Ionizable lipid-based lipid nanoparticles, methods of making and use thereof

Abstract

The application provides a kind of lipid nanoparticles based on new ionizable lipid and its preparation method and application, belong to the technical field of biomedical tissue engineering materials.The intermediate is prepared by using isooctanol, 2-butyl-1-n-octanol or 2-butyl octanoic acid and 8-bromooctanoic acid or 9-bromo-1-nonanol as raw materials, the intermediate is heated and stirred with organic amine solution in tetrahydrofuran-acetonitrile mixed solution, the reaction mixture is evaporated, extracted and purified to obtain the new ionizable lipid, and the prepared new ionizable lipid, neutral phospholipid, cholesterol and dimyristoyl glycerol-polyethylene glycol 2000 are dissolved in ethanol to obtain an ethanol phase lipid mixture, then mixed with an aqueous buffer containing mRNA, and dialyzed to obtain the lipid nanoparticles. The mRNA is delivered by using the lipid nanoparticles based on new ionizable lipid, to solve a series of difficulties encountered in the effective transcription and translation of mRNA in vivo.

Description

Technical Field

[0001] This invention belongs to the field of biomedical tissue engineering materials technology, and particularly relates to a lipid nanoparticle based on ionizable lipids, its preparation method and application. Background Technology

[0002] mRNA, short for messenger ribonucleic acid, is a single-stranded ribonucleic acid molecule transcribed from one strand of DNA as a template. It carries genetic information and directs protein synthesis. While mRNA has a large molecular weight and is highly hydrophilic, its single-stranded structure is extremely unstable and easily degraded. The limited lifespan of mRNA allows cells to rapidly alter protein synthesis to respond to their ever-changing needs, but it is difficult to meet the requirements for drug development. Furthermore, because mRNA molecules carry a negative charge, they have difficulty crossing the negatively charged cell membrane. Therefore, special modification or encapsulation delivery systems are required to achieve intracellular expression of mRNA drugs.

[0003] Lipid nanoparticles (LNPs) are a type of nanoparticle formed using lipids. Ionizable lipids are a class of lipid molecules with ionization properties, playing an important role in drug delivery systems, especially lipid-based nanoparticle drug delivery systems. These lipids can ionize under specific pH values ​​or biological conditions, thereby altering their hydrophilicity and lipophilicity balance, which in turn affects drug release, cellular absorption, and distribution in vivo. Currently, widely used LNP components mainly include the following four categories: cationic lipids, cholesterol, polyethylene glycol lipids, and accessory lipids.

[0004] Cationic lipids are key components of LNP delivery systems. Currently, the specific structures of cationic lipid molecules used by various mRNA companies differ, but they all belong to the category of positively charged cationic lipids under specific conditions. Ionization of cationic lipids is crucial for the delivery function of LNP systems. Because mRNA itself is negatively charged, the attraction between positive and negative charges binds mRNA to the LNP, improving its stability in vivo and allowing it to escape lysosomal degradation. After absorption by the cell, the low pH environment of the endosome fuses with the LNP, releasing mRNA into the cytoplasmic colloid. However, existing cationic lipids struggle to achieve a balance between delivery efficiency and cytotoxicity.

[0005] In summary, how to provide a cationic lipid with excellent delivery efficiency and good biocompatibility to synthesize excellent LNPs is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a lipid nanoparticle based on ionizable lipids, its preparation method, and its application. The invention utilizes lipid nanoparticles based on ionizable lipids to deliver mRNA, thereby solving a series of difficulties encountered in the effective transcription and translation of mRNA in vivo.

[0007] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of the present invention: A method for preparing an ionizable lipid involves preparing an intermediate from compounds A and B as raw materials, heating and stirring the intermediate with an organic amine in a tetrahydrofuran-acetonitrile mixed solution, and then evaporating, extracting, and purifying the reaction mixture to obtain the ionizable lipid. Compound A is isooctyl alcohol, 2-butyl-1-n-octyl alcohol, or 2-butyloctanoic acid; Compound B is 8-bromooctanoic acid or 9-bromo-1-nonanol.

[0008] Furthermore, the preparation method of the intermediate is as follows: compound A, carbodiimide hydrochloride and 4-dimethylaminopyridine are dissolved in dichloromethane solution, then compound B is added and stirred to obtain a reaction solution, and the reaction solution is extracted, washed, evaporated and purified to obtain the intermediate.

[0009] Furthermore, the mass ratio of compound A to compound B is (3.5-5):(5-5.5), and the mass ratio of compound A to carbodiimide hydrochloride and 4-dimethylaminopyridine is (3.5-5):4.3:(1.7-2).

[0010] More specifically, the intermediate is prepared by: Compound A was added to a dichloromethane solution dehydrated by 4A molecular sieves, followed by the sequential addition of carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP). The mixture was stirred at room temperature for 30 minutes, then compound B was added, and the mixture was stirred at room temperature for 18 hours to obtain a reaction solution. The reaction solution was diluted with dichloromethane and extracted with saturated sodium bicarbonate solution. After separation of the organic layer, the mixture was washed with brine, dried over sodium sulfate, filtered, and evaporated under vacuum. The residual liquid was purified by silica gel chromatography (0-10% ethyl acetate / n-hexane) to obtain the intermediate.

[0011] Furthermore, the ratio of the intermediate to the organic amine and the tetrahydrofuran-acetonitrile mixed solution is (2-2.5) g: (1280-1458) μL: 20 mL.

