Method for preparing nucleic acid lipid nanoparticles targeting liver and spleen by ethanol injection method

Abstract

The present invention discloses a method for preparing nucleic acid lipid nanoparticles targeting the liver and spleen by an ethanol injection method. Compared with the traditional microfluidics technology, the present invention adopts an ethanol injection method to add a lipid solution dropwise to complete the preparation of LNP under vortex mixing, thereby simplifying the operation process and reducing the equipment cost. The method of the present invention can fully mix lipids and nucleic acids to form nucleic acid lipid nanoparticles with good particle size and encapsulation rate, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of medicine and biology, and in particular relates to a method for preparing nucleic acid lipid nanoparticles targeting liver or spleen through an ethanol injection method. Background Art

[0002] As the main carrier for delivering nucleic acid drugs, lipid nanoparticles (LNPs) are widely used in fields such as mRNA vaccines and gene therapy due to their high efficiency and biocompatibility. At the same time, the targeted delivery of nucleic acid lipid nanoparticles to specific organs (such as the liver and spleen) is also a hot topic of research.

[0003] Currently, the preparation of lipid nanoparticles relies on microfluidics, which requires sophisticated equipment and strict operating conditions to control the mixing flow rate ratio of lipids and mRNA. This high-precision requirement not only makes the equipment expensive, but also increases the difficulty of operation and the training cost of technicians, making it unsuitable for large-scale production and small-scale laboratory applications. Summary of the invention

[0004] Based on the technical problems existing in the existing lipid nanoparticle (LNP) preparation method, the purpose of the present invention is to provide a method for preparing lipid nanoparticles (LNP) by ethanol injection. To achieve this purpose, the present invention adopts the following technical solutions:

[0005] The first aspect of the present invention provides a method for preparing nucleic acid lipid nanoparticles targeting liver and / or spleen by ethanol injection method, wherein the method comprises:

[0006] The lipid composition is dissolved in ethanol to obtain a lipid solution; the nucleic acid is dissolved in a buffer to obtain a nucleic acid solution; while the nucleic acid solution is stirred or vortexed, the lipid solution is added dropwise to the nucleic acid solution, the volume ratio of the nucleic acid solution to the lipid solution is 7:1-8:1, and nucleic acid lipid nanoparticles are obtained;

[0007] Taking the total molar amount of lipids in the lipid composition as 100%, the lipid composition comprises the following lipid components:

[0008] Ionizable lipids: greater than or equal to 45%, less than or equal to 50%;

[0009] PEG-modified lipids: greater than or equal to 1.5%, less than or equal to 8.5%;

[0010] Steroids: greater than or equal to 33%, less than or equal to 50%;

[0011] Auxiliary phospholipids: greater than or equal to 0% and less than or equal to 16%.

[0012] In some embodiments, based on the total molar amount of lipids in the lipid composition as 100%, the lipid composition includes the following lipid components:

[0013] Ionizable lipids: greater than or equal to 45%, less than or equal to 45.9%;

[0014] PEG-modified lipids: greater than or equal to 5.7%, less than or equal to 8.3%;

[0015] Steroids: greater than or equal to 33%, less than or equal to 46.7%;

[0016] Auxiliary phospholipids: greater than or equal to 0% and less than or equal to 15.3%.

[0017] In some embodiments, based on the total molar amount of lipids in the lipid composition as 100%, the lipid composition includes the following lipid components: ionizable lipids: 45.9%; PEG-modified lipids: 5.7%; steroids: 33%; and auxiliary phospholipids: 15.3%.

[0018] In some embodiments, the lipid composition does not contain auxiliary phospholipids; based on the total molar amount of lipids in the lipid composition as 100%, the lipid composition includes the following lipid components: ionizable lipids: 45%; PEG-modified lipids: 8.3%; steroids: 46.7%.

[0019] In some embodiments, based on the total molar amount of lipids in the lipid composition as 100%, the lipid composition includes the following lipid components: ionizable lipids: 45.3%; auxiliary phospholipids: 9%; PEG-modified lipids: 7.5%; and steroids: 38.2%.

[0020] In some embodiments, based on the total molar amount of lipids in the lipid composition as 100%, the lipid composition includes the following lipid components: ionizable lipids: 50%; auxiliary phospholipids: 10%; PEG-modified lipids: 1.5%; and steroids: 38.5%.

[0021] In some embodiments, the ionizable lipid is SM-102 and / or 8-((7-((3-(hexylthio)-2-methylpropanoyl)oxy)-n-heptyl)(2-hydroxyethyl)amine)octanoate 9-heptadecanol.

[0022] In some embodiments, the accessory phospholipid is DSPC.

[0023] In some embodiments, the PEG-modified lipid is PEG-DMG.

[0024] In some embodiments, the steroid is cholesterol.

[0025] In some embodiments, the pH of the buffer solution is 3.0-5.5, and the buffer solution is a citrate buffer solution.

[0026] In some embodiments, the method further comprises purifying and concentrating the nucleic acid lipid nanoparticles.

[0027] In some embodiments, the purifying comprises dialysis and the concentrating comprises ultrafiltration.

[0028] As a preferred embodiment, the lipid solution is added dropwise to the nucleic acid solution while the nucleic acid solution is vortexed. More preferably, the vortexing makes the liquid level after the vortex is formed to be 4 times the liquid level at rest.

