Lipid nanoparticles based on cationic lipids with branch structures at the junction and uses thereof

Abstract

The application discloses a kind of lipid nanoparticles based on cationic lipid containing branch structure at junction and application thereof, belong to medical technical field.The raw material composition of the lipid nanoparticles includes: cationic lipid containing branch structure at junction, auxiliary lipid, cholesterol, and the structural formula of the cationic lipid is as shown in formula (I).The lipid nanoparticles based on ionizable cationic lipid containing branch structure at junction provided by the application can effectively deliver nucleic acid drugs such as mRNA in animal body.Compared with commercially available ionizable lipid SM-102 and its formula, the lipid nanoparticles provided by the application have better mRNA delivery efficiency in animal body, which is conducive to improving the clinical application effect of nucleic acid drugs such as mRNA.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a kind of lipid nanoparticle based on junction containing branched structure cationic lipid and its application in mRNA vaccine and gene therapy product as delivery carrier material. BACKGROUND

[0002] mRNA drug is a kind of emerging technology in recent years, which combines molecular biology and immunology. Exogenous mRNA synthesized in vitro can encode proteins through the body's own translation system, and has unique advantages in the design of preventive and therapeutic vaccines. However, there are still many problems to be solved in mRNA vaccine design, among which the lack of safe and efficient delivery system is one of the main reasons limiting its application. How to efficiently deliver mRNA to cells and cytoplasm is a key scientific problem that needs to be solved in mRNA vaccine research.

[0003] In view of the characteristics of mRNA instability, negative charge and difficulty in being taken up by cells, scientists have developed a series of delivery systems, including lipid-based delivery systems, peptide segment-based delivery systems, polymer-based delivery systems, etc. Among them, lipid nanoparticles (LNPs) based on lipids are one of the most potential carrier materials due to their good biological safety and high delivery capacity, and currently several drugs based on lipid nanoparticles (LNPs) have been approved by FDA.

[0004] At present, the structure-activity relationship between the components of lipid nanoparticles (LNPs) is not completely clear. Lipid nanoparticles (LNPs) are usually composed of cationic lipids, phospholipids, PEG-modified lipids and cholesterol. Among them, cationic lipids are the core and soul, and their properties can affect the overall formulation and biological characteristics of LNP.

[0005] Cationic lipids are mainly composed of positively charged head groups, hydrophobic tails and hydrophobic chains connecting the two. Among them, the positively charged head group mainly participates in the wrapping of negatively charged biological macromolecules such as DNA and RNA, as well as the interaction with cell membranes and the promotion of endosome escape process. The clinically used ionizable lipids DLin-MC3-DMA, SM-102 and ALC-0315 contain tertiary amine head groups, which can undergo pH-dependent ionization. The hydrophobic tail is usually a long-chain alkyl group or a saturated / unsaturated fatty acid chain, which provides hydrophobicity to the lipid molecule, helping the stability of the liposome in the body. The hydrophobic chain connecting the head group and the tail makes the whole molecule more stable. In order to reduce the toxicity of ionizable polymer lipids, biodegradable chemical bonds are introduced to realize the degradation function, such as esters, amides and thiols, which are the first choice, and can be quickly cleared in the body, can be used multiple times and reduce side effects.

[0006] Since cationic lipids play a decisive role in the delivery of mRNA, screening high-efficiency and safe cationic lipids has important clinical significance. At present, a large number of studies focus on the optimization of head structure, tail chain structure and the development of degradable connecting fragments, and the study on the influence of the structure of the connecting fragment on the effect of the cationic lipid has not been reported. SUMMARY

[0007] The purpose of the present application is to provide a new type of cationic lipid material for preparing lipid nanoparticles, which realizes effective delivery of mRNA as a drug delivery carrier.

[0008] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] The present application provides a kind of lipid nanoparticles, the raw material composition of the lipid nanoparticles includes: the cationic lipid containing branch structure at the junction, auxiliary lipid, cholesterol, the structural formula of the cationic lipid is as shown in formula (I),

[0010]

[0011] Wherein R1 is selected from: -C(CH2)5, -CHCH2CH(CH3)2, -C(CH3)2, -CHCH3;

[0012] R2 is selected from: CH3(CH2)9CH[(CH2)7CH3]CH2O-, CH3(CH2) 11 CH[(CH2)9CH3]CH2O-, CH3(CH2) 13 CH[(CH2) 11 CH3]CH2O-, CH3(CH2)7CH[(CH2)5CH3]CH2O-, [CH3(CH2) 11 ]2N-, [CH3(CH2) 13 ]2N-.

[0013] The cationic lipid structure is composed of a tertiary amine group head, a fatty acid chain hydrophobic tail and a biodegradable connecting fragment, wherein the connecting fragment is modified with a branch structure. The tertiary amine group of the cationic lipid forms a positively charged hydrophilic end in the buffer, which can bind to a negatively charged drug through electrostatic interaction. Due to the hydrophobic supramolecular force, the lipid material self-assembles to obtain the lipid nanoparticles.

[0014] The present application shows that the lipid nanoparticles formed by self-assembly of the ionizable cationic lipid containing branch structure at the junction and other lipid materials as a drug delivery carrier have high cell transfection efficiency, and can effectively deliver drugs such as mRNA in vitro and in vivo, so as to translate them into proteins.

[0015] The preparation method of the branched cationic lipid at the connection comprises the following steps: first, 2-dimethylaminoethanol is dissolved in pyridine with glutaric anhydride derivatives to generate branched organic acid through alcoholysis reaction; then, the branched organic acid and alcohol or amine containing a fatty chain are dissolved in an organic solvent to generate ester bond or amide bond through condensation reaction under the catalysis of a catalyst, and the branched cationic lipid at the connection is separated and purified from the product.

[0016] The branched cationic lipid at the connection is constructed by using the alcohol hydroxyl group of 2-dimethylaminoethanol to react with glutaric anhydride derivatives to generate branched organic acid, and then the branched organic acid is condensed with alcohol or amine containing a fatty chain to generate ester bond or amide bond.

[0017] Preferably, the glutaric anhydride derivatives can be, but are not limited to, 1,1-cyclohexyl dihydric acid anhydride, 3-methyl glutaric anhydride, 3-isobutyl glutaric anhydride and 3,3-dimethyl glutaric anhydride.

