Lipid nanoparticle based on cationic lipid with branch structure at joint and application of lipid nanoparticle

Abstract

The invention discloses a lipid nanoparticle based on cationic lipid with a branch structure at a joint and application of the lipid nanoparticle, and belongs to the technical field of medicines. The lipid nanoparticles comprise the following raw materials: cationic lipid containing a branch structure at a joint, auxiliary lipid and cholesterol, wherein the structural formula of the cationic lipid is shown as a formula (I). According to the lipid nanoparticle based on the ionizable cationic lipid containing the branch structure at the joint, nucleic acid drugs such as mRNA and the like can be effectively delivered in animal bodies. Compared with commercially available ionizable lipid SM-102 and a formula thereof, the lipid nanoparticle provided by the invention has better mRNA delivery efficiency in animal bodies, and is beneficial to improving the clinical application effect of nucleic acid drugs such as mRNA and the like.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly to a lipid nanoparticle based on a cationic lipid with a branched structure at the junction and its application in preparing mRNA vaccines and gene therapy products as a delivery carrier material. Background Art

[0002] mRNA drugs are a technology that emerged in recent years, combining 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 that need to be solved in mRNA vaccine design. Among them, the lack of a safe and efficient delivery system is one of the main reasons restricting its application. How to efficiently deliver mRNA into cells and the cytoplasm is a key scientific problem that needs to be solved in mRNA vaccine research.

[0003] In view of the characteristics of mRNA being unstable, negatively charged, and difficult to be taken up by cells, scientists have developed a series of delivery systems, including lipid-based delivery systems, peptide-based delivery systems, polymer-based delivery systems, and so on. Among them, lipid-based lipid nanoparticles (LNPs) are one of the most promising carrier materials due to their good biosafety and high delivery ability. Currently, several drugs based on lipid nanoparticles (LNPs) have been approved by the FDA.

[0004] Currently, the structure-activity relationship among the components of lipid nanoparticles (LNPs) is not yet fully clear. Lipid nanoparticles (LNPs) usually consist of cationic lipids, phospholipids, PEG-modified lipids, and cholesterol. Among them, cationic lipids are the core and soul, and their properties in each part can affect the overall formulation and biological characteristics of LNPs.

[0005] Cationic lipids mainly consist of a positively charged head group, a hydrophobic tail, and a hydrophobic chain connecting the two. Among them, the positively charged head group mainly participates in processes such as encapsulating negatively charged biological macromolecules such as DNA and RNA, interacting with cell membranes, and promoting endosomal escape. Ionizable lipids DLin-MC3-DMA, SM-102, and ALC-0315 used clinically contain tertiary amine heads and can undergo pH-dependent ionization. The hydrophobic tail is generally a long-chain hydrocarbon group or a saturated / unsaturated fatty acid chain, which provides hydrophobicity for lipid molecules and helps the stability of liposomes 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 polymeric lipids, biodegradable chemical bonds are introduced to achieve the degradation function. Esters, amides, and thiols are preferred and can usually be rapidly cleared in the body, allowing for multiple doses and reducing side effects.

[0006] Since cationic lipids play a decisive role in the delivery of mRNA, screening for efficient and safe cationic lipids has important clinical significance. Currently, a large number of studies focus on the optimization of the head structure, tail chain structure, and the development of degradable linker segments. However, there have been no reports on the effect of linker segment structure on the performance of cationic lipids. Summary of the Invention

[0007] The object of the present invention is to provide a novel cationic lipid material for preparing lipid nanoparticles to achieve effective delivery of mRNA as a drug delivery carrier.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention provides a lipid nanoparticle, and the raw material composition of the lipid nanoparticle includes: a cationic lipid with a branched structure at the connection, a co-lipid, and cholesterol. The structural formula of the cationic lipid is shown in formula (Ⅰ),

[0010]

[0011] where R 1 is selected from: -C(CH 2 ) 5 , -CHCH 2 CH(CH 3 ) 2 , -C(CH 3 ) 2 , -CHCH 3 ;

[0012] R 2 is selected from: CH 3 (CH 2 ) 9 CH[(CH 2 ) 7 CH 3 CH 2 O-, CH 3 (CH 2 ) 11 CH[(CH 2 ) 9 CH 3 CH 2 O-, CH 3 (CH 2 ) 13 CH[(CH 2 ) 11 CH 3 CH 2 O-, CH 3 (CH 2 ) 7CH[(CH 2 ) 5 CH 3 CH 2 O-, [CH 3 (CH2) 11 2 N-, [CH 3 (CH2) 13 2 N-.

[0013] The cationic lipid structure is composed of a tertiary amine group head, a fatty acid chain hydrophobic tail, and a biodegradable linker fragment, where the linker fragment is modified with a branched structure. The tertiary amine group of the cationic lipid forms a positively charged hydrophilic end in the buffer solution and can bind to negatively charged drugs through electrostatic interaction. Due to the hydrophilic-hydrophobic supramolecular force, the lipid material undergoes self-assembly to obtain the lipid nanoparticles.