[0029] As a preferred embodiment, while the nucleic acid solution is vortexed, 500 μL of lipid solution is dripped dropwise into the 3.5 mL nucleic acid solution in the vortex state, and the process takes 3 minutes.

[0030] As a preferred embodiment, the volume ratio of the nucleic acid solution to the lipid solution is 7:1.

[0031] The second aspect of the present invention provides a nucleic acid lipid nanoparticle targeting the liver and / or spleen. The nucleic acid lipid nanoparticle targeting the liver and / or spleen is prepared by the method described in the first aspect of the present invention.

[0032] In some embodiments, the lipid nanoparticles have an average particle size of 138 to 156 nm and a potential of -2.5 to -4.4 mV.

[0033] The third aspect of the present invention provides a pharmaceutical preparation, which comprises the nucleic acid lipid nanoparticles described in the second aspect of the present invention.

[0034] The fourth aspect of the present invention provides use of the nucleic acid lipid nanoparticles described in the second aspect of the present invention and the pharmaceutical preparation described in the third aspect of the present invention in the preparation of a drug for targeting nucleic acid delivery to the liver and / or spleen.

[0035] The present invention proposes a method for preparing nucleic acid lipid nanoparticles targeting the liver or spleen based on an ethanol injection method. Compared with the microfluidics method, the present invention has a simple process, low cost, and can achieve targeted delivery to the liver and spleen.

[0036] The present invention prepares lipid nanoparticles (LNP) by ethanol injection method and introduces self-developed new ionizable lipids to optimize the formula, thereby overcoming many defects in the prior art. The specific beneficial effects are as follows:

[0037] 1. Simplify the preparation process:

[0038] The present invention adopts the ethanol injection method, and completes the preparation of LNP by adding lipid solution dropwise to the nucleic acid solution under the stirring of a vortex oscillator or a magnetic stirrer, which significantly simplifies the operation process and reduces the dependence on complex equipment. Compared with traditional microfluidics technology, this method is easier to operate and is suitable for use in laboratories with low budgets. The simplification of this method not only reduces the difficulty of operation, but also reduces the cost of equipment, so that efficient LNP preparation can be carried out in a research environment with limited resources.

[0039] 2. Highly efficient targeting:

[0040] By introducing a novel ionizable lipid developed independently and optimizing the lipid formula, the LNP prepared by the present invention shows significant liver and / or spleen targeting in animal experiments. The optimized formula can more effectively target the liver / spleen in vivo, improving the efficiency and specificity of drug delivery. This method is mainly suitable for preliminary exploration of laboratory research and provides important technical support in the research stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a flow chart of the present invention;

[0042] Figure 2 This is the fluorescent imaging of the liver and spleen of mice 24 hours after intravenous injection;

[0043] Figure 3 This is a statistical graph of the fluorescence intensity of the liver and spleen of mice 24 hours after intravenous injection. DETAILED DESCRIPTION

[0044] Definitions of Abbreviations and Key Terms in the Present Invention

[0045] Abbreviations

[0046] LNP: Lipid Nanoparticles, a nanocarrier used to deliver nucleic acid drugs.

[0047] mRNA: Messenger Ribonucleic Acid, an RNA molecule that carries genetic information.

[0048] DLS: Dynamic Light Scattering, a method used to determine the particle size and polydispersity index (PDI) of nanoparticles.

[0049] TEM: Transmission Electron Microscopy, an imaging technique used to observe the morphology of nanoparticles.

[0050] PDI: Polydispersity Index, which indicates the width of the nanoparticle size distribution.

[0051] Key Terms

[0052] Ethanol injection method: A method for preparing lipid nanoparticles by adding lipid solution dropwise under strong stirring to allow lipids to self-assemble into nanoparticles and encapsulate nucleic acids.

[0053] Self-assembly: Under appropriate conditions, lipid molecules spontaneously arrange themselves to form stable nanostructures.

[0054] Encapsulation efficiency: The percentage of nucleic acids successfully encapsulated inside lipid nanoparticles, reflecting the efficiency of the preparation method.

[0055] Nucleic acid solution: A solution containing the mRNA to be encapsulated, usually dissolved in a buffer to facilitate its stability.

[0056] Dialysis: A method of removing small molecule impurities from a mixed solution through a semipermeable membrane, used to purify lipid nanoparticles.

[0057] Ultrafiltration: A method that uses membrane separation technology to remove small molecule impurities from a solution and is used to concentrate and purify lipid nanoparticles.

[0058] Liver / Spleen Targeting: The ability of lipid nanoparticles to selectively localize and concentrate in the liver or spleen in vivo, improving the efficiency of drug delivery in specific organs.

[0059] Dynamic Light Scattering: A method that uses the scattering properties of light to determine the size and distribution of nanoparticles.

[0060] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention.

[0061] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0062] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the technical field.

[0063] The present invention provides a method for preparing lipid nanoparticles (LNP) by ethanol injection method. The following is a detailed technical scheme, including specific steps and key operating parameters.

[0064] 1. Preparation of lipid solution

[0065] 1.1 Experimental Materials:

[0066] Ionizable lipids, in the embodiments of the present invention, commercial ionizable lipid SM102 and independently developed ionizable lipid X (8-((7-((3-(hexylthio)-2-methylpropionyl)oxy)n-heptyl)(2-hydroxyethyl)amine)octanoate 9-heptadecanol ester) are used. The preparation method of ionizable lipid X refers to patent CN 114773217 A, and the structure is shown in Formula I:

[0067] (Formula I);

[0068] Cholesterol (CHOL);

[0069] DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine);

[0070] PEG-DMG (polyethylene glycol-distearoylglycerol);

[0071] Ethanol.

