A novel cationic lipid and its use in the preparation of an mRNA delivery drug

Abstract

The application discloses a kind of cationic lipids and its application in the preparation of mRNA delivery drug, belong to the biomedicine technical field.The structure formula of the cationic lipid is as shown in formula (I), with four branched chain fatty alkyl tail of hydroxyl head.Based on the cationic lipid preparation lipid nanoparticle, due to the existence of cationic lipid head hydroxyl, its pKa is in the specific interval of producing optimal transfection effect;Four branched chain fatty alkyl can form larger conical hydrophobic tail, can effectively enhance the endosome membrane damage ability of LNP in endosome escape process, improve intracellular endocytosis and endosome escape efficiency.Compared with commercially available ionizable lipids SM-102 and its formula, homologous cationic lipids and its formula, the lipid nanoparticle provided by the application has more excellent intracellular endocytosis, endosome escape efficiency, and then mediates higher mRNA delivery efficiency, effectively solves the problem of current low mRNA delivery efficiency.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a novel cationic lipid, its preparation method, and its application in the preparation of mRNA delivery drugs. Background Technology

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

[0003] Lipid nanoparticles (LNPs) based on ionizable cationic lipids are currently the mainstream mRNA delivery system due to their good biocompatibility and high delivery efficiency. LNPs typically consist of four components: ionizable cationic lipids, cholesterol, phospholipids, and PEG-modified lipids. The ionizable cationic lipids are the key component, usually composed of an amino head, a hydrophobic tail, and a linker bond. Changes in the cationic lipid structure significantly affect the delivery efficiency of LNPs. Currently, even the most efficient LNPs can only deliver ~4% of mRNA into 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-delivered drugs.

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

[0005] Since cationic lipids play a decisive role in mRNA delivery, in-depth research on the structure-activity relationship of cationic lipids and the development of efficient and safe cationic lipids, which can effectively enhance mRNA endocytosis and endosome escape after forming lipid nanoparticles and achieve efficient mRNA delivery in vivo, have important clinical significance. Summary of the Invention

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

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides a cationic lipid with the structural formula shown in formula (I).

[0009]

[0010] Where n = 2 - 4;

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

[0012] The cationic lipids provided by this invention can self-assemble with auxiliary lipids and cholesterol to form lipid nanoparticles. The tertiary amine groups of the cationic lipids form positively charged hydrophilic ends in the buffer solution, which can bind negatively charged drugs through electrostatic interactions. Due to the supramolecular forces of hydrophilicity and hydrophobicity, the lipid material self-assembles to form lipid nanoparticles encapsulating negatively charged drugs such as mRNA.

[0013] This invention demonstrates that lipid nanoparticles formed by the self-assembly of cationic lipids obtained through structural screening and other raw materials can achieve efficient drug delivery by enhancing endocytosis and endosome escape.

[0014] Studies have shown that the enhanced delivery efficiency is closely related to the presence of a four-branched aliphatic alkyl group in the cationic lipid molecule. Compared to lipid molecules with two branched aliphatic alkyl groups, lipid molecules with four branched aliphatic alkyl groups have a smaller head and a larger conical hydrophobic tail. When they bind to the membrane, they disrupt a larger area of ​​local membrane phospholipid organization, thereby generating a greater entropy stimulus to the interaction. This structure facilitates the transformation of the membrane into a hexagonal phase during endosome escape, disrupting the endosome membrane and thus enhancing endocytosis and endosome escape, ultimately significantly improving the delivery efficiency of drugs such as mRNA.

[0015] Studies have shown that the significant enhancement in delivery efficiency is also closely related to the presence of hydroxyl groups in the head. After forming lipid nanoparticles, cationic lipids with hydroxyl groups in the head have a pKa between 6 and 7, which is the most favorable range for LNP transfection.

[0016] Preferably, R1 is an aliphatic alkyl group with 8 carbon atoms and R2 is an aliphatic alkyl group with 10 carbon atoms; or R1 is an aliphatic alkyl group with 10 carbon atoms and R2 is an aliphatic alkyl group with 12 carbon atoms; or R1 is an aliphatic alkyl group with 12 carbon atoms and R2 is an aliphatic alkyl group with 14 carbon atoms.

