A multi-tailed ionizable lipid, its preparation method and application

Abstract

This invention discloses a multi-tailed ionizable lipid, its preparation method, and its applications. The structural formula of the multi-tailed ionizable lipid of this invention is as follows: [Structure formula would be inserted here]. Wherein, R1 and R2 may be the same or different, each being a hydrogen atom or an alkyl chain or alkyl ring consisting of 1-6 carbons, or R1 and R2 together form a nitrogen-containing alkyl ring; L1 and L2 may be the same or different, each being an alkyl chain or unsaturated hydrocarbon group with a length of 1-6 carbons; R is an alkyl group, alkyl ring, unsaturated hydrocarbon group, or heteroalkyl group; n = 1-6; m1 = 1-15, m2 = 1-15; x = 0-5. The multi-tailed ionizable lipid prepared by this invention has a simple synthesis method, uses readily available and common raw materials, and has a reasonable route design. Ionizable lipids can be prepared in large quantities through several addition reactions, facilitating high-throughput material screening. The obtained ionizable lipids can effectively express RNA in vitro and in vivo, exhibiting advantages such as high efficiency and low toxicity.

Description

Technical Field

[0001] This invention belongs to the field of drug carrier technology, specifically relating to a multi-tailed ionizable lipid, its preparation method, and its application. Background Technology

[0002] RNA therapy, primarily comprising antisense oligonucleotides (ASOs), small interfering RNA (siRNA), small RNA (miRNA), messenger RNA (mRNA), and circular RNA (circRNA), shows great promise in treating a wide range of diseases by manipulating different modes of action. However, due to the inherent negative charge and instability of RNA molecules, RNA struggles to overcome biological barriers and reach the cytoplasm. To overcome this problem, RNA requires safe, efficient, and stable delivery systems to protect nucleic acids from degradation and accelerate cellular uptake and efficient RNA release. Lipid nanoparticles (LNPs) are a delivery system that has successfully entered clinical trials, particularly LNPs-mRNA vaccines, which are currently used clinically to treat COVID-19, marking a significant milestone for LNP delivery systems. Furthermore, DNA, as an earlier-developed nucleic acid drug, also requires efficient delivery systems. Currently, some DNA vaccines have been approved for veterinary use, such as those for West Nile virus in horses and canine melanoma.

[0003] Cancer vaccines generally fall into four categories: tumor or immune cell vaccines, peptide vaccines, viral vector vaccines, and nucleic acid vaccines. Nucleic acid-based vaccines (DNA or RNA vaccines) are a promising candidate. First, nucleic acid vaccines can simultaneously deliver multiple antigens, such as tumor-associated antigens (TAAs) or somatic tumor mutations, triggering humoral and cellular immunity and reducing vaccine resistance. Second, unlike peptide vaccines, nucleic acid vaccines allow APCs to simultaneously or cross-present multiple epitopes of type I and II patient-specific human leukocyte antigens, thus being less restricted by human HLA types and more likely to stimulate a broader T-cell response. Finally, nucleic acid vaccines are non-infectious and are not contaminated with proteins or viruses during production, therefore they are considered to have good tolerability in both preventative and therapeutic applications. Meanwhile, lipid nanoparticles are also a key support carrier for cancer immunotherapy, playing an important role in infectious disease vaccines, cancer vaccines, and small molecule drug delivery—areas vital to national welfare and public health. Therefore, in-depth research on lipid delivery carriers has both significant scientific value and promising application prospects.

[0004] Nucleic acid delivery systems can be broadly categorized into two types: viral vectors and non-viral vectors. Viral vectors offer relatively high transfection efficiency but suffer from drawbacks such as poor safety and targeting. Over the past few decades, liposomes, representing non-viral vectors, have seen rapid development. A novel type of lipid—ionizable lipid—has been developed. This lipid can be protonated at a weakly acidic pH, becoming positively charged, while remaining neutral at physiological pH. The pH sensitivity of ionizable lipids is beneficial for in vivo mRNA delivery because neutral lipids interact less with the anion exchange membranes of blood cells, thus improving the biocompatibility of nanoparticles. When lipid nanoparticles are within the endosomes at a weakly acidic pH, ionizable lipids acquire a charge, promoting membrane instability and increasing endosome escape. Compared to traditional cationic liposomes, ionizable lipids exhibit significantly improved stability and transfection efficiency in vivo, remain electrically neutral during in vivo transport, and have low biotoxicity. This invention attempts to synthesize a novel, safe, and efficient class of ionizable lipids to address the aforementioned problems in nucleic acid delivery.