[0072] 1.2 Solution preparation: Dissolve the lipid components in ethanol to form a lipid solution, and fully stir and dissolve the above lipid components in ethanol to ensure that the components are evenly distributed.

[0073] 2. Preparation of Nucleic Acid Solution

[0074] 2.1 Experimental Materials:

[0075] mRNA

[0076] Citrate buffer

[0077] 2.2 Solution preparation: Dissolve mRNA in citrate buffer to prepare nucleic acid solution. Adjust the concentration and pH according to experimental requirements.

[0078] 3. Ethanol injection mixing

[0079] 3.1 Equipment preparation:

[0080] Vortex mixer or magnetic stirrer

[0081] 3.2 Mixing: While the nucleic acid solution is vigorously stirred or vortexed, the lipid solution is added dropwise using a syringe. Stir / oscillate at maximum speed to obtain a stable and high-level vortex, and the lipid solution is added dropwise using a syringe to ensure that the lipids and nucleic acids are fully mixed to form nanoparticles.

[0082] In the embodiments of the present invention, the strong stirring or vortexing refers to stirring with a magnetic stirrer or using a vortex oscillator to make the liquid level after the vortex is formed to be about 4 times the liquid level at rest.

[0083] 3.3 Self-assembly to form LNP: During the above mixing process, lipids and nucleic acids self-assemble to form LNP.

[0084] 4. Purification and concentration

[0085] 4.1 Purification: The mixed LNP suspension is purified by dialysis or ultrafiltration to remove the organic solvent and unencapsulated nucleic acid.

[0086] 4.2 Concentration: The purified LNP suspension can be further concentrated as needed to achieve the desired concentration.

[0087] 5. Characterization

[0088] 5.1 Particle size determination: Dynamic light scattering (DLS) was used to determine the particle size and polydispersity index (PDI) of LNPs.

[0089] 5.2 Morphology observation: Transmission electron microscopy (TEM) was used to observe the morphology and distribution of LNPs.

[0090] 5.3 Encapsulation efficiency determination: The encapsulation efficiency of LNP was determined by nucleic acid determination methods (such as fluorescence quantitative PCR).

[0091] 6. Liver-targeted testing in animal experiments

[0092] 6.1 Experimental Design: In animal models, LNPs were delivered via intravenous injection.

[0093] 6.2 Imaging analysis: Fluorescence imaging or magnetic resonance imaging techniques were used to evaluate the distribution of LNPs in vivo, with a particular focus on the accumulation of LNPs in the liver to verify their targeting.

[0094] The flowchart of the technical solution of the present invention is shown in Figure 1 .

[0095] Example 1

[0096] Preparation of lipid solution: SM-102, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), PEG-DMG (polyethylene glycol-distearoylglycerol) and cholesterol (CHOL) were dissolved in ethanol to prepare a lipid solution. The lipid concentration was 10 mg / mL, SM-102 accounted for 50 mol% of the total lipids in the nucleic acid lipid nanoparticles, DSPC accounted for 10 mol% of the total lipids in the nucleic acid lipid nanoparticles, PEG-DMG accounted for 1.5 mol% of the total lipids in the nucleic acid lipid nanoparticles, and cholesterol accounted for 38.5 mol% of the total lipids in the nucleic acid lipid nanoparticles. This formula was named Formula 1.

[0097] Preparation of nucleic acid solution: Dissolve the Luciferase mRNA to be encapsulated in 50mM sodium citrate buffer (pH 4.0) to form a nucleic acid solution with a nitrogen-to-phosphorus ratio of 6: 1. The nitrogen-to-phosphorus ratio is the molar ratio of ionizable lipid nitrogen: oligonucleotide phosphate (N:P), which represents the charge balance between the ionizable lipid cationic tertiary amine and the oligonucleotide backbone anionic phosphate group.

[0098] Ethanol injection mixing: While the nucleic acid solution is vortexed at the 10th gear by a vortex oscillator (so that the liquid level after the vortex is formed is about 4 times the liquid level at rest), 1 mL of lipid solution is dripped into the 3 mL nucleic acid solution in the vortex state for 3 minutes. During this process, the lipids self-assemble to form nanoparticles and encapsulate the nucleic acid. The entire mixing process is carried out at room temperature to ensure that the lipids and nucleic acids are fully mixed.

[0099] Purification: The mixed LNP suspension was purified by dialyzing 5 times with 20 mM sodium citrate buffer (pH 6.0) for a total of 16 h to remove the organic solvent and unencapsulated nucleic acids to obtain a purified LNP suspension.

[0100] Concentration: The LNP solution was placed in a 100 kDa ultrafiltration tube and centrifuged at 2500 rpm for 20 min to achieve concentration and further purification.

[0101] Characterization: The prepared LNPs were characterized and their particle size, encapsulation efficiency and polydispersity index (PDI) were determined. The particle size potential was measured using a particle size analyzer, and the encapsulation efficiency was measured using the Quant-iT™ RiboGreen RNA quantification kit method.