[0014] The research of the present invention shows that lipid nanoparticles formed by self-assembly of ionizable cationic lipids containing a branched structure at the junction and other lipid materials have high cell transfection efficiency as drug delivery carriers and can effectively deliver drugs such as mRNA in vitro and in vivo, enabling them to be translated into proteins.

[0015] The preparation method of the cationic lipid containing a branched structure at the junction includes: first, dissolving 2-dimethylaminoethanol and a glutaric anhydride derivative in pyridine, undergoing alcoholysis reaction to generate an organic acid with a branched structure, and then dissolving the organic acid and an alcohol or amine containing a fatty chain in an organic solvent, and undergoing a condensation reaction under the catalysis of a catalyst, and separating and purifying from the product to obtain the cationic lipid containing a branched structure at the junction.

[0016] On the molecular structure of 2-dimethylaminoethanol, one end is a tertiary amine and the other end is an alcohol hydroxyl group. The present invention utilizes its alcohol hydroxyl group to react with glutaric anhydride derivatives with different structures to generate organic acids with different branched structures, and then condenses with the hydroxyl group of an alcohol containing a fatty chain to form an ester bond, or condenses with the amino group of an amine containing a fatty chain to form an amide bond, to construct an ionizable cationic lipid containing a branched structure at the junction.

[0017] Preferably, the glutaric anhydride derivative can be, but is not limited to, 1,1-cyclohexyl diacetic anhydride, 3-methyl glutaric anhydride, 3-isobutyl glutaric anhydride, 3,3-dimethyl glutaric anhydride.

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

[0019] Preferably, stir at room temperature for 6 - 12 h to react to generate an organic acid with a branched structure. ​​

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

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

[0022] Preferably, the molar ratio of the 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 may be, but is not limited to, dichloromethane.

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

[0025] Preferably, the conditions for the condensation reaction are: stirring at room temperature for 6 - 12 h.

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

[0027] In the present invention, the lipid nanoparticles are formed by self-assembly after mixing the ionizable cationic lipid containing a branched structure at the above-mentioned junction with a co-lipid and cholesterol. The preparation method of the lipid nanoparticles may be, but is not limited to: ethanol injection method, thin film method, and sonication method. Specifically, when preparing lipid nanoparticles encapsulating a drug, the above method is used to enable the lipid material and the drug to be encapsulated to self-assemble into nanoparticles through supramolecular forces and electrostatic interactions in a buffer solution.

[0028] Among them, in the ethanol injection method, the ionizable cationic lipid, co-lipid, and cholesterol are dissolved in an appropriate amount of ethanol according to a certain ratio, and then the ethanol solution containing the lipid material is injected into the buffer solution containing the drug to be encapsulated to self-assemble into nanoparticles, and then ethanol is removed by dialysis to obtain stable nanoparticles.

[0029] Preferably, the co-lipid includes phospholipids and polyethylene glycol-functionalized lipids; the phospholipids may be, but are not limited to, distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylethanolamine (DOPE), and the polyethylene glycol-functionalized lipids may be, but are not limited to, dimyristoyl glycerol-polyethylene glycol 2000 (DMG-PEG2000).

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

[0031] The present invention 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 ionizable cationic lipids containing branched structures at the junction provided by the present invention can effectively deliver nucleic acid drugs such as mRNA, and this nanomaterial has potential application value in the development of nucleic acid drugs.

[0033] Specifically, the above use includes: adding a cationic lipid containing a branched structure at the junction, a co-lipid, and cholesterol to an acidic buffer solution containing nucleic acid, and self-assembling to form lipid nanoparticles encapsulating the nucleic acid, thereby obtaining the nucleic acid drug for delivery.

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

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

[0036] Preferably, the mass ratio of the total mass of the lipid material to the mass of mRNA is 20 - 160:1. Since mRNA molecules are relatively large, when the amount of lipid material is too small, it is difficult to effectively encapsulate and protect mRNA, thereby reducing the transfection efficiency. When the amount of lipid material is too large, it may reduce the endosomal escape efficiency, thereby reducing the transfection efficiency. Within a suitable mass ratio range, good encapsulation efficiency and transfection rate can be ensured. More preferably, the mass ratio of the total mass of the lipid material to the mass of mRNA is 40:1.

[0037] The beneficial effects of the present invention are as follows:

[0038] (1) Through two simple reactions, the present invention constructs an ionizable cationic lipid with a branched structure at the junction and applies it to the preparation of lipid nanoparticles. One end of the ionizable cationic lipid is a tertiary amine structure, which can bind negatively charged drugs through electrostatic interaction under appropriate conditions.

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

[0040] Figure 1 It is a chemical characterization diagram of a cationic lipid with a branched chain structure of isobutyl and an aliphatic chain of octyldodecanol.

[0041] Figure 2 It is a TEM diagram of the cLNP prepared in Example 7.