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

[0018] Further preferred options include cationic lipids with the structural formulas shown in formula (Y1) or (Y2). Studies have shown that the Y1 and Y2 cationic lipids exhibit high mRNA delivery efficiency in in vitro transfection, significantly superior to the commercially available SM-102. Among them, the Y1 lipid, after formulation optimization, significantly enhances endocytosis and endosome escape, demonstrating superior mRNA delivery efficiency at both in vitro and in vivo levels, showing a significantly enhanced transfection efficiency compared to the SM-102 lipid.

[0019]

[0020] The present invention also provides a method for preparing the above-mentioned 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 bibranched acrylate.

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

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

[0024] Preferably, the fatty alcohol can be, but is not limited to, 2-octyldodecyl alcohol, 2-decyl-1-tetradecyl alcohol, or 2-dodecylhexadecyl-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 can be, but is not limited to, dichloromethane.

[0027] As a preferred option, the reaction conditions are: stirring at 0°C for 4-6 hours;

[0028] In step (2), the dibranched acrylate undergoes a Michael addition reaction with the amine to form a positively charged head. Specifically, the dibranched acrylate and the amine are dissolved in an organic solvent and reacted under stirring at 30°C-80°C. After the reaction is complete, the solvent is removed by rotary evaporation, and the crude product is separated and purified by silica gel chromatography to obtain the cationic lipid.

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

[0030] Preferably, the Michael addition reaction conditions are: stirring at 30℃-80℃ for 12-48 h. More preferably, stirring at 50℃ for 24 h.

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

[0032] The preparation methods that can be used in this invention are not limited to this.

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

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

[0035] The auxiliary lipids include phospholipids and polyethylene glycol-functionalized lipids; preferably, the phospholipids may be, but are not limited to, distearylphosphatidylcholine (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 lipids, phospholipids, cholesterol, and polyethylene glycol-functionalized lipids 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. Even 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 lipids provided by this invention can efficiently deliver nucleic acid drugs such as mRNA by enhancing endocytosis and endosome escape, and have potential application value in nucleic acid drug development.

[0038] The present invention also provides the application 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 to an acidic buffer containing nucleic acids, and self-assembling to form lipid nanoparticles carrying nucleic acids, thereby preparing the nucleic acid delivery drug.

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

[0041] One method involves dissolving ionizable cationic lipids, auxiliary lipids, and cholesterol in a specific ratio in an appropriate amount of ethanol. The ethanol solution containing the lipid material is then injected into a buffer solution containing nucleic acid drugs, where they self-assemble to form nanoparticles. The ethanol is then 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. Since mRNA molecules are relatively large, insufficient lipid material makes it difficult to effectively encapsulate and protect them, thus reducing transfection efficiency. Conversely, excessive lipid material may reduce endosome escape efficiency, further decreasing transfection efficiency. Within a suitable mass ratio range, good encapsulation and transfection rates can be ensured. More preferably, the total mass ratio of lipid material to mRNA is 40:1.

[0044] The beneficial effects of this invention are as follows:

[0045] This invention provides a novel cationic lipid for the preparation of lipid nanoparticles. The cationic lipid possesses a hydroxyl head and a four-branched aliphatic alkyl tail. Lipid nanoparticles prepared based on this cationic lipid, due to the presence of the hydroxyl head, have a pKa within a specific range that produces optimal transfection efficiency. Furthermore, compared to bibranched aliphatic alkyl groups, the four-branched aliphatic alkyl group can form a larger conical hydrophobic tail, effectively enhancing the endosome membrane disruption ability of LNPs during endosome escape, improving endocytosis and endosome escape efficiency, and thus efficiently delivering mRNA and other nucleic acid drugs in vitro and in vivo. Compared to commercially available ionizable lipid SM-102 and its formulation, and homologue cationic lipids and their formulations, the lipid nanoparticles provided by this invention exhibit superior endocytosis and endosome escape efficiency, thereby mediating higher mRNA delivery efficiency and effectively solving the current problem of low mRNA delivery efficiency. Attached Figure Description

[0046] Figure 1 The NMR spectrum of cationic lipid 10A prepared in Example 1.

[0047] Figure 2 The NMR spectrum of the cationic lipid 10B prepared in Example 1.

[0048] Figure 3 The NMR spectrum of cationic lipid 1B prepared in Example 1.