[0102] Results: The particle size of LNP was 135.2nm, PDI was 0.133, potential was -3.23mV, and encapsulation efficiency was 71.3%.

[0103] The dialysis time for preparing LNP in this embodiment is relatively long, requiring overnight.

[0104] Example 2

[0105] Preparation of lipid solution: Ionizable lipid X, PEG-DMG (polyethylene glycol-distearoylglycerol) and cholesterol (CHOL) were dissolved in ethanol to prepare a lipid solution. The concentration of the lipid solution was 10 mg / mL, ionizable lipid X accounted for 48 mol% of the total lipids in the nucleic acid lipid nanoparticles, PEG-DMG accounted for 2.2 mol% of the total lipids in the nucleic acid lipid nanoparticles, and cholesterol accounted for 49.8 mol% of the total lipids in the nucleic acid lipid nanoparticles. This formula was named Formula 2.

[0106] Preparation of nucleic acid solution: Dissolve the Luciferase mRNA to be encapsulated in 50 mM sodium citrate buffer (pH 4.0) to form a nucleic acid solution with a nitrogen to phosphorus ratio of 6:1.

[0107] Ethanol injection mixing: While the nucleic acid solution is vortexed at the 10th gear on a vortex oscillator, 1 mL of lipid solution is dripped dropwise into the vortexed 3 mL nucleic acid solution for 3 minutes. During this process, the lipids self-assemble to form nanoparticles and encapsulate the nucleic acid. The entire mixing process is carried out at room temperature to ensure that the lipids and nucleic acids are fully mixed.

[0108] Purification: The mixed LNP suspension was purified by dialyzing 5 times with 20 mM sodium citrate buffer (pH 6.0) for a total of 16 h to remove the organic solvent and unencapsulated nucleic acids to obtain a purified LNP suspension.

[0109] Concentration: The LNP solution was placed in a 100 kDa ultrafiltration tube and centrifuged at 2500 rpm for 20 min to achieve concentration and further purification.

[0110] Result: flocculent precipitate appeared.

[0111] Example 3

[0112] Preparation of lipid solution: Ionizable lipid X, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), PEG-DMG (polyethylene glycol-distearoylglycerol) and cholesterol (CHOL) were dissolved in ethanol to prepare a lipid solution. The concentration of the lipid solution was 10 mg / mL, ionizable lipid X accounted for 48 mol% of the total lipids in the nucleic acid lipid nanoparticles, DSPC accounted for 9.5 mol% of the total lipids in the nucleic acid lipid nanoparticles, PEG-DMG accounted for 2 mol% of the total lipids in the nucleic acid lipid nanoparticles, and cholesterol accounted for 40.5 mol% of the total lipids in the nucleic acid lipid nanoparticles. This formula was named Formula 3.

[0113] Preparation of nucleic acid solution: Dissolve the Luciferase mRNA to be encapsulated in 50 mM sodium citrate buffer (pH 4.0) to form a nucleic acid solution with a nitrogen to phosphorus ratio of 6:1.

[0114] Ethanol injection mixing: While the nucleic acid solution is vortexed at the 10th gear on a vortex oscillator, 1 mL of lipid solution is dripped dropwise into the vortexed 3 mL nucleic acid solution for 3 minutes. During this process, the lipids self-assemble to form nanoparticles and encapsulate the nucleic acid. The entire mixing process is carried out at room temperature to ensure that the lipids and nucleic acids are fully mixed.

[0115] Purification: The mixed LNP suspension was purified by dialyzing 5 times with 20 mM sodium citrate buffer (pH 6.0) for a total of 16 h to remove the organic solvent and unencapsulated nucleic acids to obtain a purified LNP suspension.

[0116] Concentration: The LNP solution was placed in a 100 kDa ultrafiltration tube and centrifuged at 2500 rpm for 20 min to achieve concentration and further purification.

[0117] Result: flocculent precipitate appeared.

[0118] Example 4

[0119] Preparation of lipid solution: Ionizable lipid X, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), PEG-DMG (polyethylene glycol-distearoylglycerol) and cholesterol (CHOL) were dissolved in ethanol to prepare a lipid solution. The concentration of the lipid solution was 10 mg / mL, ionizable lipid X accounted for 48 mol% of the total lipids in the nucleic acid lipid nanoparticles, DSPC accounted for 16 mol% of the total lipids in the nucleic acid lipid nanoparticles, PEG-DMG accounted for 1.5 mol% of the total lipids in the nucleic acid lipid nanoparticles, and cholesterol accounted for 34.5 mol% of the total lipids in the nucleic acid lipid nanoparticles. This formula was named Formula 4.

[0120] Preparation of nucleic acid solution: Dissolve the Luciferase mRNA to be encapsulated in 50 mM sodium citrate buffer (pH 4.0) to form a nucleic acid solution with a nitrogen to phosphorus ratio of 6:1.

[0121] Ethanol injection mixing: While the nucleic acid solution is vortexed at the 10th gear on a vortex oscillator, 1 mL of lipid solution is dripped dropwise into the vortexed 3 mL nucleic acid solution for 3 minutes. During this process, the lipids self-assemble to form nanoparticles and encapsulate the nucleic acid. The entire mixing process is carried out at room temperature to ensure that the lipids and nucleic acids are fully mixed.