[0042] Figure 3 It is a physical picture of the transfection effect of the nano-drugs LNPs prepared in Examples 1-15 in mice.

[0043] Figure 4 For Figure 3 The quantitative analysis results of the transfection effect in

[0044] Figure 5 It is a physical picture of the transfection effect in mice of LNPs prepared from the lipid materials of Example 1 and Example 7 under different mixing ratios. Among them, sLNP is a lipid nanoparticle based on SM-102.

[0045] Figure 6 For Figure 5 The quantitative analysis results of the transfection effect in

[0046] Figure 7 It is the result of the cell transfection experiment of lipid nanoparticles. Among them, sLNP is a lipid nanoparticle based on SM-102, cLNP is a lipid nanoparticle of Formulation No. 5 with a branched chain structure of isobutyl and an aliphatic chain of octyldodecanol, and bLNP is a lipid nanoparticle of Formulation No. 5 without a branched chain structure and an aliphatic chain of octyldodecanol

[0047] Figure 8 It is a physical picture of the anti-cancer effect of preparing tumor drugs using lipid nanoparticles.

[0048] Figure 9 It is a tumor growth curve graph. DETAILED DESCRIPTION OF THE INVENTION

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

[0050] Unless otherwise specified, the test methods used in the following examples are all conventional methods; the materials, reagents, etc. used are, unless otherwise specified, reagents and materials that can be obtained from commercial sources.

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

[0052] EDC: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide, CAS No.: 1892-57-5;

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

[0054] CDCl 2 : 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-Cyclohexanediacetic anhydride, CAS No.: 1010-26-0;

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

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

[0060] 3,3-Dimethylglutaric 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-Dodecylhexadec-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, and its structural formula is as follows:

[0066]

[0067] DMG-PEG2000 lipid, CAS No.: 160743-62-4, and its structural formula is as follows:

[0068]

[0069] Cholesterol, CAS No.: 57 - 88 - 5, with the structural formula as follows:

[0070]

[0071] Example 1

[0072] In this example, a lipid nanoparticle based on an ionizable cationic lipid with a branched structure at the junction was prepared. The specific steps are as follows:

[0073] (1) 1,1 - Cyclohexanediacetic anhydride (1360 mg, 7.5 mmol) was slowly added to a pyridine solution (5 mL) of 2 - dimethylaminoethanol (1 g, 11.2 mmol), and the mixture was stirred overnight. Excess 2 - dimethylaminoethanol and the pyridine solution were 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 - n - octyl - 1 - dodecanol (149 mg, 0.5 mmol) were dissolved in 5 mL of CDCl 2 and stirred at room temperature for 12 h. The solvent was removed by rotary evaporation, and the crude product was separated and purified by silica gel chromatography (product eluent: n - hexane:ethyl acetate = 2:1 (volume ratio)), and then dried under vacuum to obtain product A1. The reaction process is as follows:

[0074]

[0075] (2) First, cationic lipid A1, DSPC, DMG - PEG2000, and cholesterol were dissolved in 20 μL of ethanol according to a molar ratio of 50:10:1.5:38.5 and a total mass of 120 micrograms. Under vortexing conditions, this ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kaituo Biotech), and the mixture was vigorously stirred for 20 s and then left standing for 10 minutes to prepare the nanoparticles.

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

[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. The specific steps are as follows:

[0079] (1) 1,1-Cyclohexanediacetic anhydride (1360 mg, 7.5 mmol) was slowly added to a pyridine solution (5 mL) of 2-dimethylaminoethanol (1 g, 11.2 mmol), and the mixture was stirred overnight. Excess 2-dimethylaminoethanol and the pyridine solution were 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 CDCl 2 and stirred at room temperature for 12 h. The solvent was removed by rotary evaporation, and the crude product was separated and purified by silica gel chromatography (product eluent: n-hexane:ethyl acetate = 2:1 (volume ratio)). After vacuum drying, product A2 was obtained. The reaction process is as follows:

[0080]

[0081] (2) First, cationic lipid A2, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol at a molar ratio of 50:10:1.5:38.5 and a total mass of 120 μg. Under vortexing conditions, this ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kaituo Biologics), and the mixture was vigorously stirred for 20 s and then allowed to stand for 10 minutes to prepare the nanoparticles.

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

[0083] Example 3

[0084] In this example, a lipid nanoparticle based on an ionizable cationic lipid containing a branched structure at the junction was prepared. The specific steps are as follows:

[0085] (1) 1,1-Cyclohexanediacetic anhydride (1360 mg, 7.5 mmol) was slowly added to a pyridine solution (5 mL) of 2-dimethylaminoethanol (1 g, 11.2 mmol), and the mixture was stirred overnight. Excess 2-dimethylaminoethanol and the pyridine solution were 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-dodecylhexadec-1-ol (205 mg, 0.5 mmol) were dissolved in 5 mL of CDCl 2In this process, it was stirred at room temperature for 12 h, the solvent was removed by rotary evaporation, the crude product was separated, and purified by silica gel chromatography (product eluent: n - hexane: ethyl acetate = 2:1 (volume ratio)), and product A3 was obtained after vacuum drying. The reaction process was as follows:

[0086]

[0087] (2) First, cationic lipid A3, DSPC, DMG - PEG2000, and cholesterol were dissolved in 20 μL of ethanol according to a molar ratio of 50:10:1.5:38.5 and a total mass of 120 μg. Under vortex conditions, this ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kaituo Biotech), stirred vigorously for 20 s, and then left standing for 10 minutes to prepare nanoparticles.