[0005] The inherent negative charge and instability of RNA molecules make them difficult to penetrate cells. To deliver RNA molecules to target cells, a safe, efficient, and stable delivery system is needed to protect nucleic acids from degradation and ensure the effective release of RNA molecules. Among different types of delivery systems, lipid nanoparticles have been extensively studied due to their unique properties, such as the simple chemical synthesis of lipids, the scalability of LNP processes, and strong encapsulation capacity. However, traditional nucleic acid delivery systems suffer from low efficiency, high toxicity, and poor targeting (Y. Zhang, C. Sun, C. Wang, K.E. Jankovic, Y. Dong, Lipids and Lipid Derivatives for RNA Delivery, Chem. Rev., 2021, 121, 12181-12277.). The purpose of this invention is to provide a method for preparing and using a class of multi-tailed ionizable lipids. These lipid nanoparticles can efficiently deliver mRNA, circRNA, pDNA, and siRNA within mammalian cells, specifically silencing targeted gene expression. Once a lipid carrier reaches the intracellular environment via endocytosis, achieving rapid endosome escape is a major challenge that efficient delivery systems must address. The multi-tailed ionizable lipids of this invention have 2-4 more tail groups than dual-tailed lipids. Due to the increased cross-sectional area of ​​the tail region, these lipids exhibit a more conical structure, resulting in stronger endosome disruption capabilities and enhanced delivery efficiency. The synthesis strategy for multi-tailed ionizable lipids involves rapid synthesis via orthogonal reactions, enabling the rapid synthesis of a lipid library containing numerous lipid compounds. Delivery efficiency is then determined through high-throughput cell screening. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a multi-tailed ionizable lipid, its preparation method, and its applications. This multi-tailed lipid is an ionizable lipid. The head group of this ionizable lipid is a tertiary or secondary amine group, which can acquire protons under acidic pH, thus carrying a positive charge. It can then bind to negatively charged nucleic acid molecules or small molecule drugs through electrostatic interactions, and further self-assemble with auxiliary lipids to form lipid nanoparticles, thereby delivering gene drugs. Based on the low efficiency and high toxicity encountered in current gene drug delivery methods, this multi-tailed ionizable lipid balances degradability and lipid safety while maintaining overall delivery efficiency in its chemical structure design. The chemical structure of this multi-tailed ionizable lipid contains three components: (i) an ionizable head group, (ii) a linker group, and (iii) a hydrophobic tail. Unlike the demanding and complex synthetic routes of traditional cationic lipids, the multi-tailed ionizable lipid provided by this invention has a simple chemical skeleton, a simple synthetic route, and a clear reaction mechanism. An ionizable lipid library can be obtained through Michael addition, facilitating high-throughput screening.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A multi-tailed ionizable lipid, with the following structural formula:

[0009]

[0010] Wherein, R1 and R2 may be the same or different, each being a hydrogen or an alkyl chain or alkyl ring composed of 1 to 6 carbons, or R1 and R2 together form a nitrogen-containing alkyl ring; L1 and L2 may be the same or different, each being an alkyl chain or unsaturated hydrocarbon group composed of 1 to 6 carbons; R is an alkyl, alkyl ring, unsaturated hydrocarbon group or heterohydrocarbon group; n = 1 to 6; m1 = 1 to 15, m2 = 1 to 15; x = 0 to 5.

[0011] Preferably, the structural formula of the multi-tailed ionizable lipid includes the structural formulas listed in the examples and the following structural formulas:

[0012]

[0013] The above-mentioned method for preparing multi-tailed ionizable lipids involves obtaining organic amine compounds and branched tail compounds via a Michael addition reaction.

[0014] The structure of the branched tail compound is as follows:

[0015]

[0016] Wherein, R is an alkyl group, alkyl ring, unsaturated hydrocarbon group or heterohydrocarbon group; n = 1 to 6; m1 = 1 to 15, m2 = 1 to 15; x = 0 to 5;

[0017] The organic amine compound contains at least one amino group.

