Novel cationic lipid and application thereof in preparation of mRNA delivery drugs

Abstract

The invention discloses a cationic lipid and application thereof in preparation of mRNA delivery drugs, and belongs to the technical field of biological medicines. The structural formula of the cationic lipid is shown as a formula (I), and the cationic lipid is provided with a hydroxyl head and a four-branch fatty alkyl tail. According to the lipid nanoparticles prepared on the basis of the cationic lipid, pKa of the lipid nanoparticles is in a specific interval for generating an optimal transfection effect due to the existence of a hydroxyl group at the head of the cationic lipid; the four-branch-chain fatty alkyl can form a large conical hydrophobic tail, the endosomal membrane damage capability of LNP can be effectively enhanced in the endosomal escape process, and the cell endocytosis and endosomal escape efficiency is improved. Compared with commercially available ionizable lipid SM-102 and a formula thereof, and homologue cationic lipid and a formula thereof, the lipid nanoparticle provided by the invention has more excellent cell endocytosis and endosome escape efficiency, so that higher mRNA delivery efficiency is mediated, and the problem of low mRNA delivery efficiency at present is effectively solved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a novel cationic lipid and a preparation method thereof, and application thereof in the preparation of mRNA delivery drugs. Background Art

[0002] Due to the hydrophilicity, negative charge and instability of naked mRNA, it is easily degraded by nucleases after entering the human body. Therefore, it is crucial to develop an efficient mRNA delivery system to ensure that mRNA enters cells and exerts its effect.

[0003] Lipid nanoparticles (LNPs) technology based on ionizable cationic lipids is the mainstream delivery system for mRNA delivery at present because of its good biosafety and efficient delivery ability. LNP is usually composed of four components: ionizable cationic lipids, cholesterol, phospholipids, and PEG-modified lipids. Among them, ionizable cationic lipids are key components, usually composed of amine head, hydrophobic tail, and connecting bond. Changes in the structure of cationic lipids can seriously affect the delivery efficiency of LNP. At present, even the most effective LNP can only deliver ~ 4% of mRNA to the cytoplasm (S.Liu, et al.Nature Materials, 2021, 20: 701-710.; J.Gilleron, et al.Nature Biotechnology, 2013, 31: 638-646.), which seriously hinders the further development of mRNA delivery drugs.

[0004] Many researchers have optimized the structure of ionizable lipids by combining different multi-branched tails and amine head groups to improve delivery efficiency (Jiang, AY, et al. Nature Nanotechnology, 2023, 19: 364-375.; Han, X., et al. Naure Communications, 2024, 15: 1762.). However, the structure-activity relationship between the four-branched hydrophobic tail and the amine head and its effect on delivery efficiency still need to be further explored.

[0005] Since cationic lipids play a decisive role in the delivery of mRNA, it is of great clinical significance to conduct in-depth research on the structure-activity relationship of cationic lipids, develop efficient and safe cationic lipids, effectively enhance mRNA endocytosis and endosomal escape after forming lipid nanoparticles, and achieve efficient delivery of mRNA in vivo. Summary of the invention

[0006] The purpose of the present invention is to provide a new cationic lipid having a hydroxyl head and a four-branched fatty alkyl tail. Lipid nanoparticles prepared using this material can achieve efficient delivery of nucleic acid molecules such as mRNA by enhancing cellular endocytosis and endosomal escape.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides a cationic lipid, the structural formula of which is shown in formula (I):

[0009]

[0010] Where n = 2-4;

[0011] R1 and R2 are each independently selected from aliphatic alkyl groups having 8 to 14 carbon atoms.

[0012] The cationic lipid provided by the present invention can form lipid nanoparticles by self-assembly with auxiliary lipids and cholesterol. The tertiary amine group of the cationic lipid forms a hydrophilic end with a positive charge in the buffer, which can bind negatively charged drugs through electrostatic interaction. Due to the hydrophilic and hydrophobic supramolecular forces, the lipid material self-assembles to form lipid nanoparticles encapsulating negatively charged drugs such as mRNA.

[0013] The present invention shows that lipid nanoparticles formed by self-assembly of cationic lipids obtained through structural screening and other raw materials can achieve efficient drug delivery by enhancing cellular endocytosis and endosomal escape.