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

[0089] Example 4

[0090] In this example, a lipid nanoparticle based on an ionizable cationic lipid without a branched structure at the junction was prepared. The specific steps were as follows:

[0091] (1) Glutaric anhydride (851 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 were removed by rotary evaporation to obtain product organic acid B. Under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), organic acid B (114 mg, 1 mmol) and 2 - octyl - 1 - dodecanol (149 mg, 0.5 mmol) were dissolved in 5 mL of CDCl 2 In this process, it was stirred at room temperature for 12 h, the solvent was removed by rotary evaporation, the crude product was separated, and purified by silica gel chromatography (product eluent: n - hexane: ethyl acetate = 2:1 (volume ratio)), and product B1 was obtained after vacuum drying. The reaction process was as follows:

[0092]

[0093] (2) First, dissolve cationic lipid B1, DSPC, DMG-PEG2000, and cholesterol in 20 μL of ethanol according to the molar ratio of 50:10:1.5:38.5 and a total mass of 120 μg. Under vortexing conditions, quickly inject this ethanol solution into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kaituo Biotech), stir vigorously for 20 s, and then let it stand for 10 minutes to prepare the nanoparticles.

[0094] (3) Dialyze the ethanol-sodium acetate mixed solution containing the nanoparticles prepared in (2) with 10 mM PBS solution with a neutral pH (dialysis bag Mw = 100 kDa) for 2 - 4 hours to remove ethanol and obtain the final product.

[0095] Example 5

[0096] In this example, a lipid nanoparticle based on an ionizable cationic lipid without a branched structure at the junction was prepared. The specific steps are as follows:

[0097] (1) Slowly add glutaric anhydride (851 mg, 7.5 mmol) to a pyridine solution (5 mL) of 2-dimethylaminoethanol (1 g, 11.2 mmol), stir overnight, and remove the excess 2-dimethylaminoethanol and pyridine solution by rotary evaporation to obtain the product organic acid B. Under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), dissolve organic acid B (114 mg, 1 mmol) and 2-decyl-1-tetradecanol (177 mg, 0.5 mmol) in 5 mL of CDCl 2 and stir at room temperature for 12 h. Remove the solvent by rotary evaporation, separate the crude product, and purify it by silica gel chromatography (product eluent: n-hexane:ethyl acetate = 2:1 (volume ratio)), and vacuum dry to obtain product B2. The reaction process is as follows:

[0098]

[0099] (2) First, dissolve cationic lipid B2, DSPC, DMG-PEG2000, and cholesterol in 20 μL of ethanol according to the molar ratio of 50:10:1.5:38.5 and a total mass of 120 μg. Under vortexing conditions, quickly inject this ethanol solution into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kaituo Biotech), stir vigorously for 20 s, and then let it stand for 10 minutes to prepare the nanoparticles.

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

[0101] Example 6

[0102] In this example, a lipid nanoparticle based on an ionizable cationic lipid without a branched structure at the junction was prepared. The specific steps were as follows:

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

[0104]

[0105] (2) First, 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 and a total mass of 120 micrograms. Under vortexing conditions, this ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kaituo Biotech), stirred vigorously for 20 s, and then left standing for 10 minutes to prepare the nanoparticles.

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

[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. The specific steps were as follows:

[0109] (1) 3-Isobutylglutaric anhydride (1271 mg, 7.5 mmol) was slowly added to a pyridine solution (5 mL) of 2-dimethylaminoethanol (1 g, 11.2 mmol), and the mixture was stirred overnight. Excess 2-dimethylaminoethanol and the pyridine solution were removed by rotary evaporation to obtain the 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 CDCl 2 3. The mixture was stirred at room temperature for 12 h, and the solvent was removed by rotary evaporation. The crude product was separated and purified by silica gel chromatography (product eluent: n-hexane:ethyl acetate = 2:1 (volume ratio)), and dried under vacuum to obtain product C1. The reaction process is as follows:

[0110]

[0111] As Figure 1 shown, the 1H NMR spectrum of C1 cationic lipid 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). It is consistent with the peaks of the target product, indicating the successful synthesis of the cationic lipid.

[0112] (2) First, cationic lipid C1, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol at a molar ratio of 50:10:1.5:38.5 and a total mass of 120 μg; under vortex conditions, this ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kaituo Biotech), stirred vigorously for 20 s, and then allowed to stand for 10 minutes to prepare nanoparticles.