[0018] Preferably, the molar ratio of 2-dimethylaminoethanol to glutaric anhydride derivatives is 1.5-3:1.

[0019] Preferably, the branched organic acid is generated through stirring at room temperature for 6-12 hours.

[0020] Preferably, the alcohol containing a fatty chain can be, but is not limited to, any one of 2-n-octyl-1-dodecanol, 2-decyl-1-tetradecanol, 2-dodecylhexadecan-1-ol, 2-hexyl-1-decanol, 7-tetradecanol and castor oil. More preferably, the alcohol containing a fatty chain is 2-n-octyl-1-dodecanol.

[0021] Preferably, the amine containing a fatty chain can be, but is not limited to, ditetradecylamine and didodecylamine.

[0022] Preferably, the molar ratio of the branched organic acid obtained in the first step to the alcohol or amine containing a fatty chain is 1.2-3:1.

[0023] Preferably, the organic solvent can be, but is not limited to, dichloromethane.

[0024] Preferably, the catalyst can be, but is not limited to, EDC / DMAP system.

[0025] Preferably, the condensation reaction is carried out at room temperature for 6-12 hours.

[0026] After the first step reaction, the remaining 2-dimethylaminoethanol and solvent are removed by rotary evaporation to obtain the pure product; after the second step reaction, the solvent is removed by rotary evaporation to separate the initial product, and the cationic lipid is purified by silica gel chromatography, and the eluent of silica gel chromatography is a mixture of n-hexane and ethyl acetate with a volume ratio of 2:1.

[0027] In the present application, the lipid nanoparticles are formed by self-assembly after mixing the ionizable cationic lipid with branch structure at the connection with the auxiliary lipid and cholesterol. The preparation method of the lipid nanoparticles can use, but is not limited to, ethanol injection method, film method and ultrasonic method. Specifically, when preparing the drug-loaded lipid nanoparticles, the above-mentioned methods are used to make the lipid material and the drug to be loaded in the buffer through supramolecular force and electrostatic interaction, and self-assemble to form nanoparticles.

[0028] Among them, the ethanol injection method is to dissolve the ionizable cationic lipid, the auxiliary lipid and the cholesterol in a certain amount of ethanol, then inject the ethanol solution containing the lipid material into the buffer containing the drug to be loaded, and self-assemble to form nanoparticles, and then remove the ethanol by dialysis to obtain stable nanoparticles.

[0029] Preferably, the auxiliary lipid includes phospholipid and polyethylene glycol functionalized lipid; the phospholipid can be, but is not limited to, distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylethanolamine (DOPE), and the polyethylene glycol functionalized lipid can be, but is not limited to, dimyristylglycerol-polyethylene glycol 2000 (DMG-PEG2000).

[0030] Preferably, the molar ratio of the cationic lipid, the phospholipid, the cholesterol and the polyethylene glycol functionalized lipid is 25-75:5-15:15-60:1-2. Further preferably, the molar ratio of the above-mentioned four components is 25-40:10-15:45-60:1-2. More preferably, the molar ratio of the above-mentioned four components is 39:15:45:1.

[0031] The present application also provides the use of the lipid nanoparticles as carriers in the preparation of nucleic acid drugs for delivery.

[0032] The lipid nanoparticles based on the ionizable cationic lipid with branch structure at the connection provided by the present application can effectively deliver nucleic acid drugs such as mRNA, and the nanomaterial has potential application value in the development of nucleic acid drugs.

[0033] Specifically, the use includes adding the cationic lipid with branch structure at the connection, the auxiliary lipid and the cholesterol into an acidic buffer containing nucleic acid, self-assembling to form nucleic acid-loaded lipid nanoparticles, and preparing the nucleic acid drug for delivery.

[0034] Preferably, the ionizable cationic lipid, the helper lipid, and the cholesterol are self-assembled into lipid nanoparticles in a buffer containing the nucleic acid by ethanol injection.

[0035] The delivered nucleic acid drug can be, but is not limited to, an mRNA vaccine.

[0036] Preferably, the total mass of the lipid material to the mass of the mRNA is 20-160:1. The mRNA molecule is large, and when the amount of lipid material is too small, it is difficult to effectively encapsulate and protect the mRNA, thereby reducing the transfection efficiency. When the amount of lipid material is too large, it can reduce the efficiency of endosome escape, thereby reducing the transfection efficiency. Within the appropriate mass ratio range, better encapsulation efficiency and transfection efficiency can be ensured. More preferably, the total mass of the lipid material to the mass of the mRNA is 40:1.

[0037] The present application has the following beneficial effects:

[0038] (1) The present application constructs an ionizable cationic lipid with a branched structure at the connection by a simple two-step reaction, which is applied to the preparation of lipid nanoparticles. One end of the ionizable cationic lipid is a tertiary amine structure, which can bind to negatively charged drugs through electrostatic interaction under appropriate conditions.

[0039] (2) The lipid nanoparticles based on the ionizable cationic lipid with a branched structure at the connection provided by the present application can effectively deliver nucleic acid drugs such as mRNA in animals. Compared with commercially available ionizable lipids SM-102 and its formulations, the lipid nanoparticles provided by the present application have better mRNA delivery efficiency in animals, which is conducive to improving the clinical application effect of mRNA and other nucleic acid drugs. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Chemical characterization of the cationic lipid with a branched structure of isobutyl and an octyldodecanol fatty chain.

[0041] Figure 2 TEM image of the cLNP prepared in Example 7

[0042] Figure 3 Actual image of the transfection effect of the LNPs prepared in Examples 1-15 in mice.

[0043] Figure 4 The transfection effect of the LNPs prepared in Examples 1 and 7 under different ratios of lipid materials in mice. Figure 3 The transfection effect of the LNPs prepared in Examples 1 and 7 under different ratios of lipid materials in mice.

[0044] Figure 5 The transfection effect of the LNPs prepared in Examples 1 and 7 under different ratios of lipid materials in mice.

[0045] Figure 6 For Figure 5 Quantitative analysis results of transfection effect.

