectively, add them to an appropriate amount of purified water, dissolve them and adjust the pH to 4.0 to obtain 20 mM acetate buffer.

[0080] Mixing: Using a MaxMix mixer, the organic phase (MC3 lipid solution) and the aqueous phase (20 mM citrate buffer) were mixed at a flow rate ratio of 7:1, with a total flow rate of 16 mL / min to obtain the mixed product.

[0081] Dilution: The mixture was diluted 3-fold with 20 mM Tris-HCl buffer at pH 6.0 to obtain the diluted product.

[0082] Ultrafiltration centrifugation and liquid exchange: Transfer the diluted product to an ultrafiltration tube (molecular weight cutoff of 100 kDa), and exchange the diluted product 100 times with 20 mM Tris-HCl buffer at pH 6.0 under a centrifugal force of 2000 × g to obtain the liquid exchanged product.

[0083] Ultrafiltration centrifugation concentration: Continue to concentrate the liquid exchange product at a centrifugal force of 2000 ×g to obtain a concentrated product with a lipid concentration of 8 mmol / L.

[0084] Encapsulating RNA: Add an equal volume of sucrose solution with a concentration of 320 mg / mL to the concentrated product, and add RNA solution under vortex conditions at a nitrogen-to-phosphorus ratio of 30. After incubation at 37°C for 5 min, add an equal volume of 20 mM Tris-HCl buffer with a pH of 7.4 to obtain a nucleic acid-lipid nanoparticle solution.

[0085] The three batches of nucleic acid-lipid nanoparticle solutions prepared according to the above preparation method were mixed to obtain nucleic acid-lipid nanoparticle products. Nanoparticle size analysis was performed on them, and the target nucleic acid encapsulation rate was detected by a multifunctional microplate reader.

[0086] Comparative Example 2 This comparative example provides a nucleic acid-lipid nanoparticle product, the preparation method of which is as follows: Organic phase preparation: Dlin-MC3-DMA, DSPC, Cholesterol and mPEG-DMG-2K were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 and incubated in a water bath at 40°C for 30 min to obtain a four-component lipid solution with a concentration of 8 mmol / L.

[0087] Preparation of aqueous phase: Based on the content of 1.8 g sodium acetate trihydrate and 0.4 g acetic acid per liter of purified water, weigh out sodium acetate trihydrate and acetic acid respectively, add them to an appropriate amount of purified water, dissolve them and adjust the pH to 4.0 to obtain 20 mM acetate buffer.

[0088] Mixing: Using a MaxMix mixer, the organic phase (MC3 lipid solution) and the aqueous phase (20 mM acetate buffer) were mixed at a flow rate ratio of 3:1, with a total flow rate of 16 mL / min to obtain the mixed product.

[0089] Dilution: The mixture was diluted 3-fold with 20 mM Tris-HCl buffer at pH 6.0 to obtain the diluted product.

[0090] Ultrafiltration centrifugation and liquid exchange: Transfer the diluted product to an ultrafiltration tube (molecular weight cutoff of 100 kDa), and exchange the diluted product 100 times with 20 mM Tris-HCl buffer at pH 6.0 under a centrifugal force of 2000 × g to obtain the liquid exchanged product.

[0091] Ultrafiltration centrifugation concentration: Continue to concentrate the liquid exchange product at a centrifugal force of 2000 ×g to obtain a concentrated product with a lipid concentration of 8 mmol / L.

[0092] Encapsulating RNA: Add an equal volume of sucrose solution with a concentration of 320 mg / mL to the concentrated product, and add RNA solution under vortex conditions at a nitrogen-to-phosphorus ratio of 6. After incubation at 25°C for 30 min, add an equal volume of 20 mM Tris-HCl buffer with a pH of 7.4 to obtain a nucleic acid-lipid nanoparticle solution.

[0093] The three batches of nucleic acid-lipid nanoparticle solutions prepared according to the above preparation method were mixed to obtain nucleic acid-lipid nanoparticle products. Nanoparticle size analysis was performed on them, and the target nucleic acid encapsulation rate was detected by a multifunctional microplate reader.