[0049] Figure 4 The NMR spectrum of the cationic lipid 3B prepared in Example 1.

[0050] Figure 5 The NMR spectrum of cationic lipid 5A prepared in Example 1.

[0051] Figure 6 The NMR spectrum of cationic lipid 6A prepared in Example 1.

[0052] Figure 7 This is a 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 in commercial formulations.

[0054] Figure 9 The results show the effects of lipid nanoparticle cell transfection.

[0055] Figure 10 The results are the quantitative analysis results of lipid nanoparticle cell transfection.

[0056] Figure 11 To determine the endocytosis and endosome escape of lipid nanoparticles by flow cytometry.

[0057] Figure 12 The results show the endosome escape of lipid nanoparticles as characterized by laser confocal microscopy.

[0058] Figure 13 Image and quantitative analysis of in vivo transfection effect of lipid nanoparticles.

[0059] Figure 14 A comparative graph showing the anti-tumor effects of prophylactic vaccines applied in animal models.

[0060] Figure 15 Survival results of animal models used for preventative vaccine application. Detailed Implementation

[0061] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0063] The compounds and their abbreviations used in the examples are explained below:

[0064] CDCl2: Dichloromethane, CAS No.: 75-09-2;

[0065] EtOH: Ethanol, CAS No.: 64-17-5;

[0066] Triethylamine, CAS No.: 121-44-8;

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

[0068] N,N-bis(2-hydroxyethyl)ethylenediamine, CAS No.: 3197-06-6;

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

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

[0071] 2-Dodecylhexadec-1-ol, CAS No.: 72388-18-2;

[0072] D-Insect Luciferin Potassium Salt, CAS No.: 115144-35-9;

[0073] DSPC phospholipid, CAS No.: 816-94-4, has the following structural formula:

[0074]

[0075] DMG-PEG2000 lipid, CAS No.: 160743-62-4, structural formula as follows:

[0076]

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

[0078]

[0079] Example 1: Preparation of cationic lipids

[0080] To screen for cationic lipids with the best delivery efficiency, we used the Michael addition reaction method to modularly synthesize 42 cationic lipids with different structures by combining different amine heads and branched tails of different lengths (length A < B < C).

[0081] 1. The specific steps for preparing cationic lipids are as follows:

[0082] First, 2-octyldodecyl alcohol (10.00 g, 33.5 mmol) was dissolved in 40 mL of DCM. Three molar amounts of triethylamine (10.17 g, 100.5 mmol) were added under stirring in an ice bath at 0 °C. Then, three molar amounts of acryloyl chloride (9.09 g, 100.5 mmol) were slowly added. After stirring for 4 h, the solvent was removed by rotary evaporation, and the crude product was separated. The crude product was purified by silica gel chromatography (product eluent: DCM) and then dried under vacuum to obtain di-tailed acrylate.

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

[0084]

[0085] like Figure 1 As shown, the nuclear magnetic resonance (NMR) spectrum of the cationic lipid is as follows: 1H NMR (600MHz, Chloroform-d) δ 3.97 (d, J = 5.8Hz, 4H), 3.72 (q, J = 7.0Hz, 2H), 3.63 (t, J = 5.2Hz, 4H), 2.78 (t, J = 7.3Hz, 4H), 2.64 (t, J = 5.2Hz, 4H), 2.59 (t, J = 6.8Hz, 2H), 2.50 (t, J = 6.7Hz, 2H), 2.46 (t, J = 7.3Hz, 4H), 1.63 (dt, J = 14.4, 6.9Hz, 4H), 1.27 (d, J = 13.1Hz, 68H), 0.88 (t, J = 6.9Hz, 12H), which is consistent with the peaks 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. Replacing 2-octyldodecyl alcohol (10.00 g, 33.5 mmol) in the above preparation method with 2-decyl-1-tetradecyl alcohol and 2-dodecylhexadecyl-1-ol, respectively, the ionizable cationic lipids 10B and 10C were prepared, and their structural formulas are shown below:

[0087]

[0088] 3. Replace 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-dipropylamine, N,N-bis(2-hydroxyethyl)ethylenediamine, N-(2-aminoethyl)pyrrole, and 1-(2-aminoethyl)piperidine, respectively. 0.5 mmol each of N-(2-aminoethyl)morpholine and 4-methylmorpholine-2-methylamine 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.