[0012] Furthermore, the organic amine is a mixture of N,N-diisopropyldiamine and N-hydroxyethyl-1,3-propanediamine, 3-dimethylaminopropylamine or N,N-dimethyldipropyltriamine.

[0013] Furthermore, the volume ratio of tetrahydrofuran to acetonitrile in the tetrahydrofuran-acetonitrile mixed solution is 1:1.

[0014] More specifically, the specific steps for preparing ionizable lipids from intermediates are as follows: The intermediate was mixed with N-hydroxyethyl-1,3-propanediamine, 3-dimethylaminopropylamine, or N,N-dimethyldipropyltriamine and N,N-diisopropyldiamine in a tetrahydrofuran-acetonitrile mixed solution and stirred at 63°C for 72 hours. After the reaction mixture was cooled to room temperature, it was evaporated under vacuum. The residual liquid was extracted with ethyl acetate and saturated sodium bicarbonate solution. After separation of the organic layer, it was dried over sodium sulfate, filtered, and evaporated under vacuum. The residual liquid was purified by silica gel chromatography (0-10% methanol / dichloromethane) to obtain the ionizable lipid.

[0015] The second technical solution of the present invention: An ionizable lipid prepared by the above preparation method.

[0016] The third technical solution of the present invention: A lipid nanoparticle prepared based on the aforementioned ionizable lipid.

[0017] The fourth technical solution of the present invention: A method for preparing the above-mentioned lipid nanoparticles involves dissolving the ionizable lipids, neutral phospholipids, cholesterol, and dimyristic glycerol-polyethylene glycol 2000 in ethanol to obtain an ethanol-phase lipid mixture, then mixing it with an aqueous buffer containing mRNA, allowing it to stand, and dialyzing to obtain the lipid nanoparticles.

[0018] Furthermore, the molar ratio of the ionizable lipids, neutral phospholipids, cholesterol, and dimyristic glycerol-polyethylene glycol 2000 is 50:10:38.5:1.5.

[0019] Furthermore, the volume ratio of the ethanol phase lipid mixture to the aqueous phase buffer is 1:3.

[0020] Furthermore, the aqueous buffer containing mRNA is obtained by dissolving the mRNA in 0.01M citrate buffer solution (pH=4.0).

[0021] Furthermore, the molecular weight cutoff for dialysis is 3500 Da, and the dialysis time is 2-12 hours.

[0022] The fifth technical solution of the present invention: The above-mentioned lipid nanoparticles are used in the preparation of drugs for mRNA delivery.

[0023] Compared with the prior art, the present invention has the following advantages and technical effects: This invention provides lipid nanoparticles prepared from ionizable lipids, which exhibit superior transfection efficiency and higher stability compared to commercially available ionizable lipids. The lipid nanoparticles prepared according to this invention are used to deliver mRNA, addressing a series of challenges encountered in the effective transcription and translation of mRNA in vivo. Furthermore, the preparation process of the lipid nanoparticles of this invention is relatively simple and can be mass-produced. Attached Figure Description