[0094] Comparative Example 3 This comparative example provides a nucleic acid-lipid nanoparticle product, the preparation method of which is as follows: Organic phase preparation: DOTAP-Cl, DSPC, Cholesterol and mPEG-DMG-2K were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 and incubated in a water bath at 40°C for 30 min to obtain a four-component lipid solution with a concentration of 8 mmol / L.

[0095] Preparation of aqueous phase: Weigh out sodium citrate dihydrate and sodium citrate monohydrate according to the content of 2.4 g sodium citrate dihydrate and 2.5 g sodium citrate monohydrate per liter of purified water, add them to an appropriate amount of purified water, dissolve them and adjust the pH to 4.0 to obtain 20 mM citrate buffer solution.

[0096] Mixing: Using a MaxMix mixer, the organic phase (DOTAP lipid solution) and the aqueous phase (20 mM citrate buffer) were mixed at a flow rate ratio of 3:1, with a total flow rate of 16 mL / min to obtain the mixed product.

[0097] Dilution: The mixture was diluted 3-fold with 20 mM Tris-HCl buffer at pH 6.0 to obtain the diluted product.

[0098] Ultrafiltration centrifugation and liquid exchange: Transfer the diluted product to an ultrafiltration tube (molecular weight cutoff of 100 kDa), and exchange the diluted product 100 times with 20 mM Tris-HCl buffer at pH 6.0 under a centrifugal force of 2000 × g to obtain the liquid exchanged product.

[0099] Ultrafiltration centrifugation concentration: Continue to concentrate the liquid exchange product at a centrifugal force of 2000 ×g to obtain a concentrated product with a lipid concentration of 8 mmol / L.

[0100] DNA encapsulation: Add an equal volume of sucrose solution with a concentration of 320 mg / mL to the concentrated product, and add DNA solution under vortex conditions at a nitrogen-to-phosphorus ratio of 20. After incubation at 25°C for 30 min, add an equal volume of 20 mM Tris-HCl buffer with a pH of 7.4 to obtain a nucleic acid-lipid nanoparticle solution.

[0101] The three batches of nucleic acid-lipid nanoparticle solutions prepared according to the above preparation method were mixed to obtain nucleic acid-lipid nanoparticle products. Nanoparticle size analysis was performed on them, and the target nucleic acid encapsulation rate was detected by a multifunctional microplate reader.

[0102] Comparative Example 4 This comparative example provides a nucleic acid-lipid nanoparticle product, the preparation method of which is as follows: Organic phase preparation: DOTAP-Cl, DSPC, Cholesterol and mPEG-DMG-2K were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 and incubated in a water bath at 40°C for 30 min to obtain a four-component lipid solution with a concentration of 8 mmol / L.

[0103] Preparation of aqueous phase: According to the content of 2.4 g sodium citrate dihydrate and 2.5 g sodium citrate monohydrate per liter of purified water, weigh out sodium citrate dihydrate and sodium citrate monohydrate respectively, add them to an appropriate amount of purified water, dissolve them and adjust the pH to 4.0 to obtain 20 mM citrate buffer. Mixing: Using a MaxMix mixer, the organic phase (DOTAP lipid solution) and the aqueous phase (20 mM citrate buffer) were mixed at a flow rate ratio of 3:1, with a total flow rate of 16 mL / min to obtain the mixed product.

[0104] Dilution: The mixture was diluted 3-fold with 20 mM HEPES buffer at pH 6.0 to obtain the diluted product.

[0105] Ultrafiltration centrifugation and fluid exchange: Transfer the diluted product to an ultrafiltration tube (molecular weight cutoff of 100 kDa), and perform a 100-fold volume change of the diluted product using 20 mM HEPES buffer with a pH of 6.0 at a centrifugal force of 2000 × g to obtain the fluid exchanged product.

[0106] Ultrafiltration centrifugation concentration: Continue to concentrate the liquid exchange product at a centrifugal force of 2000 ×g to obtain a concentrated product with a lipid concentration of 8 mmol / L.