[0014] Studies have shown that the enhancement of the above delivery efficiency is closely related to the presence of four-branched fatty alkyl groups in cationic lipid molecules. Compared with lipid molecules with two-branched fatty alkyl groups, lipid molecules with four-branched fatty alkyl groups have smaller heads and larger conical hydrophobic tails. When they are bound to the membrane, they will destroy a larger area of ​​local membrane phospholipid organization, thereby generating greater entropy stimulation for the interaction. This structure is conducive to the transformation of the membrane into a hexagonal phase during the endosomal escape process, destroying the endosomal membrane, thereby enhancing endocytosis and endosomal escape, and ultimately significantly improving the delivery efficiency of drugs such as mRNA.

[0015] Studies have shown that the significant enhancement of delivery efficiency is also closely related to the presence of hydroxyl groups on the head. After the cationic lipids with hydroxyl groups on the head form lipid nanoparticles, the pKa is between 6 and 7, which is most conducive to the transfection of LNPs.

[0016] Preferably, R1 is a fatty alkyl group having 8 carbon atoms, and R2 is a fatty alkyl group having 10 carbon atoms; or R1 is a fatty alkyl group having 10 carbon atoms, and R2 is a fatty alkyl group having 12 carbon atoms; or R1 is a fatty alkyl group having 12 carbon atoms, and R2 is a fatty alkyl group having 14 carbon atoms.

[0017] The n in the above structural formula (I) is an integer between 2 and 4. Preferably, the n in the structural formula (I) is 3.

[0018] Further preferably, the structural formula of the cationic lipid is as shown in formula (Y1) or formula (Y2). Studies have shown that Y1 and Y2 cationic lipids exhibit a high level of mRNA delivery efficiency in in vitro transfection, which is significantly better than commercial SM-102. Among them, after the formulation of Y1 lipid is optimized, it significantly enhances cellular endocytosis and endosomal escape, and exhibits a more excellent mRNA delivery efficiency both in vitro and in vivo, and the transfection efficiency is significantly enhanced compared to SM-102 lipid.

[0019]

[0020] The present invention also provides a method for preparing the cationic lipid, comprising the following steps:

[0021] (1) a fatty alcohol containing two branches is subjected to a substitution reaction with acryloyl chloride to prepare a dibranched acrylate;

[0022] (2) The dibranched acrylate is subjected to Michael addition reaction with N-(3-aminopropyl)diethanolamine or N,N-bis(2-hydroxyethyl)ethylenediamine, and the cationic lipid is obtained after separation and purification.

[0023] In step (1), a fatty alcohol and acryloyl chloride undergo a substitution reaction under the catalytic condition of triethylamine to prepare a dibranched acrylate. Specifically, the fatty alcohol is first dissolved in an organic solvent, a catalyst triethylamine is added under ice bath conditions, and then acryloyl chloride is slowly added, stirred for reaction, and after the reaction is completed, the solvent is removed by rotary evaporation, and a crude product is separated and purified by silica gel chromatography to obtain a dibranched acrylate.

[0024] Preferably, the fatty alcohol may be, but is not limited to, 2-octyldodecanol, 2-decyl-1-tetradecanol, and 2-dodecylhexadecanol-1-ol.

[0025] Preferably, the molar ratio of fatty alcohol, acryloyl chloride and triethylamine is 1:1.5-3:1.5-3.

[0026] Preferably, the organic solvent may be, but is not limited to, dichloromethane.

[0027] Preferably, the reaction conditions are: stirring at 0°C for 4-6h;

[0028] In step (2), the di-branched acrylate and the amine undergo Michael addition reaction to form a positively charged head. Specifically, the di-branched acrylate and the amine are dissolved in an organic solvent, stirred and reacted at 30° C.-80° C., and after the reaction is completed, the solvent is removed by rotary evaporation to separate the crude product, which is purified by silica gel chromatography to obtain the cationic lipid.

[0029] Preferably, the molar ratio of the dibranched acrylate to the amine is 2-4:1.

[0030] Preferably, the Michael addition reaction is carried out under stirring at 30°C-80°C for 12-48 hours, and more preferably, under stirring at 50°C for 24 hours.

[0031] Preferably, the eluent for silica gel chromatography is HEX and EA in a volume ratio of 3:1.

[0032] The preparation method that can be adopted by the present invention is not limited thereto.

[0033] The present invention also provides a lipid nanoparticle prepared using the above cationic lipid. Specifically, the raw material composition of the lipid nanoparticle includes: a cationic lipid with a structural formula as shown in formula (I), an auxiliary lipid and cholesterol, and the raw materials form lipid nanoparticles through self-assembly.