[0018] Preferably, the organic amine compound is one of the following compounds:

[0019]

[0020] Preferably, the branched tail compound is obtained by esterification of chlorinated acrylate with compound 1;

[0021] The structure of compound 1 is as follows:

[0022]

[0023] Wherein, R is an alkyl group, alkyl ring, unsaturated hydrocarbon group or heterohydrocarbon group; n = 1 to 6; m1 = 1 to 15, m2 = 1 to 15; x = 0 to 5.

[0024] The above-mentioned multi-tailed ionizable lipids are used in the preparation of drug carriers.

[0025] Preferably, the active ingredient of the drug includes nucleic acid molecules and protein drugs.

[0026] More preferably, the nucleic acid molecules include siRNA, miRNA, mRNA, circRNA, antisense RNA, CRISPR guide RNAs, replicable RNA, cyclic dinucleotides, polyIC, CpG ODN, plasmid DNA, and microcircular DNA; the protein drugs include colony-stimulating factors, interleukins, lymphotoxins, interferon proteins, tumor necrosis factor, antibodies, and protein antigens.

[0027] Preferably, the method for preparing the drug carrier includes the following steps:

[0028] (a) The multi-tailed ionizable lipids are mixed with cholesterol or cholesterol derivatives (β-sitosterol, etc.), auxiliary lipids, and polyethylene glycol-modified lipids in an ethanol solution to prepare a lipid mixture solution; the drug is mixed with an acidic buffer solution, and then mixed with the lipid mixture solution; the mixture is incubated at room temperature for 15 min to 1 h, and then diluted with PBS or dialyzed to obtain the drug carrier.

[0029] Alternatively (b) dissolve the multi-tailed ionizable lipids and cholesterol or cholesterol derivatives in chloroform, dry with nitrogen to evaporate the solvent, add acidic or neutral buffer solution, and sonicate for 1-20 min to prepare liposome nanoparticles for later use; mix protamine with the drug, then mix with the liposome nanoparticles, let stand for 5-30 min, add polyethylene glycol modified lipids, and place at 30-65°C for 5-20 min to obtain the drug carrier.

[0030] More preferably, in step (a), the molar ratio of the multi-tailed ionizable lipid to cholesterol or cholesterol derivatives, auxiliary lipids, and polyethylene glycol-modified lipids is 10–100:0–90:0–90:0–90; and the nitrogen-to-phosphorus ratio of the protonable amino group to the nucleic acid drug in the multi-tailed ionizable lipid is 1–100:1.

[0031] The auxiliary lipids mentioned in step (a) include at least one of the following: egg yolk lecithin, hydrogenated egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, sphingomyelin, phosphatidylethanolamine, myristoyl phosphatidylcholine, myristoyl phosphatidylglycerol, dipalmitoyl phosphatidylcholine, distearate phosphatidylcholine, dioleoyl phosphatidylethanolamine, dioleoyl lecithin, dioleoyl phosphatidylcholine, and dilauroyl phosphatidylcholine;

[0032] More preferably, the molar ratio of the multi-tailed ionizable lipid to cholesterol or cholesterol derivatives in step (b) is 1:5 to 5:1; and the mass ratio of the multi-tailed ionizable lipid to the drug is 1 to 100:1.

[0033] More preferably, the polyethylene glycol-modified lipids described in steps (a) and (b) include at least one of DSPE-PEG, C14-PEG, DMG-PEG, ALC-0159, DSPE-PEG-Maleimide, DSPE-PEG-COOH, DSPE-PEG-NH2, and chemically modified products.

[0034] More preferably, the acidic buffer solution in step (a) has a pH of 3 to 7; the acidic buffer solution is sodium acetate or sodium citrate buffer solution;

[0035] More preferably, the acidic or neutral buffer solution in step (b) has a pH of 3 to 7; the acidic or neutral buffer solution is sodium citrate, sodium acetate buffer, or DEPC water.

[0036] Compared with the prior art, the beneficial effects of the present invention are:

[0037] (1) The method for synthesizing multi-tailed ionizable lipids prepared by this invention is simple, the raw materials are common and readily available, and the route design is reasonable. Ionizable lipids can be prepared in large quantities through several addition reactions, which is convenient for high-throughput material screening; the obtained ionizable lipids can effectively express RNA in vivo and in vitro, and have the advantages of high efficiency and low toxicity.