[0046] Figure 7 Results of lipid nanoparticle cell transfection experiments, wherein sLNP is a lipid nanoparticle based on SM-102, cLNP is a lipid nanoparticle of Formula No. 5 with a branched structure of isobutyl and a fatty chain of octyldodecanol, and bLNP is a lipid nanoparticle of Formula No. 5 without a branched structure and with a fatty chain of octyldodecanol

[0047] Figure 8 Actual photos of anticancer effects of tumor drugs prepared using lipid nanoparticles.

[0048] Figure 9 Tumor growth curve. DETAILED DESCRIPTION

[0049] The application will be further described below in conjunction with specific examples. The following examples are only used to illustrate the application and are not intended to limit the scope of application. Modifications or replacements of the methods, steps or conditions of the application without departing from the spirit and essence of the application shall fall within the scope of the application.

[0050] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0051] The compounds involved in the examples and the English abbreviations are as follows:

[0052] EDC: 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide, CAS No.: 1892-57-5;

[0053] DMAP: 4-dimethylaminopyridine, CAS No.: 1122-58-3;

[0054] CDCl2: dichloromethane, CAS No.: 75-09-2;

[0055] 2-dimethylaminoethanol, CAS No.: 108-01-0;

[0056] glutaric anhydride, CAS No.: 108-55-4;

[0057] 1,1-cyclohexyl diethyl anhydride, CAS No.: 1010-26-0;

[0058] 3-methyl glutaric anhydride, CAS No.: 4166-53-4;

[0059] 3-isobutyl glutaric anhydride, CAS No.: 185815-59-2;

[0060] 3,3-dimethyl glutaric anhydride, CAS No.: 4160-82-1;

[0061] 2-n-octyl-1-dodecanol, CAS No.: 5333-42-6;

[0062] 2-decyl-1-tetradecanol, CAS No.: 58670-89-6;

[0063] 2-dodecylhexadecan-1-ol, CAS No.: 72388-18-2;

[0064] D-luciferin potassium salt, CAS No.: 115144-35-9;

[0065] DSPC phospholipid, CAS No.: 816-94-4, with the following structure:

[0066]

[0067] DMG-PEG2000 lipid, CAS No.: 160743-62-4, with the following structure:

[0068]

[0069] Cholesterol, CAS No.: 57-88-5, with the following structure:

[0070]

[0071] Example 1

[0072] This example prepared a kind of lipid nanoparticle based on junction containing branched structure of ionizable cationic lipid, specific steps are as follows:

[0073] (1) 1,1-cyclohexyl diethyl acid anhydride (1360 mg, 7.5 mmol) is slowly added to the pyridine solution (5 mL) of 2-dimethylaminoethanol (1 g, 11.2 mmol), stirred overnight, and the excess 2-dimethylaminoethanol and pyridine solution are removed by rotary evaporation to obtain product organic acid A. Under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), organic acid A (182 mg, 1 mmol) and 2-n-octyl-1-dodecanol (149 mg, 0.5 mmol) are dissolved in 5 mL of CDCl2, stirred at room temperature for 12 h, the solvent is removed by rotary evaporation, the initial product is separated, and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate = 2:1 (volume ratio)) to obtain product A1 after vacuum drying. The reaction process is as follows:

[0074]

[0075] (2) First, cationic lipid A1, DSPC, DMG-PEG2000, cholesterol were dissolved in 20 μL ethanol according to the molar ratio of 50:10:1.5:38.5, and the total mass was 120 micrograms; under the condition of vortex, the ethanol solution was quickly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg luciferase mRNA (purchased from Kaituo Biotechnology), and stirred vigorously for 20 s, then left for 10 min to prepare nanoparticles.

[0076] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) was dialyzed against 10 mM PBS solution (pH neutral) (dialysis bag Mw = 100 kDa) for 2-4 hours to remove ethanol, and the final product was obtained.

[0077] Example 2

[0078] In this example, a lipid nanoparticle based on an ionizable cationic lipid with a branched structure at the junction was prepared, and the specific steps were as follows:

[0079] (1) 1,1-cyclohexyl diethyl acid anhydride (1360 mg, 7.5 mmol) was slowly added to a pyridine solution (5 mL) of 2-dimethylaminoethanol (1 g, 11.2 mmol), stirred overnight, and the excess 2-dimethylaminoethanol and pyridine solution was removed by rotary evaporation to obtain the product organic acid A. Under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), organic acid A (182 mg, 1 mmol) and 2-decyl-1-tetradecanol (177 mg, 0.5 mmol) were dissolved in 5 mL of CDC12, stirred at room temperature for 12 h, the solvent was removed by rotary evaporation, the initial product was separated, and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate = 2:1 (volume ratio)), and the product A2 was obtained after vacuum drying. The reaction process is as follows:

[0080]

[0081] (2) First, cationic lipid A2, DSPC, DMG-PEG2000, cholesterol were dissolved in 20 μL ethanol according to the molar ratio of 50:10:1.5:38.5, and the total mass was 120 micrograms; under the condition of vortex, the ethanol solution was quickly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg luciferase mRNA (purchased from Kaituo Biotechnology), and stirred vigorously for 20 s, then left for 10 min to prepare nanoparticles.

[0082] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) is dialyzed against a 10 mM PBS solution (pH neutral) (dialysis bag Mw = 100 kDa) for 2-4 hours to remove ethanol, to obtain the final product.

[0083] Example 3

[0084] In this example, a lipid nanoparticle based on ionizable cationic lipid with branch structure at the junction is prepared, and the specific steps are as follows:

[0085] (1) 1,1-cyclohexyl diethyl acid anhydride (1360 mg, 7.5 mmol) is slowly added to a pyridine solution (5 mL) of 2-dimethylaminoethanol (1 g, 11.2 mmol), stirred overnight, and the excess 2-dimethylaminoethanol and pyridine solution is removed by rotary evaporation to obtain product organic acid A. Under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), organic acid A (182 mg, 1 mmol) and 2-dodecylhexadecan-1-ol (205 mg, 0.5 mmol) are dissolved in 5 mL of CDCl2, stirred at room temperature for 12 h, the solvent is removed by rotary evaporation, the initial product is separated, and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate = 2:1 (by volume)), and vacuum dried to obtain product A3. The reaction process is as follows:

[0086]

[0087] (2) First, the cationic lipid A3, DSPC, DMG-PEG2000, and cholesterol are dissolved in 20 μL of ethanol according to the molar ratio of 50:10:1.5:38.5, with a total mass of 120 micrograms; under the condition of vortex, the ethanol solution is quickly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kailuo Biotechnology), stirred vigorously for 20 s, and then left to stand for 10 minutes to prepare the nanoparticles.