[0034] The lipid nanoparticles can be prepared by, but are not limited to, an ethanol injection method, a thin film method, and an ultrasonic method.

[0035] The auxiliary lipids include phospholipids and polyethylene glycol functionalized lipids; preferably, 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 dimyristylglycerol-polyethylene glycol 2000 (DMG-PEG2000).

[0036] Preferably, the molar ratio of ionizable cationic lipid, phospholipid, cholesterol, and polyethylene glycol functionalized lipid is 20-70: 5-25: 15-55: 0.5-3.5. More preferably, the molar ratio of the above four components is 40-60: 5-15: 20-25: 0.5-2.5. More preferably, the molar ratio of the above four components is 60: 5: 22.5: 0.5.

[0037] The lipid nanoparticles based on the cationic lipid provided by the present invention can efficiently deliver nucleic acid drugs such as mRNA by enhancing cell endocytosis and endosomal escape, and have potential application value in the development of nucleic acid drugs.

[0038] The present invention also provides the use of the lipid nanoparticles as carriers in the preparation and delivery of nucleic acid drugs.

[0039] Specifically, the application includes: adding ionizable cationic lipids, 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.

[0040] Specifically, when preparing lipid nanoparticles, ethanol injection method, thin film method, ultrasound method and other methods are used to make lipid materials and nucleic acid drugs such as mRNA self-assemble into nanoparticles in a buffer solution through supramolecular forces and electrostatic interactions.

[0041] Among them, the ethanol injection method is to dissolve ionizable cationic lipids, auxiliary lipids, and cholesterol in a certain amount of ethanol. Then the ethanol solution containing lipid materials is injected into the buffer containing nucleic acid drugs to self-assemble into nanoparticles, and then the ethanol is removed by dialysis to obtain stable nanoparticles.

[0042] Furthermore, the delivered nucleic acid drug is an mRNA vaccine.

[0043] Preferably, the total mass ratio of lipid material to mRNA is 20-160:1. mRNA molecules are large, and when the lipid material is too little, it is difficult to effectively encapsulate and protect mRNA, thereby reducing the transfection efficiency. When the lipid material is too much, the endosomal escape efficiency may be reduced, thereby reducing the transfection efficiency. Within the appropriate mass ratio range, a good encapsulation rate and transfection rate can be guaranteed. More preferably, the total mass ratio of lipid material to mRNA is 40:1.

[0044] The present invention has the following beneficial effects:

[0045] The present invention provides a novel cationic lipid, which can be used to prepare lipid nanoparticles. The cationic lipid has a hydroxyl head and a four-branched fatty alkyl tail. The lipid nanoparticles prepared based on the cationic lipid have a pKa in a specific range that produces the best transfection effect due to the presence of a hydroxyl group on the cationic head; at the same time, compared with a di-branched fatty alkyl, a four-branched fatty alkyl can form a larger conical hydrophobic tail, which can effectively enhance the endosomal membrane destruction ability of LNP during endosomal escape, improve the efficiency of cellular endocytosis and endosomal escape, and then efficiently deliver nucleic acid drugs such as mRNA in vitro and in vivo. Compared with the commercially available ionizable lipid SM-102 and its formula, homologous cationic lipids and their formulas, the lipid nanoparticles provided by the present invention have more excellent cellular endocytosis and endosomal escape efficiency, thereby mediating a higher mRNA delivery efficiency, effectively solving the problem of low mRNA delivery efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the NMR image of the cationic lipid 10A prepared in Example 1.

[0047] Figure 2 This is the NMR image of the cationic lipid 10B prepared in Example 1.

[0048] Figure 3 This is the NMR image of the cationic lipid 1B prepared in Example 1.

[0049] Figure 4 This is the NMR image of the cationic lipid 3B prepared in Example 1.

[0050] Figure 5 This is the NMR image of the cationic lipid 5A prepared in Example 1.

[0051] Figure 6 This is the NMR image of the cationic lipid 6A prepared in Example 1.

[0052] Figure 7 This is the TEM image of the lipid nanoparticles prepared in Example 2.

[0053] Figure 8 Quantitative analysis results of in vitro transfection of LNPs prepared with different cationic lipids using commercial formulations.

[0054] Fig. 9 The results of lipid nanoparticle cell transfection.

[0055] Fig.10 Quantitative analysis results of lipid nanoparticle cell transfection.