[0038] (2) The chemical backbone of the multi-tailed ionizable lipid prepared in this invention contains a large number of ester bonds. After effectively releasing RNA in vivo, it can be rapidly hydrolyzed by enzymes, is easily metabolized and cleared in vivo, and has biodegradability. Its tail structure contains branched alkyl groups, which can increase the cross-sectional area of ​​the lipid tail, help RNA and other drugs escape from the endosomes, and thus enhance the transfection effect. The charge of the ionizable lipid can change with the pH of the environment. Under physiological conditions, it is electrically neutral, which reduces the cytotoxicity caused by excessive positive charge, thereby increasing the stability of lipid nanoparticles and helping to prolong the circulation time of the loaded nucleic acid drugs and improve the pharmacokinetic characteristics. Attached Figure Description

[0039] Figure 1 The hydrogen spectrum of target product B prepared in Example 2.

[0040] Figure 2 The hydrogen spectrum of the target product C prepared in Example 3.

[0041] Figure 3 The hydrogen spectrum of the ionizable lipid 3-5-C2C6 prepared in Example 4.

[0042] Figure 4 The hydrogen spectrum of the target product D prepared in Example 5.

[0043] Figure 5 The hydrogen spectrum of the target product E prepared in Example 6.

[0044] Figure 6 The hydrogen spectrum of the target product F prepared in Example 7.

[0045] Figure 7 The hydrogen spectrum of the ionizable lipid 3-5-CA prepared in Example 8.

[0046] Figure 8 The hydrogen spectrum of the target product H prepared in Example 10.

[0047] Figure 9 The hydrogen spectrum of target product I prepared in Example 11.

[0048] Figure 10 The hydrogen spectrum of the ionizable lipid 8-5-C8C10 prepared in Example 12.

[0049] Figure 11 The hydrogen spectrum of the ionizable lipid 14-5-C8C10 prepared in Example 13.

[0050] Figure 12 The graph shows the relative luciferase activity results of the lipid nanoparticles from Example 14 after cell transfection.

[0051] Figure 13 The graph shows the relative luciferase activity results of cell transfection with lipid nanoparticles of different neutral phospholipids in Example 15.

[0052] Figure 14 The graph shows the relative luciferase activity results of cell transfection with lipid nanoparticles of different component ratios in Example 16.

[0053] Figure 15 The graph shows the relative luciferase activity results of cell transfection with lipid nanoparticles of different nitrogen-phosphorus ratios in Example 17.

[0054] Figure 16 The graph shows the relative luciferase activity results of lipid nanoparticles with different buffer solutions for cell transfection in Example 18.

[0055] Figure 17 The graph shows the results of luciferase activity in mice after transfection of the lipid nanoparticles of Example 19.

[0056] Figure 18 This is an image of the lipid nanoparticles from Example 19 transfected in mice.

[0057] Figure 19 The graph shows the relative luciferase activity results of the lipid nanoparticles from Example 20 after cell transfection.

[0058] Figure 20 The graph shows the results of luciferase activity in mice after transfection with the lipid nanoparticles of Example 21. Detailed Implementation

[0059] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0060] The method for preparing the multi-tailed ionizable lipid of the present invention includes the following steps:

[0061] (1) Synthetic hydrophobic tail

[0062] The specific steps are as follows: In a 50 mL reaction tube, add 5 mmol of alkyl alcohol, 15 mL of N,N'-carbonyldiimidazole, 10 mmol of triethylamine (TEA), 20 mL of dichloromethane (DCM), and a magnetic stir bar sequentially. Place the reaction tube in a heating mantle at 40°C and react for 24 h until the reaction is complete. Transfer the reaction mixture to a separatory funnel, add DCM (2 x 100 mL) and saturated saline (2 x 100 mL) for extraction, and wash with 1 M HCl (2 x 20 mL). Collect the organic layer, dry it with anhydrous magnesium sulfate, and filter. The product obtained does not require further purification and can be used for the next reaction.