[0107] DNA encapsulation: Add an equal volume of sucrose solution with a concentration of 320 mg / mL to the concentrated product, and add DNA solution under vortex conditions at a nitrogen-to-phosphorus ratio of 30. After incubation at 25°C for 30 min, add an equal volume of 20 mM Tris-HCl buffer with a pH of 7.4 to obtain a nucleic acid-lipid nanoparticle solution.

[0108] The three batches of nucleic acid-lipid nanoparticle solutions prepared according to the above preparation method were mixed to obtain nucleic acid-lipid nanoparticle products. Nanoparticle size analysis was performed on them, and the target nucleic acid encapsulation rate was detected by a multifunctional microplate reader.

[0109] Comparative Example 5 This comparative example provides a nucleic acid-lipid nanoparticle product, the preparation method of which is as follows: Organic phase preparation: Dlin-MC3-DMA, DSPC, Cholesterol and mPEG-DMG-2K were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 and incubated in a water bath at 40°C for 30 min to obtain a four-component lipid solution with a concentration of 8 mmol / L.

[0110] Preparation of aqueous phase: Based on the content of 1.8 g sodium acetate trihydrate and 0.4 g acetic acid per liter of purified water, weigh out sodium acetate trihydrate and acetic acid respectively, add them to an appropriate amount of purified water, dissolve them and adjust the pH to 4.0 to obtain 20 mM acetate buffer.

[0111] Mixing: Using a MaxMix mixer, the organic phase (MC3 lipid solution) and the aqueous phase (20 mM acetate buffer) were mixed at a flow rate ratio of 3:1, with a total flow rate of 16 mL / min to obtain the mixed product.

[0112] Dilution: The mixture was diluted 3-fold with 20 mM Tris-HCl buffer at pH 6.0 to obtain the diluted product.

[0113] Ultrafiltration centrifugation and liquid exchange: Transfer the diluted product to an ultrafiltration tube (molecular weight cutoff of 100 kDa), and exchange the diluted product 100 times with 20 mM Tris-HCl buffer at pH 6.0 under a centrifugal force of 2000 × g to obtain the liquid exchanged product.

[0114] Ultrafiltration centrifugation concentration: Continue to concentrate the liquid exchange product at a centrifugal force of 2000 ×g to obtain a concentrated product with a lipid concentration of 8 mmol / L.

[0115] Encapsulating RNA: Add an equal volume of sucrose solution with a concentration of 150 mg / mL to the concentrated product, and add RNA solution under vortex conditions at a nitrogen-to-phosphorus ratio of 30. After incubation at 37°C for 5 min, add an equal volume of 20 mM Tris-HCl buffer with a pH of 7.4 to obtain a nucleic acid-lipid nanoparticle solution.

[0116] Comparative Example 6 This comparative example provides a nucleic acid-lipid nanoparticle product, the preparation method of which is as follows: Organic phase preparation: Dlin-MC3-DMA, DSPC, Cholesterol and mPEG-DMG-2K were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 and incubated in a water bath at 40°C for 30 min to obtain a four-component lipid solution with a concentration of 8 mmol / L.

[0117] Preparation of aqueous phase: Based on the content of 1.8 g sodium acetate trihydrate and 0.4 g acetic acid per liter of purified water, weigh out sodium acetate trihydrate and acetic acid respectively, add them to an appropriate amount of purified water, dissolve them and adjust the pH to 4.0 to obtain 20 mM acetate buffer.

[0118] Mixing: Using a MaxMix mixer, the organic phase (MC3 lipid solution) and the aqueous phase (20 mM acetate buffer) were mixed at a flow rate ratio of 3:1, with a total flow rate of 16 mL / min to obtain the mixed product.

[0119] Ultrafiltration centrifugation and liquid exchange: Transfer the mixed product to an ultrafiltration tube (molecular weight cutoff of 100 kDa), and use 20 mM Tris-HCl buffer with a pH of 6.0 to exchange the diluted product 300 times at a centrifugal force of 2000 × g, to obtain the liquid exchanged product.