[0056] Fig.11 The results of cellular endocytosis and endosomal escape of lipid nanoparticles were determined by flow cytometry.

[0057] Fig.12 Laser confocal imaging is used to characterize the endosomal escape of lipid nanoparticles.

[0058] Fig.13 This is a diagram and quantitative analysis of the in vivo transfection effect of lipid nanoparticles.

[0059] Fig.14 This is a comparison of the anti-tumor effects of preventive vaccines in animal models.

[0060] Fig.15 Survival outcomes in animal models for prophylactic vaccine applications. DETAILED DESCRIPTION

[0061] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0062] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0063] The compounds and English abbreviations involved in the examples are described as follows:

[0064] CDCl2: dichloromethane, CAS number: 75-09-2;

[0065] EtOH: ethanol, CAS number: 64-17-5;

[0066] Triethylamine, CAS number: 121-44-8;

[0067] N-(3-aminopropyl)diethanolamine, CAS No.: 4985-85-7;

[0068] N,N-bis(2-hydroxyethyl)ethylenediamine, CAS number: 3197-06-6;

[0069] 2-Octyldodecanol, CAS No.: 5333-42-6;

[0070] 2-Decyl-1-tetradecanol, CAS No.: 58670-89-6;

[0071] 2-Dodecylhexadecanol, CAS No.: 72388-18-2;

[0072] D-Luciferin potassium salt, CAS number: 115144-35-9;

[0073] DSPC phospholipid, CAS No.: 816-94-4, the structural formula is as follows:

[0074]

[0075] DMG-PEG2000 lipid, CAS number: 160743-62-4, the structural formula is as follows:

[0076]

[0077] Cholesterol, CAS No.: 57-88-5, the structural formula is as follows:

[0078]

[0079] Example 1: Preparation of cationic lipids

[0080] In order to screen cationic lipids with the best delivery efficiency, we used the Michael addition reaction method to combine different amine heads and side chain tails of different lengths (length A<B<C) to modularly synthesize 42 cationic lipids with different structures.

[0081] 1. The specific steps of the preparation method of cationic lipids are as follows:

[0082] First, 2-octyldodecanol (10.00 g, 33.5 mmol) was dissolved in 40 mL of DCM, and three times the molar amount of triethylamine (10.17 g, 100.5 mmol) was added under stirring in an ice bath at 0°C, and three times the molar amount of acryloyl chloride (9.09 g, 100.5 mmol) was slowly added. After stirring for 4 h, the solvent was removed by rotary evaporation to separate the crude product, which was purified by silica gel chromatography (product eluent: DCM) and vacuum dried to obtain a double-tail acrylate.

[0083] Then the obtained double-tailed acrylate (1 mmol) and N-(3-aminopropyl)diethanolamine (0.5 mmol) were dissolved in 5 mL of anhydrous ethanol, stirred at 50°C for 24 h, and then the solvent was removed by rotary evaporation to separate the crude product, which was purified by silica gel column chromatography (product eluent: HEX: EA = 3: 1) and vacuum dried to obtain an ionizable cationic lipid named 10A. The reaction flow chart is shown below:

[0084]

[0085] like Figure 1 As shown, the cationic lipid nuclear magnetic resonance (NMR) spectrum showed: 1H NMR (600 MHz, Chloroform-d) δ3.97 (d, J = 5.8 Hz, 4H), 3.72 (q, J = 7.0 Hz, 2H), 3.63 (t, J = 5.2 Hz, 4H), 2.78 (t, J = 7.3 Hz, 4H), 2.64 (t, J = 5.2 Hz, 4H), 2.59 (t, J = 6.8 Hz, 2H), 2.50 (t, J = 6.7 Hz, 2H), 2.46 (t, J = 7.3 Hz, 4H), 1.63 (dt, J = 14.4, 6.9 Hz, 4H), 1.27 (d, J = 13.1 Hz, 68H), 0.88 (t, J = 6.9 Hz, 12H), which was consistent with the peak of the target product, indicating that the cationic lipid was successfully synthesized. The structure of the cationic lipid is shown in formula (Y1).