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

[0114] As Figure 2As shown, the particle size of the nanomaterial LNPs prepared in this example was observed to be about 120 nm by transmission electron microscopy (TEM).

[0115] Example 8

[0116] In this example, a lipid nanoparticle based on an ionizable cationic lipid with a branched structure at the junction was prepared. The specific steps were as follows:

[0117] (1) 3-Isobutylglutaric anhydride (1271 mg, 7.5 mmol) was slowly added to a pyridine solution (5 mL) of 2-dimethylaminoethanol (1 g, 11.2 mmol), and the mixture was stirred overnight. Excess 2-dimethylaminoethanol and pyridine solution were 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 CDCl 2 and stirred at room temperature for 12 h. The solvent was removed by rotary evaporation, and the crude product was separated and purified by silica gel chromatography (product eluent: n-hexane:ethyl acetate = 2:1 (volume ratio)), and then dried in vacuo to obtain product C2. The reaction process was as follows:

[0118]

[0119] (2) First, cationic lipid C2, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol at a molar ratio of 50:10:1.5:38.5 and a total mass of 120 micrograms. Under vortexing conditions, this ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kaituo Biotech), and the mixture was vigorously stirred for 20 s and then allowed to stand for 10 minutes to prepare the nanoparticles.

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

[0121] Example 9

[0122] In this example, a lipid nanoparticle based on an ionizable cationic lipid with a branched structure at the junction was prepared. The specific steps were as follows:

[0123] (1) 3-Isobutylglutaric anhydride (1271 mg, 7.5 mmol) was slowly added to a pyridine solution (5 mL) of 2-dimethylaminoethanol (1 g, 11.2 mmol), and the mixture was stirred overnight. Excess 2-dimethylaminoethanol and the pyridine solution were 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-dodecylhexadec-1-ol (205 mg, 0.5 mmol) were dissolved in 5 mL of CDCl 2 and stirred at room temperature for 12 h. The solvent was removed by rotary evaporation, and the crude product was separated and purified by silica gel chromatography (product eluent: n-hexane:ethyl acetate = 2:1 (volume ratio)). After vacuum drying, product C3 was obtained. The reaction process was as follows:

[0124]

[0125] (2) First, cationic lipid C3, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol at a molar ratio of 50:10:1.5:38.5 and a total mass of 120 μg. Under vortexing conditions, this ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kaituo Biotech), and the mixture was vigorously stirred for 20 s and then allowed to stand for 10 minutes to prepare the nanoparticles.

[0126] (3) The ethanol-sodium acetate mixed solution containing the nanoparticles prepared in (2) was dialyzed against 10 mM PBS solution with neutral pH (dialysis bag Mw = 100 kDa) for 2 - 4 h 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 containing a branched structure at the junction was prepared. The specific steps were as follows:

[0129] (1) 3,3-Dimethylglutaric anhydride (1061 mg, 7.5 mmol) was slowly added to a pyridine (5 mL) solution of 2-dimethylaminoethanol (1 g, 11.2 mmol), and the mixture was stirred overnight. Excess 2-dimethylaminoethanol and the pyridine solution were removed by rotary evaporation to obtain the product organic acid D. Under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), organic acid D (142 mg, 1 mmol) and 2-n-octyldodecan-1-ol (149 mg, 0.5 mmol) were dissolved in 5 mL of CDCl 2In it, stir at room temperature for 12 h, remove the solvent by rotary evaporation, separate to obtain the crude product, and purify it by silica gel chromatography (product eluent: n - hexane: ethyl acetate = 2:1 (volume ratio)), and obtain product D1 after vacuum drying. The reaction process is as follows:

[0130]

[0131] (2) First, dissolve cationic lipid D1, DSPC, DMG - PEG2000, and cholesterol in 20 μL of ethanol according to the molar ratio of 50:10:1.5:38.5 and a total mass of 120 micrograms; under the condition of vortex, quickly inject this ethanol solution into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kaituo Biotech), stir vigorously for 20 s, and then let it stand for 10 minutes to prepare nanoparticles.

[0132] (3) Dialyze the ethanol - sodium acetate mixed solution containing nanoparticles prepared in (2) with 10 mM PBS solution with neutral pH (dialysis bag Mw = 100 kDa) for 2 - 4 hours to remove ethanol to obtain the final product.