[0024] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings: Figure 1 The molecular structural formulas of ionizable lipids 1-9 prepared in Examples 4-12 are shown below. Figure 2 The 1H NMR spectrum of ionizable lipid 1 from Example 4; Figure 3 The 1H NMR spectrum of ionizable lipid 2 from Example 5; Figure 4 The 1H NMR spectrum of ionizable lipid 3 in Example 6; Figure 5 The 1H NMR spectrum of ionizable lipid 4 from Example 7; Figure 6 The 1H NMR spectrum of ionizable lipid 5 from Example 8; Figure 7 The 1H NMR spectrum of ionizable lipid 6 from Example 9; Figure 8 The 1H NMR spectrum of ionizable lipid 7 from Example 10; Figure 9 The 1H NMR spectrum of ionizable lipid 8 from Example 11; Figure 10 The 1H NMR spectrum of ionizable lipid 9 from Example 12; Figure 11 This is a high-resolution mass spectrum of ionizable lipid 1 from Example 4; Figure 12 This is a high-resolution mass spectrum of ionizable lipid 2 from Example 5; Figure 13 This is a high-resolution mass spectrum of ionizable lipid 3 from Example 6; Figure 14 This is a high-resolution mass spectrum of ionizable lipid 4 from Example 7; Figure 15 This is a high-resolution mass spectrum of ionizable lipid 5 from Example 8; Figure 16 This is a high-resolution mass spectrum of ionizable lipid 6 from Example 9; Figure 17 This is a high-resolution mass spectrum of ionizable lipid 7 from Example 10; Figure 18 This is a high-resolution mass spectrum of ionizable lipid 8 from Example 11; Figure 19 This is a high-resolution mass spectrum of ionizable lipid 9 from Example 12; Figure 20 The encapsulation efficiency of GFP mRNA encapsulated in lipid nanoparticles prepared using ionizable lipids 1-9 prepared in Examples 4-12, respectively, and in the control group lipid nanoparticles is measured. Figure 21 The following are representative particle size distribution diagrams of lipid nanoparticles prepared in Example 13 using ionizable lipids 1-9 prepared in Examples 4-12 as raw materials, and lipid nanoparticles of the control group. Figure 22 The average particle size of lipid nanoparticles prepared in Example 13 using ionizable lipids 1-9 prepared in Examples 4-12 as raw materials and the control group lipid nanoparticles are measured. Figure 23 The results of Zeta potential measurement of lipid nanoparticles prepared using ionizable lipids 1-9 prepared in Examples 4-12 as raw materials and control group lipid nanoparticles encapsulating GFP mRNA are shown in Example 13. Figure 24 The effects of lipid nanoparticles prepared using ionizable lipids 1-9 prepared in Examples 4-12 as raw materials and control group lipid nanoparticles encapsulating GFP mRNA on the viability of DC2.4 cells at different total lipid concentrations were compared in Example 13. Figure 25 The images show fluorescence micrographs of lipid nanoparticles prepared in Example 13 using ionizable lipids 1-9 prepared in Examples 4-12 as raw materials, and control group lipid nanoparticles encapsulating GFP mRNA and transfecting DC2.4 cells. Figure 26 The transfection efficiency of lipid nanoparticles prepared using ionizable lipids 1-9 prepared in Examples 4-12 as raw materials and control group lipid nanoparticles encapsulating GFP mRNA in DC2.4 cells is shown in Example 13. Figure 27 The average fluorescence intensity of lipid nanoparticles prepared in Example 13 using ionizable lipids 1-9 prepared in Examples 4-12 as raw materials, and the control group lipid nanoparticles encapsulating GFP mRNA transfected into DC2.4 cells; Figure 28 The images show fluorescence microscopy images of lipid nanoparticles prepared in Example 13 using ionizable lipids 1-9 prepared in Examples 4-12 as raw materials, and control group lipid nanoparticles encapsulating GFP mRNA and transfecting 293T cells. Figure 29 The transfection efficiency of lipid nanoparticles prepared using ionizable lipids 1-9 prepared in Examples 4-12 as raw materials and control group lipid nanoparticles encapsulating GFP mRNA in 293T cells is shown in Example 13. Figure 30 The average fluorescence intensity of lipid nanoparticles prepared in Example 13 using ionizable lipids 1-9 prepared in Examples 4-12 as raw materials, and the control group lipid nanoparticles encapsulating GFP mRNA transfected into 293T cells. Figure 31 The image shows the average fluorescence intensity fold relative to untreated cells after lipid nanoparticles prepared using ionizable lipids 1-9 prepared in Examples 4-12 and control group lipid nanoparticles encapsulated GFP mRNA and transfected DC2.4 cells and 293T cells, respectively. Figure 32 The images show representative in vivo images of lipid nanoparticles prepared in Example 13 using ionizable lipids 5-9 prepared in Examples 8-12 as raw materials, and control group lipid nanoparticles encapsulating Fluc mRNA in mice to express luciferase catalyzing the luminescence of luciferase substrate. Figure 33 The lipid nanoparticles prepared in Example 13 using ionizable lipids 5-9 prepared in Examples 8-12 as raw materials, and the control group lipid nanoparticles encapsulated Fluc mRNA to express luciferase catalyzing the luminescence of the luciferase substrate in mice, showed relative light radiation intensity. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] All raw materials used in the embodiments of this invention were obtained through commercial purchase.

[0031] In this embodiment of the invention, room temperature refers to 25°C.

[0032] The technical solution of the present invention will be further illustrated by the following embodiments.

[0033] Example 1 3.5 g of isooctanol was added to 100 mL of dichloromethane solution dehydrated by 4A molecular sieves. Then, 4.3 g of EDCI and 1.7 g of DMAP were added sequentially. The mixture was stirred at room temperature for 30 minutes, followed by the addition of 5 g of 8-bromooctanoic acid. The mixture was stirred at room temperature for 18 hours to obtain the reaction solution. The reaction solution was diluted with dichloromethane and extracted with saturated sodium bicarbonate solution. After separation of the organic layer, the solution was washed with brine, dried over sodium sulfate, filtered, and evaporated under vacuum. The residual liquid was purified by silica gel chromatography (0-10% ethyl acetate / n-hexane) to obtain intermediate (intermediate 1).

[0034] Example 2 5 g of 2-butyl-1-n-octanol was added to 100 mL of dichloromethane solution dehydrated by 4A molecular sieve. Then, 4.3 g of EDCI and 1.7 g of DMAP were added sequentially. The mixture was stirred at room temperature for 30 minutes, followed by the addition of 5 g of 8-bromooctanoic acid. The mixture was stirred at room temperature for 18 hours to obtain the reaction solution. The reaction solution was diluted with dichloromethane and extracted with saturated sodium bicarbonate solution. After separation of the organic layer, the solution was washed with brine, dried over sodium sulfate, filtered, and evaporated under vacuum. The residual liquid was purified by silica gel chromatography (0-10% ethyl acetate / n-hexane) to obtain intermediate (intermediate 2).

[0035] Example 3 4 g of 2-butyloctanoic acid was added to 100 mL of dichloromethane solution dehydrated by 4A molecular sieve. Then, 4.3 g of EDCI and 2 g of DMAP were added sequentially, and the mixture was stirred at room temperature for 30 minutes. Then, 5.4 g of 9-bromo-1-nonanol was added, and the mixture was stirred at room temperature for 18 hours to obtain the reaction solution. The reaction solution was diluted with dichloromethane at a volume ratio of 1:3, extracted with saturated sodium bicarbonate solution, and the organic layer was separated. The mixture was washed with brine, dried over sodium sulfate, filtered, and evaporated under vacuum. The residual liquid was purified by silica gel chromatography (mobile phase: 0-10% ethyl acetate / n-hexane) to obtain intermediate (intermediate 3).