[0086] 2. Replace 2-octyldodecanol (10.00 g, 33.5 mmol) in the above preparation method with 2-decyl-1-tetradecanol and 2-dodecylhexadecanol-1-ol, respectively, to prepare ionizable cationic lipids 10B and 10C, respectively, with the following structural formulas:

[0087]

[0088] 3. Replace the N-(3-aminopropyl)diethanolamine (0.5 mmol) in the above preparation method with 1-amino-2-propanol, 2-amino-2-methyl-1.3 propanediol, 2-aminopropane-1,3-diol, N,N-dimethylethylenediamine, N,N-diethylethylenediamine, 3-diethylaminopropylamine, N,N'-di-n-propylethylenediamine, 3-aminopropyl-dipropyl-amine, N,N-bis(2-hydroxyethyl)ethylenediamine, N-(2-aminoethyl)pyrrole, 1-(2-aminoethyl)piperidine, respectively. , N-(2-aminoethyl)morpholine, and 4-methylmorpholine-2-methylamine 0.5 mmol each were used to prepare ionizable cationic lipids 1A, 1B, 1C, 2A, 2B, 2C, 3A, 3B, 3C, 4A, 4B, 4C, 5A, 5B, 5C, 6A, 6B, 6C, 7A, 7B, 7C, 8A, 8B, 8C, 9A, 9B, 9C, 11A, 11B, 11C, 12A, 12B, 12C, 13A, 13B, 13C, 14A, 14B, and 14C, respectively.

[0089] The nuclear magnetic resonance (NMR) spectra of cationic lipids 10B, 1B, 3B, 5A, and 6A are shown in Figure 2-6 shown.

[0090] Example 2: Preparation of lipid nanoparticles

[0091] This example prepares a lipid nanoparticle of a novel cationic lipid prepared in Example 1, and the specific steps are as follows:

[0092] (1) The cationic lipid, DSPC, DMG-PEG2000 and cholesterol prepared in Example 1 were dissolved in 8 μL of ethanol at a molar ratio of 50:10:1.5:38.5 with a total mass of 80 μg; the ethanol solution was quickly injected into 50 μL of 20 mM sodium acetate buffer containing 2 μg luciferase mRNA (purchased from Kaituo Biotechnology) under vortexing, stirred vigorously for 15 s, and then allowed to stand for 10 minutes to obtain nanoparticles.

[0093] (2) The ethanol-sodium acetate mixed solution containing nanoparticles obtained in (1) was dialyzed for 2 to 4 hours with a 10 mM pH-neutral PBS solution (dialysis bag Mw = 100 kDa) to remove ethanol and obtain the final product.

[0094] like Figure 7 As shown, transmission electron microscopy (TEM) observed that the particle size of the nanomaterial LNPs prepared using the cationic lipid 10A in this example was about 210 nm, which was consistent with the DLS result.

[0095] Test Example 1: Cationic lipid structure screening

[0096] BHK cells were seeded in 48-well plates (5×10 4 cells / well), and cultured in RPMI-1640 medium containing 10% FBS for 12 hours. The lipid nanoparticles containing 1 μg Luc mRNA were prepared using 42 cationic lipids as raw materials using the method of Example 2. After the lipid nanoparticles were co-incubated with the cells for 24 hours, the Luc expression was detected using the firefly Luc reporter gene detection kit according to the instructions, and the fluorescence expression was detected using an ELISA instrument to evaluate the mRNA delivery efficiency of different lipid nanoparticles, and then the cationic lipid with the best in vitro transfection effect was screened.

[0097] The results are as follows Figure 8 As shown, lipid nanoparticles prepared from cationic lipids in series 9 and 10 have good transfection efficiency, among which 10A has the best transfection efficiency, and this cationic lipid was used in all subsequent tests.

[0098] Test Example 2: Lipid Nanoparticle Cell Transfection Experiment

[0099] 1. First, LNPs containing EGFP mRNA were prepared. Ionizable cationic lipids, DSPC, cholesterol, and DMG-PEG2000 were prepared at a molar ratio of 60:5:22.5:0.5 according to the method of Example 2 to prepare lipid nanoparticles containing 2 μg EGFP mRNA. The prepared lipid nanoparticles were recorded as LNPs. At the same time, a commercial formula was introduced, and lipid nanoparticles based on cationic lipid SM-102, cationic lipid, DSPC, cholesterol, and DMG-PEG2000 were prepared at a molar ratio of 50:10:38.5:1.5 as a control, recorded as SM102-LNP.