[0063] Add 5 mmol of the product from the previous step, 10 mmol of amino alcohol, and 20 mL of DCM to a 50 mL reaction tube equipped with a magnetic stir bar. Place the reaction tube in a heating mantle at 40 °C and react for 24 h. After the reaction has cooled to room temperature, transfer the reaction mixture to a separatory funnel, add DCM (2 x 100 mL) and saturated saline (2 x 100 mL) for extraction, and wash with 1 M HCl (2 x 20 mL). Collect the organic layer, dry it with anhydrous magnesium sulfate, and filter it. Then remove the organic solvent using a rotary evaporator under reduced pressure. Separate the product by thin-layer chromatography.

[0064] (2) Synthesizing linking groups

[0065] In a three-necked flask equipped with a magnetic stir bar, 5 mmol of the hydrophobic alkyl tail of the product synthesized in the previous step, 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 min. 6.25 mmol of acryloyl chloride (premixed in 10 mL of dichloromethane) was slowly added dropwise using a constant-pressure funnel. After the acryloyl chloride addition was complete, the ice bath was removed. The reaction was allowed to proceed overnight at room temperature, then diluted with 30 mL of DCM and washed with 50 mL of 1 M HCl. The organic layer was dried over anhydrous magnesium sulfate and filtered. The product was separated by rapid chromatographic column chromatography.

[0066] (3) Reaction of head group with tail group

[0067] Select the alkyl tail synthesized in step (2) and 100 mg of amine, and add them sequentially to a 3 mL reaction flask lined with tetrafluoroethylene. The reaction is heated at 90 °C for 48 h. After the reaction is completed, the product can be directly used for cell transfection experiments or separated by rapid chromatography column separation.

[0068] The ionizable lipid library synthesized in this invention involves simple reaction steps and mild conditions, allowing for large-scale preparation within one week. This ionizable lipid can efficiently transfect mRNA, meeting the delivery requirements of next-generation RNA vaccines. The preferred ionizable lipids exhibit transfection effects comparable to, or even better than, several commercially available lipid products.

[0069] Example 1:

[0070] In a 50 mL reaction tube, 5 mmol of 3-nonanol, 15 mmol of N,N'-carbonyldiimidazole, 10 mmol of TEA, 20 mL of DCM, and a magnetic stir bar were added sequentially. The reaction tube was placed in a heating mantle at 40 °C and reacted for 24 h. The reaction progress was monitored using thin-layer chromatography (TLC). Once the reaction was complete, the reaction mixture was transferred to a separatory funnel, and DCM (2 x 100 mL) and saturated saline (2 x 100 mL) were added for extraction. The mixture was washed with 1 M HCl (2 x 20 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. Product A was obtained and could be used for the next reaction without further purification.

[0071] Example 2:

[0072]

[0073] In a 50 mL reaction tube equipped with a magnetic stir bar, 5 mmol of intermediate product A, 10 mmol of 5-amino-1-pentanol, and 20 mL of DCM were added sequentially. The reaction tube was placed in a heating mantle at 40 °C and reacted for 24 h. The reaction progress was monitored using TLC. Once the reaction was complete, the reaction mixture was transferred to a separatory funnel, and DCM (2 x 100 mL) and saturated saline (2 x 100 mL) were added for extraction. The mixture was washed with 1 M HCl (2 x 20 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography to obtain the target product B in 85% yield.

[0074] The proton NMR spectrum of the obtained product is as follows Figure 1 As shown, the proton NMR data are as follows:

[0075] 1 H NMR (400MHz, CDCl3): 4.90 (t, J=5.6Hz, 1H), 4.61-4.56 (m, 1H), 3.55-3.51 (m, 2H), 3.1 1-3.06(m,2H),2.83(s,1H),1.53-1.41(m,8H),1.35-1.19(m,10H),0.82-0.78(m,6H).

[0076] Example 3:

[0077]

[0078] In a three-necked flask equipped with a magnetic stir bar, 5 mmol of intermediate B, 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 min. 6.25 mmol of acryloyl chloride (premixed in 10 mL of DCM) was slowly added dropwise using a constant-pressure funnel. After the acryloyl chloride addition was complete, the ice bath was removed. The reaction solution was allowed to react at room temperature for 24 h. The reaction progress was monitored using TLC. Once the reaction was complete, the solution was diluted with DCM (2 x 50 mL) and washed with 1 M HCl (2 x 20 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography to obtain the target product C in 90% yield.