[0122] Purification: The mixed LNP suspension was purified by dialyzing 5 times with 20 mM sodium citrate buffer (pH 6.0) for a total of 16 h to remove the organic solvent and unencapsulated nucleic acids to obtain a purified LNP suspension.

[0123] Concentration: The LNP solution was placed in a 100 kDa ultrafiltration tube and centrifuged at 2500 rpm for 20 min to achieve concentration and further purification.

[0124] Result: flocculent precipitate appeared.

[0125] Example 5

[0126] Preparation of lipid solution: Ionizable lipid X, PEG-DMG (polyethylene glycol-distearoylglycerol) and cholesterol (CHOL) were dissolved in ethanol to prepare a lipid solution. The concentration of the lipid solution was 12.1 mg / mL, ionizable lipid X accounted for 45 mol% of the total lipids in the nucleic acid lipid nanoparticles, PEG-DMG accounted for 8.3 mol% of the total lipids in the nucleic acid lipid nanoparticles, and cholesterol accounted for 46.7 mol% of the total lipids in the nucleic acid lipid nanoparticles. This formula was named Formula 5.

[0127] Preparation of nucleic acid solution: Dissolve the Luciferase mRNA to be encapsulated in 50 mM sodium citrate buffer (pH 4.0) to form a nucleic acid solution with a nitrogen to phosphorus ratio of 6:1.

[0128] Ethanol injection mixing: While the nucleic acid solution is vortexed at the 10th gear on a vortex oscillator, 1 mL of lipid solution is dripped dropwise into the vortexed 3 mL nucleic acid solution for 3 minutes. During this process, the lipids self-assemble to form nanoparticles and encapsulate the nucleic acid. The entire mixing process is carried out at room temperature to ensure that the lipids and nucleic acids are fully mixed.

[0129] Purification: The mixed LNP suspension was purified by dialyzing 5 times with 20 mM sodium citrate buffer (pH 6.0) for a total of 16 h to remove the organic solvent and unencapsulated nucleic acids to obtain a purified LNP suspension.

[0130] Concentration: The LNP solution was placed in a 100 kDa ultrafiltration tube and centrifuged at 2500 rpm for 20 min to achieve concentration and further purification.

[0131] Characterization: The prepared LNPs were characterized and their particle size, encapsulation efficiency and polydispersity index (PDI) were determined. The particle size potential was measured using a particle size analyzer, and the encapsulation efficiency was measured using the Quant-iT™ RiboGreen RNA quantification kit method.

[0132] The solution is clear and free of particles. The particle size of LNP is 163.5nm, PDI is 0.175, potential is -12.31mV, and encapsulation efficiency is 61.83%. It takes a long time to purify and remove ethanol.

[0133] Example 6

[0134] Preparation of lipid solution: Ionizable lipid X, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), PEG-DMG (polyethylene glycol-distearoylglycerol) and cholesterol (CHOL) were dissolved in ethanol to prepare a lipid solution. The concentration of the lipid solution was 13.3 mg / mL, ionizable lipid X accounted for 45.3 mol% of the total lipids in the nucleic acid lipid nanoparticles, DSPC accounted for 9 mol% of the total lipids in the nucleic acid lipid nanoparticles, PEG-DMG accounted for 7.5 mol% of the total lipids in the nucleic acid lipid nanoparticles, and cholesterol accounted for 38.2 mol% of the total lipids in the nucleic acid lipid nanoparticles. This formula was named Formula VI.

[0135] Preparation of nucleic acid solution: Dissolve the Luciferase mRNA to be encapsulated in 50 mM sodium citrate buffer (pH 4.0) to form a nucleic acid solution with a nitrogen to phosphorus ratio of 6:1.

[0136] Ethanol injection mixing: While the nucleic acid solution is vortexed at the 10th gear on a vortex oscillator, 1 mL of lipid solution is dripped dropwise into the vortexed 3 mL nucleic acid solution for 3 minutes. During this process, the lipids self-assemble to form nanoparticles and encapsulate the nucleic acid. The entire mixing process is carried out at room temperature to ensure that the lipids and nucleic acids are fully mixed.

[0137] Purification: The mixed LNP suspension was purified by dialyzing 5 times with 20 mM sodium citrate buffer (pH 6.0) for a total of 16 h to remove the organic solvent and unencapsulated nucleic acids to obtain a purified LNP suspension.

[0138] Concentration: The LNP solution was placed in a 100 kDa ultrafiltration tube and centrifuged at 2500 rpm for 20 min to achieve concentration and further purification.

[0139] Characterization: The prepared LNPs were characterized and their particle size, encapsulation efficiency and polydispersity index (PDI) were determined. The particle size potential was measured using a particle size analyzer, and the encapsulation efficiency was measured using the Quant-iT™ RiboGreen RNA quantification kit method.

[0140] The solution was clear and free of particles, the particle size of LNP was 159.0nm, PDI was 0.196, potential was -10.39mV, and encapsulation efficiency was 63.71%. A longer purification time was required to remove ethanol.

[0141] Example 7

[0142] Preparation of lipid solution: Ionizable lipid X, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), PEG-DMG (polyethylene glycol-distearoylglycerol) and cholesterol (CHOL) were dissolved in ethanol to prepare a lipid solution. The concentration of the lipid solution was 14.0 mg / mL, ionizable lipid X accounted for 45.9 mol% of the total lipids in the nucleic acid lipid nanoparticles, DSPC accounted for 15.3 mol% of the total lipids in the nucleic acid lipid nanoparticles, PEG-DMG accounted for 5.7 mol% of the total lipids in the nucleic acid lipid nanoparticles, and cholesterol accounted for 33 mol% of the total lipids in the nucleic acid lipid nanoparticles. This formula was named Formula 7.