[0100] 2. Inoculate BHK / Hela cells in 48-well plates (1×10 4Cells / well), cultured with RPMI 1640 / DMEM medium at 37°C and 5% CO2 for 12 hours without treatment and with +10% FPS treatment (+10% FPS was used to observe the transfection effect of cells under serum conditions). The original culture medium was replaced with fresh culture medium, and the cells were treated with LNPs containing 2μg eGFP mRNA. After 24 hours of co-incubation, the transfection was observed and recorded using a fluorescence microscope. The cells were then washed with 100μL PBS, digested with 100μL trypsin for 3 minutes, and collected by centrifugation at 1000rpm for 5 minutes. After the cells were suspended in 0.5mL PBS and filtered through a nylon mesh (45μm), the fluorescence was measured by flow cytometry, and the data were recorded and analyzed using FlowJo software.

[0101] like Fig. 9 , Fig.10 As shown in the figure, under the green fluorescence channel, compared with the commercial SM102-LNP group, the LNP group had a stronger green fluorescence signal within 24 hours, indicating that this group expressed more green fluorescent protein. This shows that the material can promote the transfection of mRNA into the cytoplasm and its translation into protein to a greater extent, thus exerting its efficacy.

[0102] Test Example 3: Lipid Nanoparticle Cellular Endocytosis and Endosomal Escape Experiment

[0103] 1. First, LNPs containing Cy5 / EGFP mRNA were prepared. Ionizable cationic lipids, DSPC, cholesterol, and DMG-PEG2000 were prepared at a molar ratio of 60:5:22.5:0.5 according to the method of Example 2 to prepare lipid nanoparticles containing 1 μg Cy5 / EGFP mRNA. The prepared lipid nanoparticles were recorded as LNPs. At the same time, a commercial formula was introduced, and lipid nanoparticles based on cationic lipid SM-102, cationic lipids, DSPC, cholesterol, and DMG-PEG2000 were prepared at a molar ratio of 50:10:38.5:1.5 as a control, recorded as SM102-LNP.

[0104] 2. Inoculate BHK cells in a 48-well plate to a volume of 5 × 10 4 / well, cultured for 12h at 37°C and 5% CO2, using RPMI 1640 medium. Then the original medium was replaced with fresh medium, and the cells were treated with LNPs containing 1μg Cy5 / EGFP mRNA. After incubation for 0.5h, 1h, 2h, and 4h, the cells were washed with 100μL PBS, digested with 100μL trypsin for 3min, and then centrifuged at 1000rpm for 5min to collect the cells. After resuspending the cells with 0.5mL PBS, the cells were filtered using a nylon mesh (45μm), and then the fluorescence was detected and the data were recorded using flow cytometry, and the data were analyzed using flowJo software.

[0105] like Fig.11 As shown, the Cy5 mRNA in the LNP group encapsulating Cy5 mRNA can be effectively internalized by cells, and the average fluorescence intensity of Cy5 is significantly enhanced. At each time point, the eFGP protein expression in the LNP group encapsulating EGFP mRNA is ahead of the commercial SM-102, and the endosomal escape effect is significantly enhanced. The prepared 10A LNP has a high level of endocytosis and endosomal escape, which significantly enhances the delivery effect of mRNA.

[0106] Test Example 4: Confocal laser scanning imaging of lipid nanoparticle endocytosis

[0107] 1. First, LNPs containing Cy5 mRNA were prepared. Ionizable cationic lipids, DSPC, cholesterol, and DMG-PEG2000 were prepared at a molar ratio of 60:5:22.5:0.5 according to the method of Example 2 to prepare lipid nanoparticles containing 0.5 μg Cy5 mRNA. The prepared lipid nanoparticles were recorded as LNPs. At the same time, a commercial formula was introduced, and lipid nanoparticles based on cationic lipid SM-102, cationic lipid, DSPC, cholesterol, and DMG-PEG2000 were prepared at a molar ratio of 50:10:38.5:1.5 as a control, recorded as SM102-LNP.

[0108] 2. Inoculate BHK cells in a glass bottom dish to a concentration of 1×10 4 / well, cultured for 12 hours at 37°C and 5% CO2, using RPMI 1640 medium containing 10% FBS. The original medium was then replaced with fresh medium, and the cells were treated with LNPs containing 0.5μg Cy5 mRNA. After 4 hours of co-incubation, the cell nuclei and lysosomes were stained. The cells were then observed and recorded using a confocal scanning laser microscope, and the microscope parameters remained unchanged during the experiment.

[0109] like Fig.12 As shown, the co-localization degree of LNP and lysosome was lower at 4 h, and mRNA had successfully escaped from the endosome.