[0036] Example 4 2.01 g of intermediate 1 prepared in Example 1 was mixed with 235 μL (236 mg) of N-hydroxyethyl-1,3-propanediamine solution and 775.44 mg (1045 μL) of N,N-diisopropyldiamine in 20 mL of THF-MeCN (1:1) mixed solution and stirred at 63 °C for 72 hours. After the reaction mixture was cooled to room temperature, it was evaporated under vacuum. The residual liquid was extracted with ethyl acetate and saturated sodium bicarbonate solution. After separation of the organic layer, it was dried with sodium sulfate, filtered and evaporated under vacuum. The residual liquid was purified by silica gel chromatography (0-10% methanol / dichloromethane) to obtain an ionizable lipid, denoted as ionizable lipid 1.

[0037] Example 5 2.4 g of intermediate 2 prepared in Example 2 was mixed with 236 mg (235 μL) of N-hydroxyethyl-1,3-propanediamine solution and 775.44 mg (1045 μL) of N,N-diisopropyldiamine in 20 mL of THF-MeCN (1:1) mixed solution and stirred at 63 °C for 72 hours. After the reaction mixture was cooled to room temperature, it was evaporated under vacuum. The residual liquid was extracted with ethyl acetate and saturated sodium bicarbonate solution. After separation of the organic layer, it was dried with sodium sulfate, filtered and evaporated under vacuum. The residual liquid was purified by silica gel chromatography (0-10% methanol / dichloromethane) to obtain an ionizable lipid, denoted as ionizable lipid 2.

[0038] Example 6 2.44 g of intermediate 3 prepared in Example 3 was mixed with 236 mg (235 μL) of N-hydroxyethyl-1,3-propanediamine solution and 775.44 mg (1045 μL) of N,N-diisopropyldiamine in 20 mL of THF-MeCN (1:1) mixed solution and stirred at 63 °C for 72 hours. After the reaction mixture was cooled to room temperature, it was evaporated under vacuum. The residual liquid was extracted with ethyl acetate and saturated sodium bicarbonate solution. After separation of the organic layer, it was dried with sodium sulfate, filtered and evaporated under vacuum. The residual liquid was purified by silica gel chromatography (0-10% methanol / dichloromethane) to obtain an ionizable lipid, denoted as ionizable lipid 3.

[0039] Example 7 2.01 g of intermediate 1 prepared in Example 1 was mixed with 306.54 mg (413 μL) of 3-dimethylaminopropylamine solution and 775.44 mg (1045 μL) of N,N-diisopropyldiamine in 20 mL of THF-MeCN (1:1) mixed solution and stirred at 63 °C for 72 hours. After the reaction mixture was cooled to room temperature, it was evaporated under vacuum. The residual liquid was extracted with ethyl acetate and saturated sodium bicarbonate solution. After the organic layer was separated, it was dried with sodium sulfate, filtered, and evaporated under vacuum. The residual liquid was purified by silica gel chromatography (0-10% methanol / dichloromethane) to obtain an ionizable lipid, designated as ionizable lipid 4.

[0040] Example 8 2.4 g of intermediate 2 prepared in Example 2 was mixed with 306.54 mg (413 μL) of 3-dimethylaminopropylamine solution and 775.44 mg (1045 μL) of N,N-diisopropyldiamine in 20 mL of THF-MeCN (1:1) mixed solution and stirred at 63 °C for 72 hours. After the reaction mixture was cooled to room temperature, it was evaporated under vacuum. The residual liquid was extracted with ethyl acetate and saturated sodium bicarbonate solution. After separation of the organic layer, it was dried with sodium sulfate, filtered, and evaporated under vacuum. The residual liquid was purified by silica gel chromatography (0-10% methanol / dichloromethane) to obtain an ionizable lipid, designated as ionizable lipid 5.

[0041] Example 9 2.44 g of intermediate 3 prepared in Example 3 was mixed with 306.54 mg (413 μL) of 3-dimethylaminopropylamine solution and 775.44 mg (1045 μL) of N,N-diisopropyldiamine in 20 mL of THF-MeCN (1:1) mixed solution and stirred at 63 °C for 72 hours. After the reaction mixture was cooled to room temperature, it was evaporated under vacuum. The residual liquid was extracted with ethyl acetate and saturated sodium bicarbonate solution. After separation of the organic layer, it was dried with sodium sulfate, filtered and evaporated under vacuum. The residual liquid was purified by silica gel chromatography (0-10% methanol / dichloromethane) to obtain an ionizable lipid, designated as ionizable lipid 6.

[0042] Example 10 2.01 g of intermediate 1 prepared in Example 1 was mixed with 318.56 mg (361 μL) of N,N-dimethyldipropyltriamine solution and 775.44 mg (1045 μL) of N,N-diisopropyldiamine in 20 mL of THF-MeCN (1:1) mixed solution and stirred at 63 °C for 72 hours. After the reaction mixture was cooled to room temperature, it was evaporated under vacuum. The residual liquid was extracted with ethyl acetate and saturated sodium bicarbonate solution. After separation of the organic layer, it was dried with sodium sulfate, filtered, and evaporated under vacuum. The residual liquid was purified by silica gel chromatography (0-10% methanol / dichloromethane) to obtain an ionizable lipid, designated as ionizable lipid 7.