[0143] Preparation of nucleic acid solution: Dissolve the Luciferase mRNA to be encapsulated in 50 mM sodium citrate buffer (pH 4.0) to form a nucleic acid solution with a nitrogen to phosphorus ratio of 6:1.

[0144] Ethanol injection mixing: While the nucleic acid solution is vortexed at the 10th gear on a vortex oscillator, 1 mL of lipid solution is dripped dropwise into the vortexed 3 mL nucleic acid solution for 3 minutes. During this process, the lipids self-assemble to form nanoparticles and encapsulate the nucleic acid. The entire mixing process is carried out at room temperature to ensure that the lipids and nucleic acids are fully mixed.

[0145] Purification: The mixed LNP suspension was purified by dialyzing 5 times with 20 mM sodium citrate buffer (pH 6.0) for a total of 16 h to remove the organic solvent and unencapsulated nucleic acids to obtain a purified LNP suspension.

[0146] Concentration: The LNP solution was placed in a 100 kDa ultrafiltration tube and centrifuged at 2500 rpm for 20 min to achieve concentration and further purification.

[0147] Characterization: The prepared LNPs were characterized and their particle size, encapsulation efficiency and polydispersity index (PDI) were determined. The particle size potential was measured using a particle size analyzer, and the encapsulation efficiency was measured using the Quant-iT™ RiboGreen RNA quantification kit method.

[0148] The solution was clear and free of particles, the particle size of LNP was 190.4nm, PDI was 0.139, potential was -10.45mV, and encapsulation efficiency was 63.46%. A longer purification time was required to remove ethanol.

[0149] Example 8

[0150] Preparation of lipid solution: Ionizable lipid X, PEG-DMG (polyethylene glycol-distearoylglycerol) and cholesterol (CHOL) were dissolved in ethanol to prepare a lipid solution. The concentration of the lipid solution was 12.1 mg / mL, ionizable lipid X accounted for 45 mol% of the total lipids in the nucleic acid lipid nanoparticles, PEG-DMG accounted for 8.3 mol% of the total lipids in the nucleic acid lipid nanoparticles, and cholesterol accounted for 46.7 mol% of the total lipids in the nucleic acid lipid nanoparticles (Formula 5).

[0151] Preparation of nucleic acid solution: Dissolve the Luciferase mRNA to be encapsulated in 50 mM sodium citrate buffer (pH 4.0) to form a nucleic acid solution with a nitrogen to phosphorus ratio of 6:1.

[0152] Ethanol injection mixing: While the nucleic acid solution is vortexed at the 10th gear on a vortex oscillator, 500 μL of lipid solution is dripped into the 3.5 mL nucleic acid solution in a vortex state for 3 minutes. During this process, the lipids self-assemble to form nanoparticles and encapsulate the nucleic acid. The entire mixing process is carried out at room temperature to ensure that the lipids and nucleic acids are fully mixed.

[0153] Purification: The mixed LNP suspension was purified by dialyzing three times against 20 mM sodium citrate buffer (pH 6.0) for 4 h to remove the organic solvent and unencapsulated nucleic acid to obtain a purified LNP suspension.

[0154] Concentration: The LNP solution was placed in a 100 kDa ultrafiltration tube and centrifuged at 2500 rpm for 20 min to achieve concentration and further purification.

[0155] Characterization: The prepared LNPs were characterized and their particle size, encapsulation efficiency and polydispersity index (PDI) were determined. The particle size potential was measured using a particle size analyzer, and the encapsulation efficiency was measured using the Quant-iT™ RiboGreen RNA quantification kit method.

[0156] Liver targeting test: In animal models, LNPs were delivered by intravenous injection and the distribution of LNPs in vivo was evaluated using imaging technology. Particular attention was paid to the accumulation of LNPs in the liver to verify their targeting, which was mainly achieved by optimizing the lipid formulation. The results are shown in Tables 1 and Figure 2 .

[0157] Example 9

[0158] Preparation of lipid solution: Ionizable lipid X, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), PEG-DMG (polyethylene glycol-distearoylglycerol) and cholesterol (CHOL) were dissolved in ethanol to prepare a lipid solution. The concentration of the lipid solution was 13.3 mg / mL, ionizable lipid X accounted for 45.3 mol% of the total lipids in the nucleic acid lipid nanoparticles, DSPC accounted for 9 mol% of the total lipids in the nucleic acid lipid nanoparticles, PEG-DMG accounted for 7.5 mol% of the total lipids in the nucleic acid lipid nanoparticles, and cholesterol accounted for 38.2 mol% of the total lipids in the nucleic acid lipid nanoparticles (Formula VI).

[0159] Preparation of nucleic acid solution: Dissolve the Luciferase mRNA to be encapsulated in 50 mM sodium citrate buffer (pH 4.0) to form a nucleic acid solution with a nitrogen to phosphorus ratio of 6:1.