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

[0090] Example 2: Preparation of lipid nanoparticles

[0091] This embodiment prepares a novel cationic lipid nanoparticle based on the cationic lipid prepared in Example 1. The specific steps are as follows:

[0092] (1) The cationic lipids, 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 and a total mass of 80 micrograms. Under vortex conditions, the ethanol solution was rapidly injected into 50 μL of 20 mM sodium acetate buffer containing 2 μg luciferase mRNA (purchased from Kaituo Biotechnology), 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 prepared in (1) was dialyzed with 10mM pH neutral PBS solution (dialysis bag Mw=100kDa) for 2-4 hours to remove ethanol and obtain the final product.

[0094] like Figure 7 As shown, transmission electron microscopy (TEM) revealed that the particle size of the LNPs nanomaterials prepared using cationic lipid 10A in this embodiment was approximately 210 nm, consistent with the DLS results.

[0095] Test Example 1: Screening of Cationic Lipid Structures

[0096] BHK cells were seeded in 48-well plates (5 × 10⁶ cells per well). 4 Cells / well were cultured in RPMI-1640 medium containing 10% FBS for 12 hours. Using the method in Example 2, lipid nanoparticles containing 1 μg of Luc mRNA were prepared from 42 different cationic lipids. After co-incubating the lipid nanoparticles with cells for 24 hours, Luc expression was detected using a firefly Luc reporter gene assay kit according to the manufacturer's instructions. Fluorescence expression was detected using a microplate reader to evaluate the mRNA delivery efficiency of different lipid nanoparticles, thereby screening for cationic lipids with the best in vitro transfection effect.

[0097] The results are as follows Figure 8 As shown, lipid nanoparticles prepared from cationic lipids of series 9 and 10 have good transfection efficiency, with 10A exhibiting the best transfection efficiency. All subsequent tests used this cationic lipid.

[0098] Test Example 2: Cell Transfection Experiment with Lipid Nanoparticles

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

[0100] 2. Seed BHK / HeLa cells in 48-well plates (1×10⁻⁶ cells / well). 4Cells (cells / well) were cultured in RPMI 1640 / DMEM medium at 37°C and 5% CO2 for 12 hours in untreated and +10% FPS conditions (+10% FPS was used to observe the transfection effect in the presence of serum). The original medium was replaced with fresh medium, and cells were treated with LNPs containing 2 μg eGFP mRNA. After co-incubation for 24 hours, transfection was observed and recorded using a fluorescence microscope. Cells were then washed with 100 μL PBS, digested with 100 μL trypsin for 3 minutes, and collected by centrifugation at 1000 rpm for 5 minutes. Cells were resuspended in 0.5 mL PBS and filtered through a nylon mesh (45 μm). Fluorescence was measured by flow cytometry, and data were recorded and analyzed using FlowJo software.

[0101] like Figure 9 , Figure 10 As shown, under the green fluorescence channel, compared with the commercial SM102-LNP group, the LNP group showed a stronger green fluorescence signal within 24 hours, indicating that the group expressed more green fluorescent protein. This suggests that the material can better promote mRNA transfection into the cytoplasm for translation into protein and exert its effect.

[0102] Test Example 3: Cellular Endocytosis and Endosome Escape Experiment of Lipid Nanoparticles

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

[0104] 2. BHK cells were seeded into 48-well plates to reach 5 × 10⁶ cells / well. 4Cells were cultured in RPMI 1640 medium at 37°C and 5% CO2 for 12 h. The medium was then replaced with fresh medium, and cells were treated with LNPs containing 1 μg Cy5 / EGFP mRNA. After co-incubation for 0.5 h, 1 h, 2 h, and 4 h, cells were washed with 100 μL PBS, digested with 100 μL trypsin for 3 min, and then centrifuged at 1000 rpm for 5 min to collect cells. Cells were resuspended in 0.5 mL PBS, filtered through a 45 μm nylon mesh, and fluorescence was detected and recorded by flow cytometry. Data were analyzed using FlowJo software.