[0133] Example 11

[0134] In this example, a lipid nanoparticle based on an ionizable cationic lipid containing a branched structure at the junction was prepared. The specific steps are as follows:

[0135] (1) Slowly add 3,3 - dimethylglutaric anhydride (1061 mg, 7.5 mmol) to a pyridine (5 mL) solution of 2 - dimethylaminoethanol (1 g, 11.2 mmol), stir overnight, remove the excess 2 - dimethylaminoethanol and pyridine solution by rotary evaporation to obtain product organic acid D. Under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), dissolve organic acid D (142 mg, 1 mmol) and 2 - decyl - 1 - tetradecanol (177 mg, 0.5 mmol) in 5 mL of CDCl 2 In it, stir at room temperature for 12 h, remove the solvent by rotary evaporation, separate to obtain the crude product, and purify it by silica gel chromatography (product eluent: n - hexane: ethyl acetate = 2:1 (volume ratio)), and obtain product D2 after vacuum drying. The reaction process is as follows:

[0136]

[0137] (2) First, dissolve cationic lipid D2, DSPC, DMG-PEG2000, and cholesterol in 20 μL of ethanol according to a molar ratio of 50:10:1.5:38.5 and a total mass of 120 micrograms. Under vortexing conditions, quickly inject this ethanol solution into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kaituo Biotech), stir vigorously for 20 s, and then let it stand for 10 minutes to prepare nanoparticles.

[0138] (3) Dialyze the ethanol-sodium acetate mixed solution containing nanoparticles prepared in (2) with 10 mM PBS solution with a neutral pH (dialysis bag Mw = 100 kDa) for 2 - 4 hours to remove ethanol and obtain the final product.

[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. The specific steps are as follows:

[0141] (1) Slowly add 3,3-dimethylglutaric anhydride (1061 mg, 7.5 mmol) to a pyridine solution (5 mL) of 2-dimethylaminoethanol (1 g, 11.2 mmol), stir overnight, and remove the excess 2-dimethylaminoethanol and pyridine solution by rotary evaporation to obtain product organic acid D. Under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), dissolve organic acid D (142 mg, 1 mmol) and 2-dodecylhexadec-1-ol (205 mg, 0.5 mmol) in 5 mL of CDCl 2 and stir at room temperature for 12 h. Remove the solvent by rotary evaporation, separate the crude product, and purify it by silica gel chromatography (product eluent: n-hexane:ethyl acetate = 2:1 (volume ratio)), and obtain product D3 after vacuum drying. The reaction process is as follows:

[0142]

[0143] (2) First, dissolve cationic lipid D3, DSPC, DMG-PEG2000, and cholesterol in 20 μL of ethanol according to a molar ratio of 50:10:1.5:38.5 and a total mass of 120 micrograms. Under vortexing conditions, quickly inject this ethanol solution into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kaituo Biotech), stir vigorously for 20 s, and then let it stand for 10 minutes to prepare nanoparticles.

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

[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. The specific steps are as follows:

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

[0148]

[0149] (2) First, cationic lipid E1, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol according to a molar ratio of 50:10:1.5:38.5 and a total mass of 120 micrograms. Under vortexing conditions, this ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kaituo Biotech), and the mixture was vigorously stirred for 20 s and then left standing for 10 minutes to prepare the nanoparticles.

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

[0151] Example 14

[0152] In this example, a lipid nanoparticle based on an ionizable cationic lipid with a branched structure at the junction was prepared. The specific steps are as follows:

[0153] (1) 3-Methylglutaric anhydride (957 mg, 7.5 mmol) was slowly added to a pyridine solution (5 mL) of 2-dimethylaminoethanol (1 g, 11.2 mmol), and the mixture was stirred overnight. Excess 2-dimethylaminoethanol and the pyridine solution were removed by rotary evaporation to obtain the product organic acid E. Under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), organic acid E (128 mg, 1 mmol) and 2-decyl-1-tetradecanol (177 mg, 0.5 mmol) were dissolved in 5 mL of CDCl 2 3. The mixture was stirred at room temperature for 12 h, and the solvent was removed by rotary evaporation. The crude 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, cationic lipid E2, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol at a molar ratio of 50:10:1.5:38.5 and a total mass of 120 micrograms. Under vortexing conditions, the ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kaituo Biotech), and the mixture was vigorously stirred for 20 s and then allowed 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 with 10 mM PBS solution with a neutral pH (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. The specific steps are as follows:

[0159] (1) 3-Methylglutaric anhydride (957 mg, 7.5 mmol) was slowly added to a pyridine solution (5 mL) of 2-dimethylaminoethanol (1 g, 11.2 mmol), and the mixture was stirred overnight. Excess 2-dimethylaminoethanol and the pyridine solution were removed by rotary evaporation to obtain the product organic acid E. Under the catalysis of EDC (191 mg, 1.2 mmol) and DMAP (20 mg, 0.2 mmol), organic acid E (128 mg, 1 mmol) and 2-dodecylhexadec-1-ol (205 mg, 0.5 mmol) were dissolved in 5 mL of CDCl2, and the mixture was stirred at room temperature for 12 h. The solvent was removed by rotary evaporation, and the crude product was separated and purified by silica gel chromatography (product eluent: n-hexane:ethyl acetate = 2:1 (volume ratio)). After vacuum drying, product E3 was obtained. The reaction process is as follows:

[0160]

[0161] (2) First, cationic lipid E3, DSPC, DMG-PEG2000, and cholesterol were dissolved in 20 μL of ethanol at a molar ratio of 50:10:1.5:38.5 and a total mass of 120 μg. Under vortex conditions, this ethanol solution was rapidly injected into 60 μL of 20 mM sodium acetate buffer containing 3 μg of luciferase mRNA (purchased from Kaituo Biotech), and the mixture was vigorously stirred for 20 s and then allowed 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 with a neutral pH (dialysis bag Mw = 100 kDa) for 2 - 4 hours to remove ethanol, obtaining the final product.