[0043] Example 11 2.4 g of intermediate 2 prepared in Example 2 was mixed with 318.56 mg (361 μL) of N,N-dimethyldipropyltriamine solution and 775.44 mg (1045 μL) of N,N-diisopropyldiamine in 20 mL of THF-MeCN (1:1) mixed solution and stirred at 63 °C for 72 hours. After the reaction mixture was cooled to room temperature, it was evaporated under vacuum. The residual liquid was extracted with ethyl acetate and saturated sodium bicarbonate solution. After separation of the organic layer, it was dried with sodium sulfate, filtered, and evaporated under vacuum. The residual liquid was purified by silica gel chromatography (0-10% methanol / dichloromethane) to obtain an ionizable lipid, designated as ionizable lipid 8.

[0044] Example 12 2.44 g of intermediate 3 prepared in step 3 was mixed with 318.56 mg (361 μL) of N,N-dimethyldipropyltriamine solution and 775.44 mg (1045 μL) of N,N-diisopropyldiamine in 20 mL of THF-MeCN (1:1) mixed solution and stirred at 63 °C for 72 hours. After the reaction mixture was cooled to room temperature, it was evaporated under vacuum. The residual liquid was extracted with ethyl acetate and saturated sodium bicarbonate solution. After separation of the organic layer, it was dried with sodium sulfate, filtered, and evaporated under vacuum. The residual liquid was purified by silica gel chromatography (0-10% methanol / dichloromethane) to obtain an ionizable lipid, designated as ionizable lipid 9.

[0045] The molecular structures of ionizable lipids 1-9 prepared in Examples 4-12 are shown in [reference needed]. Figure 1 The 1H NMR spectra of the ionizable lipids 1-9 prepared in Examples 4-12 are shown below. Figures 2-10 High-resolution mass spectra of ionizable lipids 1-9 prepared in Examples 4-12 are shown below. Figures 11-19 .

[0046] Example 13 Lipid nanoparticles were prepared using ionizable lipids 1-9 prepared in Examples 4-12 as raw materials, and the steps are as follows: (1) Preparation of the ethanol phase ① Using ethanol as a solvent, prepare 10 mg / mL neutral phospholipid solution, 10 mg / mL cholesterol solution, 10 mg / mL dimyristoylglycerol-polyethylene glycol 2000 solution, and 20 mg / mL ionizable lipid No. 1 solution respectively. According to the molar ratio of ionizable lipid No. 1, neutral phospholipid, cholesterol, and dimyristoylglycerol-polyethylene glycol 2000 of 50:10:38.5:1.5, mix 264.426 μL of ionizable lipid No. 1 solution, 94.818 μL of neutral phospholipid solution, 178.63 μL of cholesterol solution, and 45.16 μL of dimyristoylglycerol-polyethylene glycol 2000 solution, and then add 416.96 μL of ethanol (to make the concentration of ionizable lipids in the ethanol phase of each group the same) to obtain ethanol phase 1.

[0047] ② Using ethanol as a solvent, prepare 10 mg / mL neutral phospholipid solution, 10 mg / mL cholesterol solution, 10 mg / mL dimyristoylglycerol-polyethylene glycol 2000 solution, and 20 mg / mL ionizable lipid No. 2 solution respectively. Mix 314.925 μL of the prepared ionizable lipid No. 2 solution, 94.818 μL of neutral phospholipid solution, 178.63 μL of cholesterol solution, and 45.16 μL of dimyristoylglycerol-polyethylene glycol 2000 solution, and then add 366.45 μL of ethanol to obtain ethanol phase 2.

[0048] ③ Using ethanol as a solvent, prepare 10 mg / mL neutral phospholipid solution, 10 mg / mL cholesterol solution, 10 mg / mL dimyristoylglycerol-polyethylene glycol 2000 solution, and 20 mg / mL ionizable lipid No. 3 solution respectively. According to the molar ratio of ionizable lipid No. 3, neutral phospholipid, cholesterol, and dimyristoylglycerol-polyethylene glycol 2000 of 50:10:38.5:1.5, mix 327.54 μL of ionizable lipid No. 3 solution, 94.818 μL of neutral phospholipid solution, 178.63 μL of cholesterol solution, and 45.16 μL of dimyristoylglycerol-polyethylene glycol 2000 solution, and then add 353.83 μL of ethanol to obtain ethanol phase 3.

[0049] ④ Using ethanol as a solvent, prepare 10 mg / mL neutral phospholipid solution, 10 mg / mL cholesterol solution, 10 mg / mL dimyristoylglycerol-polyethylene glycol 2000 solution, and 20 mg / mL ionizable lipid No. 4 solution respectively. According to the molar ratio of ionizable lipid No. 4, neutral phospholipid, cholesterol, and dimyristoylglycerol-polyethylene glycol 2000 of 50:10:38.5:1.5, mix 183.3 μL of ionizable lipid No. 4 solution, 94.818 μL of neutral phospholipid solution, 178.63 μL of cholesterol solution, and 45.16 μL of dimyristoylglycerol-polyethylene glycol 2000 solution, and then add 498.08 μL of ethanol to obtain ethanol phase 4.

[0050] ⑤ Using ethanol as a solvent, prepare 10 mg / mL neutral phospholipid solution, 10 mg / mL cholesterol solution, 10 mg / mL dimyristoylglycerol-polyethylene glycol 2000 solution, and 20 mg / mL ionizable lipid No. 5 solution respectively. Mix 216.97 μL of the prepared ionizable lipid No. 5 solution, 94.818 μL of neutral phospholipid solution, 178.63 μL of cholesterol solution, and 45.16 μL of dimyristoylglycerol-polyethylene glycol 2000 solution, then add 464.41 μL of ethanol to obtain ethanol phase 5.