[0088] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) is dialyzed against a 10 mM PBS solution (pH neutral) (dialysis bag Mw = 100 kDa) for 2-4 hours to remove ethanol, to obtain the final product.

[0089] Example 4

[0090] In this example, a lipid nanoparticle based on ionizable cationic lipid with branch structure at the junction is prepared, and the specific steps are as follows:

[0091] (1) Glutaric anhydride (851 mg, 7.5 mmol) was slowly added to a solution of 2-dimethylaminoethanol (1 g, 11.2 mmol) in pyridine (5 mL) and stirred overnight. The excess 2-dimethylaminoethanol and pyridine solution were removed by rotary evaporation to obtain the product organic acid B. Organic acid B (114 mg, 1 mmol) and 2-n-octyl-1-dodecanol (149 mg, 0.5 mmol) were dissolved in 5 mL of CDC12 under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), stirred at room temperature for 12 h, the solvent was removed by rotary evaporation, the initial product was separated and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate = 2: 1 (volume ratio)), and the product B1 was obtained after vacuum drying. The reaction process is as follows:

[0092]

[0093] (2) First, the cationic lipid B1, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol according to the molar ratio of 50:10:1.5:38.5, with a total mass of 120 micrograms; the ethanol solution was quickly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kailituo Biotechnology) under the condition of vortex, and then stirred vigorously for 20 s, and then left to stand for 10 minutes to prepare the nanoparticles.

[0094] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) was dialyzed against 10 mM PBS solution (pH neutral) (dialysis bag Mw = 100 kDa) for 2-4 hours to remove ethanol, and the final product was obtained.

[0095] Example 5

[0096] In this example, a lipid nanoparticle based on an ionizable cationic lipid with no branched structure at the junction was prepared, and the specific steps were as follows:

[0097] (1) Glutaric anhydride (851 mg, 7.5 mmol) was slowly added to a solution of 2-dimethylaminoethanol (1 g, 11.2 mmol) in pyridine (5 mL) and stirred overnight. The excess 2-dimethylaminoethanol and pyridine solution were removed by rotary evaporation to obtain the product organic acid B. Organic acid B (114 mg, 1 mmol) and 2-decyl-1-tetradecanol (177 mg, 0.5 mmol) were dissolved in 5 mL of CDC12 under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), stirred at room temperature for 12 h, the solvent was removed by rotary evaporation, the initial product was separated and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate = 2: 1 (volume ratio)), and the product B2 was obtained after vacuum drying. The reaction process is as follows:

[0098]

[0099] (2) First, the cationic lipid B2, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol according to the molar ratio of 50:10:1.5:38.5, with a total mass of 120 micrograms; the ethanol solution was quickly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kailituo Biotechnology) under the condition of vortex, and then stirred vigorously for 20 s, and then left to stand for 10 minutes to prepare the nanoparticles.

[0100] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) was dialyzed against 10 mM PBS solution (pH neutral) (dialysis bag Mw = 100 kDa) for 2-4 hours to remove ethanol, and the final product was obtained.

[0101] Example 6

[0102] In this example, a lipid nanoparticle based on an ionizable cationic lipid with no branched structure at the junction was prepared, and the specific steps were as follows:

[0103] (1) Glutaric anhydride (851 mg, 7.5 mmol) was slowly added to a solution of 2-dimethylaminoethanol (1 g, 11.2 mmol) in pyridine (5 mL) and stirred overnight. The excess 2-dimethylaminoethanol and pyridine solution were removed by rotary evaporation to obtain the product organic acid B. Organic acid B (114 mg, 1 mmol) and 2-dodecylhexadecan-1-ol (205 mg, 0.5 mmol) were dissolved in 5 mL of CDC12 under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), stirred at room temperature for 12 h, the solvent was removed by rotary evaporation, the initial product was separated and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate = 2: 1 (volume ratio)), and the product B3 was obtained after vacuum drying. The reaction process is as follows:

[0104]

[0105] (2) First, the cationic lipid B3, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol according to the molar ratio of 50:10:1.5:38.5, with a total mass of 120 micrograms; the ethanol solution was quickly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kailituo Biotechnology) under the condition of vortex, and then stirred vigorously for 20 s, and then left to stand for 10 minutes to prepare the nanoparticles.

[0106] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) was dialyzed against 10 mM PBS solution (pH neutral) (dialysis bag Mw = 100 kDa) for 2-4 hours to remove ethanol, and the final product was obtained.

[0107] Example 7

[0108] In this example, a lipid nanoparticle based on an ionizable cationic lipid with a branched structure at the junction was prepared, and the specific steps were as follows:

[0109] (1) 3-Isobutyl glutaric anhydride (1271 mg, 7.5 mmol) was slowly added to a solution of 2-dimethylaminoethanol (1 g, 11.2 mmol) in pyridine (5 mL) and stirred overnight. The excess 2-dimethylaminoethanol and pyridine solution was removed by rotary evaporation to obtain product organic acid C. Under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), organic acid C (170 mg, 1 mmol) and 2-n-octyl-1-dodecanol (149 mg, 0.5 mmol) were dissolved in 5 mL of CDCl2, stirred at room temperature for 12 h, the solvent was removed by rotary evaporation, the initial product was separated and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate = 2:1 (by volume)), and the product C1 was obtained after vacuum drying. The reaction process is as follows:

[0110]

[0111] As shown in Figure 1 , the C1 cationic lipid nuclear magnetic resonance (NMR) spectrum shows: 1H NMR (600 MHz, Chloroform-d): δ 4.18 (td, J = 5.8, 2.8 Hz, 2H), 3.96 (d, J = 5.7 Hz, 2H), 2.58 (t, J = 5.9 Hz, 2H), 2.37 (dt, J = 10.4, 4.3 Hz, 4H), 2.29 (s, 6H), 1.61 (dt, J = 13.3, 6.7 Hz, 2H), 1.27 (d, J = 9.4 Hz, 34H), 0.88 (dd, J = 8.6, 6.4 Hz, 12H). Consistent with the peaks of the target product, it can be seen that the synthesis of the cationic lipid is successful.