[0105] like Figure 11 As shown, the Cy5 mRNA encapsulated in the LNP group was effectively internalized by cells, and the average fluorescence intensity of Cy5 was significantly enhanced. At all time points, the eFGP protein expression in the LNP group encapsulated in EGFP mRNA was superior to that of commercial SM-102, and the endosome escape effect was significantly enhanced. The prepared 10A LNPs exhibited high levels of endocytosis and endosome escape, significantly enhancing the mRNA delivery effect.

[0106] Test Example 4: Laser confocal evaluation of lipid nanoparticle endocytosis

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

[0108] 2. Seed BHK cells in glass dishes to reach a density of 1×10⁶ cells / mL. 4 Cells were cultured in RPMI 1640 medium containing 10% FBS at 37°C and 5% CO2 for 12 h. The medium was then replaced with fresh medium, and cells were treated with LNPs containing 0.5 μg Cy5 mRNA. After co-incubation for 4 h, cell nuclei and lysosomes were stained. Cells were then observed and recorded using a confocal scanning laser microscope, with microscope parameters remaining constant throughout the experiment.

[0109] like Figure 12 As shown, at 4h, the colocalization of LNP with lysosomes was even lower, indicating that the mRNA had successfully escaped from the endosome.

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

[0111] The mice were injected subcutaneously with PBS solution containing 1 μg of Luc mRNA-encapsulated LNP (lipid nanoparticles prepared under the formulation conditions of Test Example 2). Six hours later, the mice were injected intraperitoneally with luciferase substrate D-fluorescein potassium salt (10 mg / mL, 200 μL), and the fluorescence signal was observed using a small animal in vivo imaging system.

[0112] like Figure 13 As shown, we observed a significant fluorescent signal in the mice at 6 hours, indicating that the lipid nanoparticles were successfully taken up by the cells at the injection site and in the surrounding area. At the same time, the vector successfully delivered luciferase mRNA into the cytoplasm of the mice and successfully translated it into a large amount of protein.

[0113] It is evident that the aforementioned 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 mixed in a molar ratio of 60:5:22.5:0.5, and lipid nanoparticles containing 5 μg of OVA / Luc mRNA were prepared according to the method in Example 2. The prepared lipid nanoparticles were denoted as LNP(mOVA) / LNP(mLuc). Simultaneously, a commercially available formulation was introduced. Lipid nanoparticles prepared based on cationic lipid SM-102, and cationic lipid, DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 50:10:38.5:1.5 were used as a control and denoted as SM102-LNP(mOVA).

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

[0117] like Figure 14 As shown, LNP(mOVA) exhibits superior tumor prevention efficacy compared to SM102-LNP(mOVA). This demonstrates that the lipid nanoparticles prepared above possess excellent mRNA delivery efficiency and in vivo therapeutic effects.

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

[0119] 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. The application of lipid nanoparticles as carriers in the preparation and delivery of nucleic acid drugs, characterized in that, The raw material composition of the lipid nanoparticles includes: cationic lipids, phospholipids, cholesterol, and polyethylene glycol-functionalized lipids, with a molar ratio of 60:5:22.5:0.5; the raw materials form lipid nanoparticles through self-assembly. The structural formula of the cationic lipid is shown in formula (Y1). (Y1)。 2. The application as described in claim 1, characterized in that, The method for preparing the cationic lipid includes the following steps: (1) A fatty alcohol containing two branches is subjected to a substitution reaction with acryloyl chloride to prepare a bibranched acrylate. (2) The dibranched acrylate was subjected to a Michael addition reaction with N-(3-aminopropyl)diethanolamine, and after separation and purification, the cationic lipid was obtained.

3. The application as described in claim 2, characterized in that, In step (1), the reaction conditions are: stirring at 0℃ for 4-6 h; in step (2), the Michael addition reaction conditions are: stirring at 30℃-80℃ for 12-48 h.

4. The application as described in claim 1, characterized in that, The phospholipid is distearylphosphatidylcholine or dioleoylphosphatidylethanolamine, and the polyethylene glycol functionalized lipid is dimyristylglycerol-polyethylene glycol 2000.

5. The application as described in claim 1, characterized in that, The application includes: adding cationic lipids, phospholipids, polyethylene glycol-functionalized lipids, and cholesterol to an acidic buffer containing nucleic acids, and self-assembling to form lipid nanoparticles carrying nucleic acids, thereby preparing the nucleic acid delivery drug.

6. The application as described in claim 5, 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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