[0051] ⑥ Using ethanol as a solvent, prepare 10 mg / mL neutral phospholipid solution, 10 mg / mL cholesterol solution, 10 mg / mL dimyristoylglycerol-polyethylene glycol 2000 solution, and 20 mg / mL ionizable lipid 6 solution respectively. Mix 225.38 μL of the prepared ionizable lipid 6 solution, 94.818 μL of neutral phospholipid solution, 178.63 μL of cholesterol solution, and 45.16 μL of dimyristoylglycerol-polyethylene glycol 2000 solution, then add 456.01 μL of ethanol to obtain ethanol phase 6.

[0052] ⑦ Using ethanol as a solvent, prepare 10 mg / mL neutral phospholipid solution, 10 mg / mL cholesterol solution, 10 mg / mL dimyristoylglycerol-polyethylene glycol 2000 solution, and 20 mg / mL ionizable lipid No. 7 solution respectively. According to the molar ratio of ionizable lipid No. 7, neutral phospholipid, cholesterol, and dimyristoylglycerol-polyethylene glycol 2000 of 50:10:38.5:1.5, mix 184.50 μL of ionizable lipid No. 7 solution, 94.818 μL of neutral phospholipid solution, 178.63 μL of cholesterol solution, and 45.16 μL of dimyristoylglycerol-polyethylene glycol 2000 solution, and then add 603.09 μL of ethanol to obtain ethanol phase 7.

[0053] ⑧ Using ethanol as a solvent, prepare 10 mg / mL neutral phospholipid solution, 10 mg / mL cholesterol solution, 10 mg / mL dimyristoylglycerol-polyethylene glycol 2000 solution, and 20 mg / mL ionizable lipid No. 8 solution respectively. Mix 218.17 μL of the prepared ionizable lipid No. 8 solution, 94.818 μL of neutral phospholipid solution, 178.63 μL of cholesterol solution, and 45.16 μL of dimyristoylglycerol-polyethylene glycol 2000 solution, then add 596.43 μL of ethanol to obtain ethanol phase 8.

[0054] ⑨ Using ethanol as a solvent, prepare 10 mg / mL neutral phospholipid solution, 10 mg / mL cholesterol solution, 10 mg / mL dimyristoylglycerol-polyethylene glycol 2000 solution, and 20 mg / mL ionizable lipid No. 9 solution respectively. Mix 226.58 μL of the prepared ionizable lipid No. 9 solution, 94.818 μL of neutral phospholipid solution, 178.63 μL of cholesterol solution, and 45.16 μL of dimyristoylglycerol-polyethylene glycol 2000 solution, then add 561.01 μL of ethanol to obtain ethanol phase 9.

[0055] (2) Preparation of the aqueous phase 5.94 mL of GFP mRNA (1 mg / mL) was diluted with 21.06 mL of citrate buffer (0.01 M, pH=4.0) to obtain an aqueous buffer.

[0056] (3) Preparation of lipid nanoparticles 30 μL of the ethanol phases 1-9 prepared in step (1) were rapidly mixed with 90 μL of the aqueous buffer containing mRNA obtained in step (2), vortexed for 15 seconds, and then allowed to stand for 10 minutes. The mixture was then dialyzed in PBS for two hours using a dialysis tube with a molecular weight cutoff of 3500 Da to obtain lipid nanoparticles.

[0057] Control group: GFP mRNA was encapsulated using commercially available ionizable cationic lipids (purchased from McLean Pharmaceuticals, hereinafter the same). The specific steps are as follows: (1) Using ethanol as a solvent, prepare 10 mg / mL neutral phospholipid solution, 10 mg / mL cholesterol solution, 10 mg / mL dimyristoylglycerol-polyethylene glycol 2000 solution, and 20 mg / mL control lipid solution respectively. According to the molar ratio of control lipid, neutral phospholipid, cholesterol and dimyristoylglycerol-polyethylene glycol 2000 of 50:10:38.5:1.5, mix 192.63 μL of control lipid solution, 94.818 μL of neutral phospholipid solution, 178.63 μL of cholesterol solution and 45.16 μL of dimyristoylglycerol-polyethylene glycol 2000 solution, and then add 488.76 μL of ethanol to obtain the ethanol phase of control lipid.

[0058] (2) Dilute 330 μL of GFP mRNA (1 mg / mL) with 2.67 mL of citrate buffer (0.01 M, pH=4.0) to obtain an aqueous buffer.

[0059] (3) Mix 30 μL of the control group lipid ethanol phase with 90 μL of aqueous buffer containing mRNA, vortex for 15 seconds, let stand for 10 minutes, and dialyze in PBS for two hours using a dialysis tube with a molecular weight cutoff of 3500 Da to obtain control group lipid nanoparticles.

[0060] Performance testing I. Determination of Encapsulation Efficiency The assay was performed using the Quant-iT™ RiboGreen™ RNA kit.

[0061] The encapsulation efficiency of GFP mRNA prepared from lipid nanoparticles (prepared using ionizable lipids 1-9 from Examples 4-12) and the control group lipid nanoparticles in Example 13 is shown in the figure. Figure 20 .

[0062] Depend on Figure 20It can be seen that the lipid nanoparticles prepared based on ionizable lipids in this invention have excellent encapsulation efficiency, with ionizable lipids No. 4-8 being superior to commercially available ionizable lipids. High encapsulation efficiency of lipid nanoparticles means that more mRNA was successfully encapsulated within the nanoparticles during the preparation process. This is generally an ideal characteristic because high encapsulation efficiency can improve the efficacy of lipid nanoparticles in the field of mRNA therapy.