[0112] (2) First, the cationic lipid C1, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol according to the molar ratio of 50:10:1.5:38.5, with a total mass of 120 micrograms; under the condition of vortex, the ethanol solution was quickly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kailuo Biotechnology); after stirring vigorously for 20 s, it was left to stand for 10 minutes to prepare the nanoparticles.

[0113] (3) The ethanol-sodium acetate mixed solution containing the nanoparticles prepared in (2) was dialyzed against 10 mM PBS solution (pH neutral) (dialysis bag Mw = 100 kDa) for 2-4 hours to remove ethanol, and the final product was obtained.

[0114] As shown in Figure 2 , transmission electron microscopy (TEM) observation showed that the particle size of the nanomaterial LNPs prepared in this embodiment was about 120 nm.

[0115] Example 8

[0116] In this example, a kind of lipid nanoparticles based on the ionizable cationic lipid containing branched structure at the junction was prepared, and the specific steps were as follows:

[0117] (1) 3-isobutyl glutaric anhydride (1271 mg, 7.5 mmol) was slowly added to a solution of 2-dimethylaminoethanol (1 g, 11.2 mmol) in pyridine (5 mL), stirred overnight, and the excess 2-dimethylaminoethanol and pyridine solution was removed by rotary evaporation to obtain the product organic acid C. Under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), organic acid C (170 mg, 1 mmol) and 2-decyl-1-tetradecanol (177 mg, 0.5 mmol) were dissolved in 5 mL of CDCl2, stirred at room temperature for 12 h, the solvent was removed by rotary evaporation, the initial product was separated, and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate = 2:1 (volume ratio)), and vacuum dried to obtain product C2. The reaction process was as follows:

[0118]

[0119] (2) First, the cationic lipid C2, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol according to the molar ratio of 50:10:1.5:38.5, with a total mass of 120 micrograms; the ethanol solution was quickly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kailuo Biotechnology) under vortex conditions, and stirred vigorously for 20 s, then left to stand for 10 minutes to prepare the nanoparticles.

[0120] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) was dialyzed against 10 mM PBS solution (pH neutral) (dialysis bag Mw = 100 kDa) for 2-4 hours to remove ethanol, and the final product was obtained.

[0121] Example 9

[0122] In this example, a kind of lipid nanoparticles based on the ionizable cationic lipid containing branched structure at the junction was prepared, and the specific steps were as follows:

[0123] (1) 3-isobutyl glutaric anhydride (1271 mg, 7.5 mmol) was slowly added to a solution of 2-dimethylaminoethanol (1 g, 11.2 mmol) in pyridine (5 mL) and stirred overnight. The excess 2-dimethylaminoethanol and pyridine solution were removed by rotary evaporation to obtain product organic acid C. Organic acid C (170 mg, 1 mmol) and 2-dodecylhexadecan-1-ol (205 mg, 0.5 mmol) were dissolved in 5 mL of CDC12 under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), stirred at room temperature for 12 h, the solvent was removed by rotary evaporation, the initial product was separated and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate = 2:1 (volume ratio)), and the product C3 was obtained after vacuum drying. The reaction process is as follows:

[0124]

[0125] (2) First, the cationic lipid C3, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol according to the molar ratio of 50:10:1.5:38.5, with a total mass of 120 micrograms; the ethanol solution was quickly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kailituo Biotechnology) under the condition of vortex, and then stirred vigorously for 20 s, and then left to stand for 10 minutes to prepare the nanoparticles.

[0126] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) was dialyzed against 10 mM PBS solution (pH neutral) (dialysis bag Mw = 100 kDa) for 2-4 hours to remove ethanol, and the final product was obtained.

[0127] Example 10

[0128] In this example, a lipid nanoparticle based on an ionizable cationic lipid with a branched structure at the junction was prepared, and the specific steps were as follows:

[0129] (1) 3,3-dimethylglutaric anhydride (1061 mg, 7.5 mmol) was slowly added to a solution of 2-dimethylaminoethanol (1 g, 11.2 mmol) in pyridine (5 mL) and stirred overnight. The excess 2-dimethylaminoethanol and pyridine solution was removed by rotary evaporation to obtain product organic acid D. Organic acid D (142 mg, 1 mmol) and 2-n-octyl-1-dodecanol (149 mg, 0.5 mmol) were dissolved in 5 mL of CDC12 under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), stirred at room temperature for 12 h, the solvent was removed by rotary evaporation, the initial product was separated and purified by silica gel chromatography (eluent: n-hexane: ethyl acetate = 2:1 (volume ratio)), and the product D1 was obtained after vacuum drying. The reaction process is as follows:

[0130]

[0131] (2) First, cationic lipid D1, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol according to the molar ratio of 50:10:1.5:38.5, with a total mass of 120 micrograms; the ethanol solution was quickly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kailituo Biotechnology) under the condition of vortex, and then stirred vigorously for 20 s, and then left to stand for 10 minutes to prepare the nanoparticles.

[0132] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) was dialyzed against 10 mM PBS solution (dialysis bag Mw = 100 kDa) for 2-4 hours to remove ethanol, and the final product was obtained.

[0133] Example 11

[0134] In this example, a lipid nanoparticle based on an ionizable cationic lipid with a branched structure at the junction was prepared, and the specific steps were as follows:

[0135] (1) 3,3-dimethylglutaric anhydride (1061 mg, 7.5 mmol) was slowly added to a solution of 2-dimethylaminoethanol (1 g, 11.2 mmol) in pyridine (5 mL) and stirred overnight. The excess 2-dimethylaminoethanol and pyridine solution was removed by rotary evaporation to obtain product organic acid D. Organic acid D (142 mg, 1 mmol) and 2-decyl-1-tetradecanol (177 mg, 0.5 mmol) were dissolved in 5 mL of CDC12 under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), stirred at room temperature for 12 h, the solvent was removed by rotary evaporation, the initial product was separated and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate = 2: 1 (volume ratio)), and the product D2 was obtained after vacuum drying. The reaction process is as follows:

[0136]

[0137] (2) First, the cationic lipid D2, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol according to the molar ratio of 50:10:1.5:38.5, with a total mass of 120 micrograms; the ethanol solution was quickly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kailituo Biotechnology) under the condition of vortex, and then stirred vigorously for 20 s, and then left to stand for 10 minutes to prepare the nanoparticles.