[0120] Ultrafiltration centrifugation concentration: Continue to concentrate the liquid exchange product at a centrifugal force of 2000 ×g to obtain a concentrated product with a lipid concentration of 8 mmol / L.

[0121] Encapsulating RNA: Add an equal volume of sucrose solution with a concentration of 320 mg / mL to the concentrated product, and add RNA solution under vortex conditions at a nitrogen-to-phosphorus ratio of 30. After incubation at 37°C for 5 min, add an equal volume of 20 mM Tris-HCl buffer with a pH of 7.4 to obtain a nucleic acid-lipid nanoparticle solution.

[0122] The three batches of nucleic acid-lipid nanoparticle solutions prepared according to the above preparation method were mixed to obtain nucleic acid-lipid nanoparticle products. Nanoparticle size analysis was performed on them, and the target nucleic acid encapsulation rate was detected by a multifunctional microplate reader.

[0123] The three batches of nucleic acid-lipid nanoparticle solutions prepared according to the above preparation method were mixed to obtain nucleic acid-lipid nanoparticle products. Nanoparticle size analysis was performed on them, and the target nucleic acid encapsulation rate was detected by a multifunctional microplate reader.

[0124] The average particle size, PDI, and encapsulation efficiency of the final nucleic acid-lipid nanoparticle products from the above examples and comparative examples are shown in Table 1. The particle size distribution of nucleic acid-lipid nanoparticles in the three batches of nucleic acid-lipid nanoparticle solutions from Examples 1, 3, Comparative Examples 1 and 3 is shown in Table 1. Figures 1-4 .

[0125] Table 1 According to Table 1, the lipid nanoparticles of Examples 1 and 2 have an average particle size of approximately 90 nm and a polydispersity index (PDI) ≤ 0.15. The lipid nanoparticles of Examples 3 and 4 have an average particle size of approximately 117 nm and a PDI < 0.25. All examples have an encapsulation efficiency of over 90%, with Examples 3 and 4 reaching 99.2%. Comparative Examples 1, 2, 5, and 6, although all have a PDI < 0.15, have slightly larger average particle sizes and significantly lower encapsulation efficiencies. Comparative Examples 3 and 4, while having encapsulation efficiencies comparable to Examples 3 and 4, have significantly larger average particle sizes, significantly higher PDIs, and poorer batch-to-batch reproducibility. Compared to the comparative examples, each example, while ensuring encapsulation efficiency, also achieves smaller particle sizes, better uniformity, and batch-to-batch reproducibility of the nucleic acid-lipid nanoparticles, resulting in better quality.

[0126] Furthermore, the preparation process of the nucleic acid-lipid nanoparticle products in each embodiment does not involve hazardous chemicals, ensuring high safety, simple operation, and low difficulty in scale-up production, thus effectively improving production efficiency. More importantly, compared with traditional preparation methods, each embodiment places nucleic acid encapsulation at the end of the preparation process, significantly reducing the waste and damage of nucleic acid drug active molecules in traditional preparation methods, improving the flexibility and stability of lipid nanoparticle formulations, and effectively solving the problems of narrow application range, high storage requirements, and short shelf life of single formulations.

[0127] The above embodiments are merely preferred examples of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications or improvements made without departing from the spirit of the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A method of preparing a nucleic acid-lipid nanoparticle, characterized by: It comprises the following steps: (1) using a microfluidic mixing device to mix a lipid solution with a solvent being an alcohol solvent and a first buffer solution with a pH value of 3.6-4.5 according to a flow rate ratio of the lipid solution to the first buffer solution being (2-4):1 and a total flow rate of 15-25 mL / min to obtain a mixture, the lipid solution containing cationic lipids, auxiliary phospholipids, cholesterol and PEGylated lipids in a molar ratio of (40-60):(5-15):(35-40):(1-2), and the total lipid concentration of the lipid solution being 5-25 mmol / L; (2) using a second buffer solution with a pH value of 5.5-6.5 to exchange the alcohol solvent in the mixture to remove the alcohol solvent in the mixture, and concentrating the obtained exchange product to obtain a concentrated product; (3) mixing and incubating the concentrated product and nucleic acids in the presence of a protective agent according to a nitrogen-phosphorus ratio of 15-35 at 25-37℃ to obtain the nucleic acid-lipid nanoparticle.