[0163] Test Example 1: Analysis of the particle size and surface potential of lipid nanoparticles

[0164] The lipid nanoparticles prepared in Examples 1 - 15 were tested using 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.nm, and the distribution coefficient PDI = 0.148; the ζ potential was 0.501 mv, indicating that the nanoparticles were electrically neutral.

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

[0167] The average particle size of the nanomaterial LNP prepared in Example 3 is 116.4 nm, and the distribution coefficient PDI = 0.300; the ζ potential is -5.43 mv, indicating that the nanoparticles are weakly negatively charged.

[0168] The average particle size of the nanomaterial LNP prepared in Example 4 is 164.0 nm, and the distribution coefficient PDI = 0.121; the ζ potential is 1.323 mv, indicating that the nanoparticles are electrically neutral.

[0169] The average particle size of the nanomaterial LNP prepared in Example 5 is 123.6 nm, and the distribution coefficient PDI = 0.173; the ζ potential is 1.117 mv, indicating that the nanoparticles are electrically neutral.

[0170] The average particle size of the nanomaterial LNP prepared in Example 6 is 117.7 nm, and the distribution coefficient PDI = 0.198; the ζ potential is -3.570 mv, indicating that the nanoparticles are electrically neutral.

[0171] The average particle size of the nanomaterial LNP prepared in Example 7 is 121.6 nm, and the distribution coefficient PDI = 0.156; the ζ potential is -3.137 mv, indicating that the nanoparticles are electrically neutral.

[0172] The average particle size of the nanomaterial LNP prepared in Example 8 is 96.8 nm, and the distribution coefficient PDI = 0.161; the ζ potential is -7.767 mv, indicating that the nanoparticles are weakly negatively charged.

[0173] The average particle size of the nanomaterial LNP prepared in Example 9 is 254.9 nm, and the distribution coefficient PDI = 0.725; the ζ potential is -5.613 mv, indicating that the nanoparticles are weakly negatively charged.

[0174] The average particle size of the nanomaterial LNP prepared in Example 10 is 154.3 nm, and the distribution coefficient PDI = 0.181; the ζ potential is -6.447 mv, indicating that the nanoparticles are weakly negatively charged.

[0175] The average particle size of the nanomaterial LNP prepared in Example 11 is 113.3 nm, and the distribution coefficient PDI = 0.109; the ζ potential is -2.156 mv, indicating that the nanoparticles are electrically neutral.

[0176] The average particle size of the nanomaterial LNP prepared in Example 12 is 131.8 nm, and the distribution coefficient PDI = 0.138; the ζ potential is -4.480 mv, indicating that the nanoparticles are electrically neutral.

[0177] The average particle size of the nanomaterial LNP prepared in Example 13 is 175.1 nm, and the distribution coefficient PDI = 0.160; the ζ potential is -4.313 mv, indicating that the nanoparticles are electrically neutral.

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

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

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

[0181] 1. The PBS solution of LNP after dialysis was injected into mice by subcutaneous injection. After 6 h, the luciferase substrate D-luciferin potassium salt (10 mg / mL, 200 μL) was injected into the abdominal cavity, and the fluorescence signal was observed by a small animal in vivo imager.

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

[0183] It can be seen that 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 mice was quantitatively analyzed by a small animal in vivo imager. As Figure 4 shown, it can be seen that the branched structure at the connection site has a great influence on the performance of the liposome. Among them, 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 lipid nanoparticles, we designed 5 different formulations as shown in Table 1 to explore the optimal molar ratio and maximize the mRNA delivery efficiency of lipid nanoparticles.

[0187] Table 1

[0188]

[0189] Dissolve ionizable cationic lipid A1 / C1 with a branched structure, DSPC, cholesterol, and DMG-PEG2000 in 20 μL of ethanol according to the molar ratios shown in Table 1 and a total mass of 40 micrograms; under vortexing conditions, quickly inject this ethanol solution into 60 μL of 20 mM sodium acetate buffer containing 1 μg luciferase mRNA, stir vigorously for 20 s, and then let it stand for 10 minutes to prepare nanoparticles.

[0190] Inject the PBS solution of the dialyzed LNP into the mice by subcutaneous injection. After 6 h, inject luciferase substrate potassium D-luciferin (10 mg / mL, 200 μL) by intraperitoneal injection, and observe the fluorescence signal with a small animal in vivo imager.