[0138] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) was dialyzed against 10 mM PBS solution (pH neutral) (dialysis bag Mw = 100 kDa) for 2-4 hours to remove ethanol, and the final product was obtained.

[0139] Example 12

[0140] In this example, a lipid nanoparticle based on an ionizable cationic lipid with a branched structure at the junction was prepared, and the specific steps were as follows:

[0141] (1) 3,3-dimethylglutaric anhydride (1061 mg, 7.5 mmol) was slowly added to a solution of 2-dimethylaminoethanol (1 g, 11.2 mmol) in pyridine (5 mL) and stirred overnight. The excess 2-dimethylaminoethanol and pyridine solution was removed by rotary evaporation to obtain product organic acid D. Organic acid D (142 mg, 1 mmol) and 2-dodecylhexadecan-1-ol (205 mg, 0.5 mmol) were dissolved in 5 mL of CDC12 under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), stirred at room temperature for 12 h, the solvent was removed by rotary evaporation, the initial product was separated and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate = 2:1 (volume ratio)), and the product D3 was obtained after vacuum drying. The reaction process is as follows:

[0142]

[0143] (2) First, the cationic lipid D3, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol according to the molar ratio of 50:10:1.5:38.5, with a total mass of 120 micrograms; the ethanol solution was quickly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kailituo Biotechnology) under the condition of vortex, and then stirred vigorously for 20 s, and then left to stand for 10 minutes to prepare the nanoparticles.

[0144] (3) The ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) was dialyzed against 10 mM PBS solution (pH neutral) (dialysis bag Mw = 100 kDa) for 2-4 hours to remove ethanol, and the final product was obtained.

[0145] Example 13

[0146] In this example, a lipid nanoparticle based on an ionizable cationic lipid with a branched structure at the junction was prepared, and the specific steps were as follows:

[0147] (1) 3-methyl glutaric anhydride (957 mg, 7.5 mmol) was slowly added to a solution of 2-dimethylaminoethanol (1 g, 11.2 mmol) in pyridine (5 mL) and stirred overnight. The excess 2-dimethylaminoethanol and pyridine solution was removed by rotary evaporation to obtain the product organic acid E. Organic acid E (128 mg, 1 mmol) and 2-n-octyl-1-dodecanol (149 mg, 0.5 mmol) were dissolved in 5 mL of CDC12 under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), stirred at room temperature for 12 h, the solvent was removed by rotary evaporation, the initial product was separated and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate = 2: 1 (volume ratio)), and the product E1 was obtained after vacuum drying. The reaction process is as follows:

[0148]

[0149] (2) First, the cationic lipid E1, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol according to the molar ratio of 50:10:1.5:38.5, with a total mass of 120 micrograms; the ethanol solution was quickly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kailituo Biotechnology) under the condition of vortex, and then stirred vigorously for 20 s, and then left to stand for 10 minutes to prepare the nanoparticles.

[0150] (3) The ethanol-sodium acetate mixed solution containing the nanoparticles prepared in (2) was dialyzed against 10 mM PBS solution (pH neutral) (dialysis bag Mw = 100 kDa) for 2-4 hours to remove ethanol, and the final product was obtained.

[0151] Example 14

[0152] This example prepared a lipid nanoparticle based on an ionizable cationic lipid with a branched structure at the junction, and the specific steps are as follows:

[0153] (1) 3-methyl glutaric anhydride (957 mg, 7.5 mmol) was slowly added to a solution of 2-dimethylaminoethanol (1 g, 11.2 mmol) in pyridine (5 mL) and stirred overnight. The excess 2-dimethylaminoethanol and pyridine solution were removed by rotary evaporation to obtain the product organic acid E. Organic acid E (128 mg, 1 mmol) and 2-decyl-1-tetradecanol (177 mg, 0.5 mmol) were dissolved in 5 mL of CDC12 under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), stirred at room temperature for 12 h, the solvent was removed by rotary evaporation, the initial product was separated and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate = 2:1 (volume ratio)), and the product E2 was obtained after vacuum drying. The reaction process is as follows:

[0154]

[0155] (2) First, the cationic lipid E2, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol according to the molar ratio of 50:10:1.5:38.5, with a total mass of 120 micrograms; the ethanol solution was quickly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kailituo Biotechnology) under the condition of vortex, and then stirred vigorously for 20 s, and then left to stand for 10 minutes to prepare the nanoparticles.

[0156] (3) The ethanol-sodium acetate mixed solution containing the nanoparticles prepared in (2) was dialyzed against 10 mM PBS solution (pH neutral) (dialysis bag Mw = 100 kDa) for 2-4 hours to remove ethanol, and the final product was obtained.

[0157] Example 15

[0158] In this example, a lipid nanoparticle based on an ionizable cationic lipid with a branched structure at the junction was prepared, and the specific steps were as follows:

[0159] (1) 3-methyl glutaric anhydride (957 mg, 7.5 mmol) was slowly added to a solution of 2-dimethylaminoethanol (1 g, 11.2 mmol) in pyridine (5 mL) and stirred overnight. The excess 2-dimethylaminoethanol and pyridine solution was removed by rotary evaporation to obtain the product organic acid E. Organic acid E (128 mg, 1 mmol) and 2-dodecylhexadecan-1-ol (205 mg, 0.5 mmol) were dissolved in 5 mL of CDC12 under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), stirred at room temperature for 12 h, the solvent was removed by rotary evaporation, the initial product was separated and purified by silica gel chromatography (product eluent: n-hexane: ethyl acetate = 2:1 (volume ratio)), and the product E3 was obtained after vacuum drying. The reaction process is as follows:

[0160]

[0161] (2) First, the cationic lipid E3, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol according to the molar ratio of 50:10:1.5:38.5, with a total mass of 120 micrograms; the ethanol solution was quickly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kailituo Biotechnology) under the condition of vortex, and then stirred vigorously for 20 s, and then left to stand for 10 minutes to prepare the nanoparticles.