[0110] Test Example 5: In vivo transfection experiment of lipid nanoparticles

[0111] The PBS solution of LNP (lipid nanoparticles prepared under the conditions of Test Example 2) encapsulating 1 μg Luc mRNA after dialyzation was injected into the body of mice by subcutaneous injection. After 6 hours, the luciferase substrate D-luciferin potassium salt (10 mg / mL, 200 μL) was injected intraperitoneally, and the fluorescence signal was observed by small animal in vivo imaging.

[0112] like Fig.13 As shown, we observed that at 6 hours, there was an obvious fluorescent signal in the mouse body, indicating that the lipid nanoparticles were successfully taken up by the injection site and surrounding cells. At the same time, the vector successfully delivered luciferase mRNA to the cytoplasm of the mouse and was successfully translated into a large amount of protein.

[0113] It can be seen that the above-mentioned lipid nanoparticles can effectively and efficiently deliver mRNA in animals and have potential clinical application value.

[0114] Application Example 1: Application of lipid nanoparticles in preventive tumor vaccines

[0115] 1. First, LNPs containing OVA mRNA / Luc mRNA were prepared. Ionizable cationic lipid 10A, DSPC, cholesterol, and DMG-PEG2000 were used at a molar ratio of 60:5:22.5:0.5. Lipid nanoparticles containing 5 μg OVA / Luc mRNA were prepared according to the method of Example 2. The prepared lipid nanoparticles were recorded as LNP(mOVA) / LNP(mLuc). At the same time, a commercial formula was introduced, and lipid nanoparticles based on cationic lipid SM-102, cationic lipid, DSPC, cholesterol, and DMG-PEG2000 were prepared at a molar ratio of 50:10:38.5:1.5 as a control, recorded as SM102-LNP(mOVA).

[0116] 2. On day 1, 800,000 B16-OVA tumor cells were implanted into mice by subcutaneous injection. On days 0, 7, and 14, lipid nanoparticles containing 5 μg OVA / Luc mRNA LNP and SM102-LNP prepared according to the above formula No. 13 were injected into mice by subcutaneous injection.

[0117] like Fig.14 As shown, LNP (mOVA) showed a better tumor prevention effect than SM102-LNP (mOVA). It can be seen that the lipid nanoparticles prepared above have excellent mRNA delivery efficiency and in vivo efficacy.

[0118] like Fig.15 As shown, mice treated with 10A LNP(mOVA) exhibited a longer survival period.

[0119] 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 cationic lipid, characterized in that The structural formula of the cationic lipid is shown in formula (I), Where n = 2-4; R1 and R2 are each independently selected from aliphatic alkyl groups having 8 to 14 carbon atoms.

2. The cationic lipid according to claim 1, wherein When R1 is a fatty alkyl group having 8, 10 or 12 carbon atoms, R2 is a fatty alkyl group having 10, 12 or 14 carbon atoms.

3. The cationic lipid according to claim 2, wherein The structural formula of the cationic lipid is shown in formula (Y1) or formula (Y2), 4. The method for preparing a cationic lipid as claimed in claim 1, wherein The following steps are involved: (1) a fatty alcohol containing two branches is subjected to a substitution reaction with acryloyl chloride to prepare a dibranched acrylate; (2) The dibranched acrylate is subjected to Michael addition reaction with N-(3-aminopropyl)diethanolamine or N,N-bis(2-hydroxyethyl)ethylenediamine, and the cationic lipid is obtained after separation and purification.

5. The preparation method according to claim 4, characterized in that: In step (1), the reaction conditions are: stirring at 0°C for 4-6 hours; in step (2), the Michael addition reaction conditions are: stirring at 30°C-80°C for 12-48 hours.

6. A lipid nanoparticle, characterized in that: The raw materials thereof include: the cationic lipids, auxiliary lipids and cholesterol as described in any one of claims 1 to 3, and the raw materials form lipid nanoparticles through self-assembly.

7. The lipid nanoparticle according to claim 6, characterized in that The auxiliary lipids include phospholipids and polyethylene glycol functionalized lipids; the molar ratio of the cationic lipids, phospholipids, cholesterol and polyethylene glycol functionalized lipids is 20-70: 5-25: 15-55: 0.5-3.

5.

8. Use of the lipid nanoparticles as claimed in claim 6 or 7 as carriers in the preparation and delivery of nucleic acid drugs.

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

10. 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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