[0063] II. Determination of DLS Particle Size Distribution Representative particle size distributions of lipid nanoparticles prepared using ionizable lipids 1-9 (prepared in Examples 4-12) and control group lipid nanoparticles encapsulating GFP mRNA in Example 13 are shown in the figure. Figure 21 The results of the average particle size determination are shown in [the table below]. Figure 22 In the figure, LNP / GFP mRNA 1 represents lipid nanoparticles prepared using ionizable lipid 1 prepared in Example 4 as the raw material, LNP / GFP mRNA 2 represents lipid nanoparticles prepared using ionizable lipid 2 prepared in Example 4 as the raw material, and so on. The size of lipid nanoparticles has a significant impact on their uptake by cells. Generally, the size of lipid nanoparticles should be between 50-200 nm to ensure effective uptake and transfection by cells. On the one hand, the size of lipid nanoparticles should be small enough to pass through cell membrane pores or enter intracellular vesicles. On the other hand, the size of lipid nanoparticles should not be too small, as excessively small nanoparticles may be cleared by phagocytes (such as macrophages), thus failing to exert their function. Appropriate lipid nanoparticle size and surface charge can improve their application effects in gene therapy, drug delivery, and other fields. Figure 21 and 22 It can be seen that the lipid nanoparticles prepared by this invention have a particle size between 100-200 nm and a particle size distribution coefficient of less than 0.2, indicating that the lipid nanoparticles prepared by this invention have a uniform particle size distribution and a size suitable for entering cells for transfection.

[0064] III. Measurement of Zeta potential In Example 13, the zeta potential measurements of lipid nanoparticles prepared using ionizable lipids 1-9 (prepared in Examples 4-12) and the control group lipid nanoparticles encapsulating GFP mRNA are shown in the figure. Figure 23 .Depend on Figure 23 It can be seen that the surface of the lipid nanoparticles prepared by this invention tends to be neutral. On the one hand, this indicates that the positively charged cationic lipids and the negatively charged RNA successfully bind. On the other hand, the electrically neutral nanoparticles can effectively avoid the adsorption of non-specific plasma proteins in vivo, thereby reducing clearance and facilitating in vivo application.

[0065] IV. Cytotoxicity Assay CCK-8 assay for cytotoxicity DC2.4 cells were digested, centrifuged, and counted. The cell suspension was then seeded into 96-well plates at a density of 5 × 10⁶ cells / well. 3 Cells / well (100uL per well) were cultured for 12 hours at 37°C, 5% CO2, and 90% humidity.

[0066] Lipid nanoparticle solutions with different concentration gradients (total lipid concentrations of 60, 30, and 15 µM) were prepared (lipid nanoparticles prepared from ionizable lipids 1-9 and control group lipid nanoparticles). Each solution was added to a 96-well plate in triplicate at each concentration and incubated for 24 hours at 37°C, 5% CO2, and 90% humidity.

[0067] Add 10 μL of CCK-8 solution to each well and incubate for 1 hour at 37°C, 5% CO2, and 90% humidity. Measure the absorbance at 450 nm using a microplate reader, and calculate cell viability using untreated cells as a blank control.

[0068] In Example 13, the relative cell viability of lipid nanoparticles prepared using ionizable lipids 1-9 (prepared in Examples 4-12) and the control group lipid nanoparticles encapsulating GFP mRNA at different total lipid concentrations is shown in the figure. Figure 24 Good biocompatibility means it causes fewer toxic or immune responses in vivo, which facilitates the application of nanoparticles in the medical field. Figure 24 It can be seen that the lipid nanoparticles prepared based on ionizable lipids in this invention have no significant effect on the survival of DC2.4 cells, indicating that they have good biocompatibility and are a promising drug delivery system that can be applied in the medical field to drug delivery, gene therapy and tumor treatment.

[0069] Application Example 1 Transfection procedure: The lipid nanoparticles prepared in Example 13 were directly added to the DC2.4 cell culture medium; Incubation: Cells were incubated at 37°C, 5% CO2, and 90% humidity for 24 hours so that LNPs could interact with the cell membrane, be taken up by the cell, release nucleic acids, and express the corresponding proteins.

[0070] In Example 13, fluorescence micrographs of lipid nanoparticles prepared using ionizable lipids 1-9 (prepared in Examples 4-12) and control group lipid nanoparticles encapsulating GFP mRNA transfected into DC2.4 cells are shown below. Figure 25 .

[0071] The transfection efficiency of DC2.4 cells was quantified by flow cytometry. The transfection efficiency of lipid nanoparticles prepared using ionizable lipids 1-9 (prepared in Examples 4-12) and the control group lipid nanoparticles encapsulating GFP mRNA in DC2.4 cells was measured in Example 13. Figure 26 .

[0072] In Example 13, the average fluorescence intensity of lipid nanoparticles prepared using ionizable lipids 1-9 (prepared in Examples 4-12) and the control group lipid nanoparticles encapsulating GFP mRNA transfected into DC2.4 cells is shown in the figure. Figure 27 .

[0073] Application Example 2 Transfection procedure: The lipid nanoparticles prepared in Example 13 were directly added to the 293T cell culture medium; Incubation: Cells were incubated at 37°C, 5% CO2, and 90% humidity for 24 hours so that LNPs could interact with the cell membrane, be taken up by the cell, release nucleic acids, and express the corresponding proteins.