[0162] (3) The ethanol-sodium acetate mixed solution containing the nanoparticles prepared in (2) was dialyzed against 10 mM PBS solution (dialysis bag Mw = 100 kDa) for 2-4 hours to remove ethanol, and the final product was obtained.

[0163] Test Example 1: Particle size and surface potential analysis of lipid nanoparticles

[0164] The lipid nanoparticles prepared in Examples 1-15 were tested by a Malvern particle size analyzer, and the results are as follows:

[0165] The average particle size of the nanomaterial LNP prepared in Example 1 was 112.1 nm, the distribution coefficient PDI = 0.148; the zeta potential was 0.501 mv, and the nanoparticles were electrically neutral.

[0166] The average particle size of the nanomaterial LNP prepared in Example 2 was 115.1 nm, the distribution coefficient PDI = 0.116; the zeta potential was 0.350 mv, and the nanoparticles were electrically neutral.

[0167] The average particle size of the nanomaterial LNP prepared in Example 3 was 116.4 nm, the distribution coefficient PDI = 0.300; the zeta potential was -5.43 mv, and it can be seen that the nanoparticles were weakly negatively charged.

[0168] The average particle size of the nanomaterial LNP prepared in Example 4 was 164.0 nm, the distribution coefficient PDI = 0.121; the zeta potential was 1.323 mv, and it can be seen that the nanoparticles were electrically neutral.

[0169] The average particle size of the nanomaterial LNP prepared in Example 5 was 123.6 nm, the distribution coefficient PDI = 0.173; the zeta potential was 1.117 mv, and it can be seen that the nanoparticles were electrically neutral.

[0170] The average particle size of the nanomaterial LNP prepared in Example 6 was 117.7 nm, the distribution coefficient PDI = 0.198; the zeta potential was -3.570 mv, and it can be seen that the nanoparticles were electrically neutral.

[0171] The average particle size of the nanomaterial LNP prepared in Example 7 was 121.6 nm, the distribution coefficient PDI = 0.156; the zeta potential was -3.137 mv, and it can be seen that the nanoparticles were electrically neutral.

[0172] The average particle size of the nanomaterial LNP prepared in Example 8 was 96.8 nm, the distribution coefficient PDI = 0.161; the zeta potential was -7.767 mv, and it can be seen that the nanoparticles were weakly negatively charged.

[0173] The average particle size of the nanomaterial LNP prepared in Example 9 was 254.9 nm, the distribution coefficient PDI = 0.725; the zeta potential was -5.613 mv, and it can be seen that the nanoparticles were weakly negatively charged.

[0174] The average particle size of the nanomaterial LNP prepared in Example 10 was 154.3 nm, the distribution coefficient PDI = 0.181; the zeta potential was -6.447 mv, and it can be seen that the nanoparticles were weakly negatively charged.

[0175] The average particle size of the nanomaterial LNP prepared in Example 11 was 113.3 nm, the distribution coefficient PDI = 0.109; the zeta potential was -2.156 mv, and it can be seen that the nanoparticles were electrically neutral.

[0176] The average particle size of the nanomaterial LNP prepared in Example 12 was 131.8 nm, the distribution coefficient PDI = 0.138; the zeta potential was -4.480 mv, and it can be seen that the nanoparticles were electrically neutral.

[0177] The average particle size of the nanomaterial LNP prepared in Example 13 was 175.1 nm, the distribution coefficient PDI = 0.160; the zeta potential was -4.313 mv, and it can be seen that the nanoparticles were electrically neutral.

[0178] The average particle size of the nanomaterial LNP prepared in Example 14 is 143.3 nm, the distribution coefficient PDI = 0.222; the zeta potential is -1.090 mv, and it can be seen that the nanoparticles are electrically neutral.

[0179] The average particle size of the nanomaterial LNP prepared in Example 15 is 125.8 nm, the distribution coefficient PDI = 0.212; the zeta potential is -4.007 mv, and it can be seen that the nanoparticles are electrically neutral.

[0180] Test Example 2: In vivo transfection experiment of lipid nanoparticles

[0181] 1. The PBS solution of the dialyzed LNP was injected into the mouse by subcutaneous injection, and 6h later, the luciferase substrate D-luciferin potassium salt (10 mg / mL, 200 μL) was injected into the mouse by intraperitoneal injection, and the fluorescence signal was observed by a small animal live imaging instrument.

[0182] As shown in Figure 3 , we observed that at 6h, there was a significant fluorescence signal in the mouse, indicating that the lipid nanoparticles were successfully taken up by the cells at the injection site and surrounding cells, and the vector successfully delivered the luciferase mRNA to the cytoplasm of the mouse cells and successfully translated into a large amount of protein.

[0183] As can be seen, the above lipid nanoparticles can effectively deliver mRNA in animals, and have potential clinical application value.

[0184] The transfection effect of the nanomaterial LNP prepared in each example in the mouse was quantitatively analyzed by a small animal live imaging instrument. As shown in Figure 4 , it can be seen that the branched structure at the connection site has a huge impact on the performance of the liposome, and the liposomes in Examples 1 and 7 have the best transfection effect and will be used in subsequent experiments.

[0185] 2. Optimization of lipid nanoparticle formulation and in vivo transfection experiment

[0186] In order to improve the delivery efficiency of the lipid nanoparticles, we designed 5 different formulations, as shown in Table 1, to explore the best molar ratio to maximize the mRNA delivery efficiency of the lipid nanoparticles.

[0187] Table 1

[0188]

[0189] The ionizable cationic lipids A1 / C1, DSPC, cholesterol, DMG-PEG2000 containing branched structure were dissolved in 20 μL ethanol according to the molar ratio shown in Table 1, and the total mass was 40 μg. Under the condition of vortex, the ethanol solution was quickly injected into 60 μL of 20 mM sodium acetate buffer containing 1 μg of luciferase mRNA, and then stirred vigorously for 20 s, and then left for 10 min to prepare the nanoparticles.