2. The method of claim 1, wherein the nucleic acid-lipid nanoparticle is prepared by: The pH value of the first buffer solution is 3.8-4.2; And / or, the pH value of the second buffer solution is 5.8-6.

2.

3. The method of claim 1, wherein the nucleic acid-lipid nanoparticle is prepared by: The first buffer solution is an acetate buffer solution or a citrate buffer solution; And / or, the second buffer solution is a Tris-HCl buffer solution; And / or, the buffer salt concentration of the first buffer solution and the second buffer solution is independently 10-50 mM.

4. The method of claim 1, wherein the nucleic acid-lipid nanoparticle is prepared by: The lipid solution contains cationic lipids, auxiliary phospholipids, cholesterol and PEGylated lipids; And / or, the alcohol solvent is ethanol and / or tert-butyl alcohol; And / or, the protective agent is one or more of sucrose, sodium chloride and mannitol; And / or, the nitrogen-phosphorus ratio is 20-30.

5. The method of claim 1, wherein the nucleic acid-lipid nanoparticle is prepared by: When the nucleic acid is RNA, the cationic lipids are Dlin-MC3-DMA, and the first buffer solution is an acetate buffer solution.

6. The method of claim 1, wherein: When the nucleic acid is DNA, the cationic lipids are DOTAP-Cl, the second buffer solution is a citrate buffer solution, the total flow rate is greater, and the nitrogen-phosphorus ratio is smaller.

7. The method of claim 1, wherein the nucleic acid-lipid nanoparticle is prepared by: In step (2), the exchange and the concentration are both performed by ultrafiltration centrifugation using an ultrafiltration tube with a molecular weight cut-off of 50-150 kDa.

8. The method of claim 7, wherein the nucleic acid-lipid nanoparticle is prepared by: In step (2), the centrifugal force used is 1500 ×g-3000 ×g; And / or, in step (2), the mixture is diluted 2-5 times using the second buffer solution, and then the second buffer solution is used for 80-120 times volume exchange to obtain the exchange product; And / or, in step (2), the total lipid concentration of the concentrated product is 5-25 mmol / L.

9. The method of claim 1, wherein the nucleic acid-lipid nanoparticle is prepared by: In step (3), the concentrated product is mixed with a sucrose solution with a concentration of 160-400 mg / mL according to a volume ratio of (0.8-1.2):1, and then mixed with a nucleic acid solution with a concentration of 0.5-1 mg / mL for incubation; And / or, in step (3), the incubation liquid obtained by the incubation is mixed with a third buffer solution with a pH value of 7-7.5 for storage.

10. The method of preparing a nucleic acid-lipid nanoparticle according to any one of claims 1 to 9, wherein: The preparation method comprises the following steps: (1) Provide a lipid solution with a total lipid concentration of 5-25 mmol / L and an alcohol solvent, and provide a first buffer solution with a pH of 3.6-4.5; (2) The lipid solution and the first buffer solution are mixed at a flow rate ratio of (2~4):1 and a total flow rate of 15~25 mL / min to obtain a mixture; (3) Dilute the mixture 2 to 5 times with a second buffer solution to obtain a diluted product; (4) First, the diluted product is replaced with the second buffer solution at a volume of 80 to 120 times to obtain the replacement product. Then, the replacement product is concentrated to a total lipid concentration of 5 to 25 mmol / L to obtain the concentrated product. Both the replacement and the concentration are carried out by ultrafiltration centrifugation using an ultrafiltration tube with a molecular weight cutoff of 50 to 150 kDa. (5) The concentrated product is mixed with a sucrose solution with a concentration of 160~400 mg / mL at a volume ratio of (0.8~1.2):1, and then mixed with a nucleic acid solution with a concentration of 0.5~1 mg / mL at 25~37℃ and incubated. The resulting incubation solution is mixed with a third buffer solution with a pH of 7~7.5 and stored.

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