[0191] As Figure 5 shown, we observed that there were obvious fluorescence signals in the mice at 6 h, and there were obvious differences among different groups (where sLNP is a lipid nanoparticle prepared based on a commercial formulation (the same as formulation No. 1 in Table 1, cationic lipid:DSPC:cholesterol:DMG-PEG2000 = 50:10:38.5:1.5) and the commercial cationic lipid SM-102). This shows that adjusting the ratio of the four components can significantly affect and improve the delivery efficiency of lipid nanoparticles for mRNA.

[0192] We quantitatively analyzed the in vivo transfection effect of this formulation in mice with a small animal in vivo imager. As Figure 6 shown, it can be seen that the adjustment of the ratio and the optimization of the formulation can significantly improve the delivery efficiency of lipid nanoparticles. The lipid nanoparticles (cLNP) based on cationic lipid C1 under formulation No. 5 showed the best efficacy, better than the commercial lipid nanoparticles.

[0193] Subsequent experiments will be completed using cationic lipid C1 and formulation No. 5. At the same time, lipid nanoparticles (bLNP) based on cationic lipid B1 without a branched structure under formulation No. 5 and lipid nanoparticles (sLNP) based on cationic lipid SM-102 under the commercial formulation will be introduced as controls.

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

[0195] Seed mouse kidney cells (BHK cells) in a white and transparent 48-well plate. Before LNP transfection of the cells, the cells adhere and grow to reach 1×10 5 / well. Before transfection, add LNPs containing 2μg EGFP mRNA to 300μL Opti-MEM and incubate for 10min; wash the cells once with 1mL Opti-MEM. Then drip the transfection mixture onto the medium. Observe the transfection status (fluorescence microscope) after 16-24 hours.

[0196] like Figure 7 As shown in the figure, under the green fluorescence channel, the cLNP group and the sLNP group had stronger green fluorescence signals within 24 hours than the bLNP group, indicating that the cLNP group expressed more green fluorescent protein. This shows that compared with lipid nanoparticles with cationic lipids without branches at the junction, the branched structure at the junction can promote mRNA transfection into the cytoplasm and translation into protein to a greater extent, thereby exerting its efficacy.

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

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

[0199] like Figure 8 , 9 As shown in the figure, cLNP and sLNP showed the same anti-tumor effect, and were significantly better than bLNP. It can be seen that lipid nanoparticles based on ionizable cationic lipids with branched structures at the junction have better mRNA delivery efficiency and efficacy than ionizable cationic lipid nanoparticles without branched structures at the junction.

[0200] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A lipid nanoparticle, characterized in that: The raw material composition of the lipid nanoparticles includes: a cationic lipid having a branched structure at the connection, an auxiliary lipid, and cholesterol. The structural formula of the cationic lipid is shown in formula (I). Wherein, R1 is selected from: -C(CH2)5, -CHCH2CH(CH3)2, -C(CH3)2, -CHCH3; 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-.

2. The lipid nanoparticle according to claim 1, characterized in that The preparation method of the cationic lipid with a branched structure at the connection point comprises: firstly dissolving 2-dimethylaminoethanol and a glutaric anhydride derivative in pyridine, causing an alcoholysis reaction to generate an organic acid with a branched structure, then dissolving the organic acid and an alcohol or amine containing a fatty chain in an organic solvent, causing a condensation reaction under the catalytic action of a catalyst, and separating and purifying the product to obtain the cationic lipid with a branched structure at the connection point.

3. The lipid nanoparticle according to claim 2, characterized in that The glutaric anhydride derivative is any one of 1,1-cyclohexyldiacetic anhydride, 3-methylglutaric anhydride, 3-isobutylglutaric anhydride and 3,3-dimethylglutaric anhydride.

4. The lipid nanoparticle according to claim 2, characterized in that The alcohol containing a fatty chain is any one of 2-octyl-1-dodecanol, 2-decyl-1-tetradecanol, 2-dodecylhexadecanol-1-ol, 2-hexyl-1-decanol, 7-tetradecanol and castor oil; the amine containing a fatty chain is ditetradecylamine or didodecylamine.

5. The lipid nanoparticle according to claim 1, characterized in that The auxiliary lipids include phospholipids and polyethylene glycol functionalized lipids; the phospholipids are distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine, and the polyethylene glycol functionalized lipids are dimyristylglycerol-polyethylene glycol 2000.

6. The lipid nanoparticle according to claim 5, characterized in that The molar ratio of cationic lipid, phospholipid, cholesterol and polyethylene glycol functionalized lipid is 25-75:5-15:15-60:1-2.

7. Use of the lipid nanoparticles as described in any one of claims 1 to 6 as a carrier in the preparation and delivery of nucleic acid drugs.

8. The use according to claim 7, characterized in that The application comprises: adding cationic lipids with branched structures at the connection points, auxiliary lipids and cholesterol into an acidic buffer containing nucleic acids, self-assembling to form lipid nanoparticles encapsulating nucleic acids, and preparing the nucleic acid delivery drug.

9. The use according to claim 8, characterized in that The delivered nucleic acid drug is an mRNA vaccine, and the ratio of the total mass of lipid material to the mass of mRNA is 20-160:1.

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