[0190] The PBS solution of LNP after dialysis was injected into the mouse body by subcutaneous injection, and 6 h later, luciferase substrate D-luciferin potassium salt (10 mg / mL, 200 μL) was injected into the mouse body by intraperitoneal injection, and the fluorescence signal was observed by a small animal live imaging instrument.

[0191] As shown in Figure 5 , we observed that there was obvious fluorescence signal in the mouse body at 6 h, and there was obvious difference between different groups (wherein sLNP is a lipid nanoparticle prepared based on a commercial formula (same as formula 1 in Table 1, cationic lipid: DSPC: cholesterol: DMG-PEG2000 = 50:10:38.5:1.5) and commercial cationic lipid SM-102). It is shown that by adjusting the ratio of the four components, the delivery efficiency of the mRNA by the lipid nanoparticle can be obviously affected and improved.

[0192] By a small animal live imaging instrument, we quantitatively analyzed the in vivo transfection effect of the formula in mice, as shown in Figure 6 , it can be seen that the adjustment of the ratio and the optimization of the formula can significantly improve the delivery efficiency of the lipid nanoparticle. The lipid nanoparticle based on C1 cationic lipid (cLNP) under formula 5 shows the best curative effect, better than the commercial lipid nanoparticle.

[0193] Subsequent experiments will use cationic lipid C1 and formula 5, and introduce lipid nanoparticles based on cationic lipid B1 without branched structure (bLNP) under formula 5 and lipid nanoparticles based on cationic lipid SM-102 under commercial formula (sLNP) as controls.

[0194] Test Example 3: Lipid Nanoparticle Cell Transfection Experiment

[0195] Mouse kidney cells (BHK cells) were plated in a white and transparent 48-well plate. Before LNP transfection of the cells, the cells were adhered and grown to 1 × 10 5 / well. Before transfection, LNPs containing 2 μg EGFP mRNA were added to 300 μL Opti-MEM and incubated for 10 min; the cells were then washed once with 1 mL Opti-MEM. The transfection mixture was then dropped onto the medium. Transfection was observed 16-24 hours later (fluorescence microscopy).

[0196] like Figure 7 As shown, under the green fluorescence channel, the cLNP group and sLNP group exhibited a stronger green fluorescence signal than the bLNP group within 24 hours, indicating that the cLNP group expressed more green fluorescent protein. This suggests that, compared to lipid nanoparticles with cationic lipids without branched junctions, lipid nanoparticles with branched junctions can more effectively promote mRNA transfection into the cytoplasm for protein translation.

[0197] Application Example 1: Application of lipid nanoparticles in tumor vaccines

[0198] On day 1, mice were injected with 800,000 B16-OVA tumor cells via subcutaneous injection. On days 5, 8, and 11, mice were injected with lipid nanoparticles cLNP, bLNP, and commercially available sLNP containing 5 μg of OVA mRNA prepared according to the above formula No. 5 via subcutaneous injection.

[0199] like Figure 8 , 9 As shown, cLNP and sLNP exhibited equivalent tumor-suppressing effects, with cLNP being significantly superior to bLNP. This demonstrates that lipid nanoparticles based on ionizable cationic lipids with branched junctions exhibit better mRNA delivery efficiency and therapeutic efficacy than ionizable cationic lipid nanoparticles without branched junctions.

[0200] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A lipid nanoparticle characterized in that, The raw material composition of the lipid nanoparticle comprises: a cationic lipid with a branched structure at the connection, an auxiliary lipid, and cholesterol, wherein the structure of the cationic lipid is shown as formula (I), (Ⅰ)， R1 is selected from -C(CH2)5, -CHCH2CH(CH3)2, -C(CH3)2, and -CHCH3. R2is selected from the group consisting of CH3(CH2)9CH[(CH2)7CH3]CH2O-, CH3(CH2) 11 CH[(CH2) 13 CH[(CH2) 11 CH3]CH2O-, CH3(CH2)7CH[(CH2)5CH3]CH2O-.

2. The lipid nanoparticle of claim 1, wherein, The preparation method of the cationic lipid with a branched structure at the connection comprises: first, dissolving 2-dimethylaminoethanol and a glutaric anhydride derivative in pyridine to generate an organic acid with a branched structure through alcoholysis, then dissolving the organic acid and an alcohol containing a fatty chain in an organic solvent, and generating a condensation reaction under the catalysis of a catalyst, and separating and purifying the product to obtain the cationic lipid with a branched structure at the connection.

3. The lipid nanoparticle of claim 2, wherein, The glutaric anhydride derivative is any one of 1,1-cyclohexyl dihydric acid anhydride, 3-methyl glutaric anhydride, 3-isobutyl glutaric anhydride, and 3,3-dimethyl glutaric anhydride.

4. The lipid nanoparticle of claim 2, wherein, The alcohol containing a fatty chain is any one of 2-n-octyl-1-dodecanol, 2-decyl-1-tetradecanol, 2-dodecylhexadecan-1-ol, 2-hexyl-1-decanol, and 7-tetradecanol.

5. The lipid nanoparticle of claim 1, wherein, The auxiliary lipid comprises a phospholipid and a polyethylene glycol functionalized lipid; the phospholipid is distearoyl phosphatidylcholine or dioleoyl phosphatidylethanolamine, and the polyethylene glycol functionalized lipid is dimyristyl glycerol-polyethylene glycol 2000.

6. The lipid nanoparticle of claim 5, wherein, The molar ratio of the cationic lipid, the phospholipid, the cholesterol, and the polyethylene glycol functionalized lipid is 25-75:5-15:15-60:1-2.

7. The lipid nanoparticle as claimed in any one of claims 1-6 as a carrier for preparing a nucleic acid drug delivery.

8. Use according to claim 7, wherein the compound is ###0002### The application comprises: adding the cationic lipid with a branched structure at the connection, the auxiliary lipid, and the cholesterol into an acid buffer containing the nucleic acid, and self-assembling to form a nucleic acid-loaded lipid nanoparticle, thereby preparing the nucleic acid drug delivery.

9. Use according to claim 8, wherein the compound is ###0002### The nucleic acid drug delivery is an mRNA vaccine, and the total mass of the lipid material to the mass of the mRNA is 20-160:1.

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