[0074] In Example 13, fluorescence micrographs of lipid nanoparticles prepared using ionizable lipids 1-9 (prepared in Examples 4-12) and control group lipid nanoparticles encapsulating GFP mRNA transfected into 293T cells are shown. Figure 28 .

[0075] The transfection efficiency of DC2.4 cells was quantified by flow cytometry. The transfection efficiency of lipid nanoparticles prepared using ionizable lipids 1-9 (prepared in Examples 4-12) and the control group lipid nanoparticles encapsulating GFP mRNA in 293T cells was measured in Example 13. Figure 29 .

[0076] In Example 13, the average fluorescence intensity of lipid nanoparticles prepared using ionizable lipids 1-9 (prepared in Examples 4-12) and the control group lipid nanoparticles encapsulating GFP mRNA transfected into 293T cells is shown in the figure. Figure 30 .

[0077] In Example 13, the average fluorescence intensity fold-down of lipid nanoparticles prepared using ionizable lipids 1-9 (prepared in Examples 4-12) and control group lipid nanoparticles encapsulated with GFP mRNA and transfected into DC2.4 and 293T cells, relative to untreated cells, is shown in the following heatmap: Figure 31 .

[0078] Depend on Figures 25-31It can be seen that the lipid nanoparticles No. 2 and Nos. 5-9 obtained in this invention have comparable or higher transfection efficiencies than the control group of ionizable cationic lipids, and can achieve highly efficient green fluorescent protein expression. High transfection efficiency of lipid nanoparticles means that they can effectively deliver mRNA into target cells during transfection and achieve highly efficient protein expression. This is generally an ideal characteristic, especially for fields such as gene therapy and gene expression research.

[0079] Application Example 3 Lipid nanoparticles were prepared by replacing the aqueous buffer in Example 13 with an equal volume of Fluc mRNA aqueous buffer. The preparation method was the same as in Example 13, and commercially available ionizable cationic lipids encapsulating Fluc mRNA were used as a control group.

[0080] Injection administration: The above lipid nanoparticles were injected intramuscularly into the left inner thigh of mice. Eight hours after administration, the luciferase substrate was injected intraperitoneally. Within 10-15 minutes, the bioluminescence in mice was observed using small animal in vivo imaging.

[0081] In Example 13, representative in vivo imaging images of lipid nanoparticles prepared using ionizable lipids 5-9 (prepared in Examples 8-12) as raw materials, and control group lipid nanoparticles encapsulating Fluc mRNA, were shown. Figure 32 In the figure, LNP / Flu mRNA No. 5 represents lipid nanoparticles prepared from ionizable lipid No. 5 prepared in Example 8, and so on, the same below.

[0082] Figure 24 The lipid nanoparticles prepared in Example 13 using ionizable lipids 5-9 (prepared in Examples 8-12) and the control group lipid nanoparticles encapsulating Fluc mRNA were expressed in mice. The relative light intensity of the luciferase-catalyzed luciferase substrate luminescence is shown in the figure. Figure 33 .

[0083] Depend on Figure 32 and Figure 33 As can be seen, compared with the mice given the control group of nanoparticles, the mice given the lipid nanoparticles obtained in this invention showed almost no luciferase expression in their livers, but exhibited highly efficient luciferase expression activity at the injection site. The results indicate that the nanoparticles obtained in this invention can efficiently express the corresponding proteins in vivo and have broad application prospects for in vivo mRNA delivery.

[0084] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing lipid nanoparticles, characterized in that, Ionizable lipids, neutral phospholipids, cholesterol, and dimyristic glycerol-polyethylene glycol 2000 were dissolved in ethanol to obtain an ethanol-phase lipid mixture, which was then mixed with an aqueous buffer containing mRNA, allowed to stand, and dialyzed to obtain the lipid nanoparticles. The molar ratio of the ionizable lipids, neutral phospholipids, cholesterol, and dimyristic glycerol-polyethylene glycol 2000 is 50:10:38.5:1.5; The method for preparing the ionizable lipid involves preparing an intermediate using compound A and compound B as raw materials, heating and stirring the intermediate with an organic amine in a tetrahydrofuran-acetonitrile mixed solution, and then evaporating, extracting, and purifying the reaction mixture to obtain the ionizable lipid. The organic amine is a mixture of N,N-diisopropyl diamine and N,N-dimethyldipropyltriamine; Compound A is isooctyl alcohol; Compound B is 8-bromooctanoic acid; The preparation method of the intermediate is as follows: Compound A, carbodiimide hydrochloride, and 4-dimethylaminopyridine were dissolved in dichloromethane solution, and then compound B was added and stirred to obtain a reaction solution. The reaction solution was extracted, washed, evaporated, and purified to obtain the intermediate. The mass ratio of compound A to compound B was 3.5:5, and the mass ratio of compound A to carbodiimide hydrochloride and 4-dimethylaminopyridine was 3.5:4.3:1.

7. The ratio of the intermediate to the organic amine and the tetrahydrofuran-acetonitrile mixed solution is (2-2.5) g : (1280-1458) μL : 20 mL; The structural formula of the ionizable lipid is: .

2. The method for preparing lipid nanoparticles according to claim 1, characterized in that, The volume ratio of the ethanol phase lipid mixture to the aqueous phase buffer is 1:

3.

3. The use of lipid nanoparticles prepared by the preparation method according to any one of claims 1-2 in the preparation of mRNA delivery drugs.

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