[0160] Ethanol injection mixing: While the nucleic acid solution is vortexed at the 10th gear on a vortex oscillator, 500 μL of lipid solution is dripped into the 3.5 mL nucleic acid solution in a vortex state for 3 minutes. During this process, the lipids self-assemble to form nanoparticles and encapsulate the nucleic acid. The entire mixing process is carried out at room temperature to ensure that the lipids and nucleic acids are fully mixed.

[0161] Purification: The mixed LNP suspension was purified by dialyzing three times against 20 mM sodium citrate buffer (pH 6.0) for 4 h to remove the organic solvent and unencapsulated nucleic acid to obtain a purified LNP suspension.

[0162] Concentration: The LNP solution was placed in a 100 kDa ultrafiltration tube and centrifuged at 2500 rpm for 20 min to achieve concentration and further purification.

[0163] Characterization: The prepared LNPs were characterized and their particle size, encapsulation efficiency and polydispersity index (PDI) were determined. The particle size potential was measured using a particle size analyzer, and the encapsulation efficiency was measured using the Quant-iT™ RiboGreen RNA quantification kit method.

[0164] Organ targeting test: In animal models, LNPs were delivered by intravenous injection and the distribution of LNPs in vivo was evaluated using imaging technology. Particular attention was paid to the accumulation of LNPs in the liver / spleen to verify their targeting, which was mainly achieved by optimizing the lipid formulation. The results are shown in Table 1 and Figure 2 .

[0165] Example 10

[0166] Preparation of lipid solution: Ionizable lipid X, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), PEG-DMG (polyethylene glycol-distearoylglycerol) and cholesterol (CHOL) were dissolved in ethanol to prepare a lipid solution. The concentration of the lipid solution was 14.0 mg / mL, ionizable lipid X accounted for 45.9 mol% of the total lipids in the nucleic acid lipid nanoparticles, DSPC accounted for 15.3 mol% of the total lipids in the nucleic acid lipid nanoparticles, PEG-DMG accounted for 5.7 mol% of the total lipids in the nucleic acid lipid nanoparticles, and cholesterol accounted for 33 mol% of the total lipids in the nucleic acid lipid nanoparticles (Formula 7).

[0167] Preparation of nucleic acid solution: Dissolve the Luciferase mRNA to be encapsulated in 50 mM sodium citrate buffer (pH 4.0) to form a nucleic acid solution with a nitrogen to phosphorus ratio of 6:1.

[0168] Ethanol injection mixing: While the nucleic acid solution is vortexed at the 10th gear on a vortex oscillator, 500 μL of lipid solution is dripped into the 3.5 mL nucleic acid solution in a vortex state for 3 minutes. During this process, the lipids self-assemble to form nanoparticles and encapsulate the nucleic acid. The entire mixing process is carried out at room temperature to ensure that the lipids and nucleic acids are fully mixed.

[0169] Purification: The mixed LNP suspension was purified by dialyzing three times against 20 mM sodium citrate buffer (pH 6.0) for 4 h to remove the organic solvent and unencapsulated nucleic acid to obtain a purified LNP suspension.

[0170] Concentration: The LNP solution was placed in a 100 kDa ultrafiltration tube and centrifuged at 2500 rpm for 20 min to achieve concentration and further purification.

[0171] Characterization: The prepared LNPs were characterized and their particle size, encapsulation efficiency and polydispersity index (PDI) were determined. The particle size potential was measured using a particle size analyzer, and the encapsulation efficiency was measured using the Quant-iT™ RiboGreen RNA quantification kit method.

[0172] Organ targeting test: In animal models, LNPs are delivered by intravenous injection, and imaging techniques are used to evaluate the distribution of LNPs in vivo. Particular attention is paid to the accumulation of LNPs in the spleen to verify their targeting, which is mainly achieved by optimizing the lipid formulation.

[0173] The LNP data obtained in Examples 8-10 are as follows:

[0174] Table 1 Characterization data of LNP

[0175]

[0176] The LNP particle size, potential, and encapsulation efficiency of the three formulations were qualified, and they were targeted after in vivo injection (see Figure 2 , Figure 3 ).

[0177] Embodiment 11

[0178] Preparation of lipid solution: SM-102, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), PEG-DMG (polyethylene glycol-distearoylglycerol) and cholesterol (CHOL) were dissolved in ethanol to prepare a lipid solution. The lipid concentration was 10 mg / mL, SM-102 accounted for 50 mol% of the total lipids in the nucleic acid lipid nanoparticles, DSPC accounted for 10 mol% of the total lipids in the nucleic acid lipid nanoparticles, PEG-DMG accounted for 1.5 mol% of the total lipids in the nucleic acid lipid nanoparticles, and cholesterol accounted for 38.5 mol% of the total lipids in the nucleic acid lipid nanoparticles (Formula 1).

[0179] Preparation of nucleic acid solution: Dissolve the Luciferase mRNA to be encapsulated in 50 mM sodium citrate buffer (pH 4.0) to form a nucleic acid solution with a nitrogen to phosphorus ratio of 6:1.

[0180] Ethanol injection mixing: While the nucleic acid solution is vortexed at the 10th gear on a vortex oscillator, 500 μL of lipid solution is dripped into the 3.5 mL nucleic acid solution in a vortex state for 3 minutes. During this process, the lipids self-assemble to form nanoparticles and encapsulate the nucleic acid. The entire mixing process is carried out at room temperature to ensure that the lipids and nucleic acids are fully mixed.

[0181] Purification: The mixed LNP suspension was purified by dialyzing three times against 20 mM sodium citrate buffer (pH 6.0) for 4 h to remove the organic solvent and unencapsulated nucleic acid to obtain a purified LNP suspension.

[0182] Concentration: The LNP solution was placed in a 100 kDa ultrafiltration tube and centrifuged at 2500 rpm for 20 min to achieve concentration and further purification.

[0183] Characterization: The prepared LNPs were characterized and their particle size, encapsulation efficiency and polydispersity index (PDI) were determined. The particle size potential was measured using a particle size analyzer, and the encapsulation efficiency was measured using the Quant-iT™ RiboGreen RNA quantification kit method.

[0184] Results: The particle size of LNP was 123.1 nm, PDI was 0.183, potential was -7.23 mV, and encapsulation efficiency was 76.3%.

[0185] Test Example 1

[0186] The present invention compares the effects of different ratios of water phase to ethanol phase in different formulations on drug properties. The results are shown in Tables 2 to 4.

[0187] The preparation methods of the lipid nanoparticles in Table 2 are the same as those in Example 8, with the only difference being the ratio of the aqueous phase to the ethanol phase.

[0188] Table 2 Effects of different ratios of water phase to ethanol phase on drugability of liver target formula (Formula 5)

[0189]

[0190] The preparation methods of the lipid nanoparticles in Table 3 are the same as those in Example 9, with the only difference being the ratio of the aqueous phase to the ethanol phase.

[0191] Table 3 Effects of different ratios of water phase to ethanol phase on drugability of liver / spleen target formulation (Formula 6)

[0192]

[0193] The preparation method of each lipid nanoparticle in Table 4 is the same as that in Example 10, the only difference being the ratio of the aqueous phase to the ethanol phase.

[0194] Table 4 Effect of different ratios of water phase to ethanol phase on drugability of spleen target formula (formulation 7)

[0195]

[0196] Test Example 2

[0197] The present invention compares the effects of stirring / vortex mixing on the drugability of LNP, and the results are shown in Tables 5 to 7.

[0198] The preparation method of each lipid nanoparticle in Table 5 is the same as that in Example 8, the only difference being the mixing method.

[0199] Table 5 Effect of stirring / vortex mixing of liver target formulation on the drugability of LNP

[0200]

[0201] The preparation method of each lipid nanoparticle in Table 6 is the same as that in Example 9, the only difference is the mixing method.

[0202] Table 6 Effect of stirring / vortex mixing of liver / spleen target formulations on the drugability of LNPs

[0203]

[0204] The preparation method of each lipid nanoparticle in Table 7 is the same as that in Example 10, the only difference being the mixing method.

[0205] Table 7 Effect of stirring / vortex mixing of spleen-targeting formulations on the drugability of LNPs

[0206]

[0207] Regardless of the stirring / vortexing method of the magnetic stirrer, the liquid level after the vortex is formed is about 4 times the liquid level when it is static.

[0208] Test Example 3

[0209] The present invention compares the effects of different dropping speeds on the drugability of LNP. 500 μL of ethanol phase was dropped into 3.5 mL of water phase in a vortex state, and the results of the effects of different completion times on the drugability of LNP are shown in Table 8.

[0210] The preparation method of each lipid nanoparticle in Table 8 is the same as that in Example 8, the only difference being the dropping speed.

[0211] Table 8 Effect of droplet velocity on drugability of LNP

[0212]

[0213] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing spleen-targeted nucleic acid lipid nanoparticles by ethanol injection, characterized in that: The method includes: The lipid composition is dissolved in ethanol to obtain a lipid solution; the nucleic acid is dissolved in a buffer to obtain a nucleic acid solution; while the nucleic acid solution is stirred or vortexed, the lipid solution is added dropwise to the nucleic acid solution, the volume ratio of the nucleic acid solution to the lipid solution is 7:1, and nucleic acid lipid nanoparticles are obtained; Taking the total molar amount of lipids in the lipid composition as 100%, the lipid composition comprises the following lipid components: Ionizable lipids: 45.9%; PEG-modified lipids: 5.7%; Steroids: 33%; Auxiliary phospholipids: 15.3%; The ionizable lipid is 8-((7-((3-(hexylthio)-2-methylpropionyl)oxy)n-heptyl)(2-hydroxyethyl)amine)octanoate 9-heptadecanol ester.

2. The method according to claim 1, characterized in that The pH of the buffer solution is 3.0-5.5, and the buffer solution is a citrate buffer solution.

3. The method according to claim 1 or 2, characterized in that: The method also includes purifying and concentrating the nucleic acid lipid nanoparticles.

4. The method according to claim 3, characterized in that The purification includes dialysis, and the concentration includes ultrafiltration.

5. A nucleic acid lipid nanoparticle targeting spleen, characterized in that: The spleen-targeted nucleic acid lipid nanoparticles are prepared by the method described in any one of claims 1-4.

6. The nucleic acid lipid nanoparticle according to claim 5, characterized in that The average particle size of the lipid nanoparticles is 138-156 nm, and the potential is -2.55--4.4 mV.

7. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the nucleic acid lipid nanoparticles according to claim 5 or 6.

8. Use of the nucleic acid lipid nanoparticles according to claim 5 or 6, or the pharmaceutical preparation according to claim 7, in the preparation of a drug for targeting nucleic acid delivery to the spleen.

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