[0079] The proton NMR spectrum of the obtained product is as follows Figure 2 As shown, the proton NMR data are as follows:

[0080] 1 H NMR (400MHz, CDCl3): 6.37 (d, J = 17.2, 1H), 6.08 (q, J = 6.8 Hz, 1H), 5.79 (d, J = 10.4, 1H), 4.69-4.62 (m, 2H), 4.13 (t, J = 6. 4Hz,2H),3.16-3.13(m,2H),1.70-1.63(m,2H),1.55-1.36(m,8H),1.27-1.24(m,8H),0.85(dd,J=5.6Hz,J=3.6Hz,6H).

[0081] Example 4:

[0082]

[0083] 100 mg of 1-(2-aminoethyl)pyrrolidine and two stoichiometric amounts of intermediate C were added to a 5 mL reaction flask equipped with a magnetic inlet (the flask cap had a tetrafluoroethylene liner). The reaction was carried out at 90 °C for 48 h. After the reaction was complete, the product was separated by thin-layer chromatography to obtain the ionizable lipid 3-5-C2C6.

[0084] The proton NMR spectrum of the obtained product is as follows Figure 3 As shown, the proton NMR data are as follows:

[0085] 1H NMR(400MHz, CDCl3):4.78-4.65(m,4H),4.08-4.03(m,4H),3.19-3.02(m,8H),2.81-2.77(m,2H) ,2.63-2.42(m,10H),1.80-1.48(m,20H),1.39-1.25(m,20H),0.87(dd,J=6.8Hz,J=5.2Hz,12H).

[0086] Example 5:

[0087]

[0088] In a 50 mL reaction tube, 5 mmol of 1-adamantine alcohol, 15 mmol of N,N'-carbonyldiimidazole, 10 mmol of TEA, 20 mL of DCM, and a magnetic stir bar were added sequentially. The reaction tube was placed in a heating mantle at 40 °C and reacted for 24 h. The reaction progress was monitored using TLC. After the reaction was complete, the reaction mixture was transferred to a separatory funnel, and DCM (2 x 100 mL) and saturated saline (2 x 100 mL) were added for extraction. The mixture was washed with 1 M HCl (2 x 20 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography to obtain the target product D in 80% yield.

[0089] The proton NMR spectrum of the obtained product is as follows Figure 4 As shown, the proton NMR data are as follows:

[0090] 1 H NMR (400MHz, CDCl3): 8.03(s,1H),7.32(s,1H),6.98(s,1H),2.22-2.19(m,9H),1.68-1.66(m,6H).

[0091] Example 6:

[0092]

[0093] In a 50 mL reaction tube equipped with a magnetic stir bar, 5 mmol of intermediate product D, 10 mmol of 5-amino-1-pentanol, and 20 mL of DCM were added sequentially. The reaction tube was placed in a heating mantle at 40 °C and reacted for 24 h. The reaction progress was monitored using TLC. Once the reaction was complete, the reaction mixture was transferred to a separatory funnel, and DCM (2 x 100 mL) and saturated saline (2 x 100 mL) were added for extraction. The mixture was washed with 1 M HCl (2 x 20 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography to obtain the target product E in 76% yield.

[0094] The proton NMR spectrum of the obtained product is as follows Figure 5 As shown, the proton NMR data are as follows:

[0095] 1 H NMR (400MHz, CDCl3): 4.64 (s, 1H), 3.61 (t, J = 6.8Hz, 2H), 3.11-3.06 (m, 2H), 2.13-1.98 (m, 10H), 1.63-1.34 (m, 12H).

[0096] Example 7:

[0097]

[0098] In a three-necked flask equipped with a magnetic stir bar, 5 mmol of intermediate product E, 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 min. 6.25 mmol of acryloyl chloride (premixed in 10 mL of DCM) was slowly added dropwise using a constant-pressure funnel. After the acryloyl chloride addition was complete, the ice bath was removed. The reaction solution was allowed to react at room temperature for 24 h. The reaction progress was monitored using TLC. Once the reaction was complete, the solution was diluted with DCM (2 x 50 mL) and washed with 1 M HCl (2 x 20 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography to obtain the target product F in 88% yield.

[0099] The proton NMR spectrum of the obtained product is as follows Figure 6 As shown, the proton NMR data are as follows:

[0100] 1 H NMR (400MHz, CDCl3): 6.39 (dd, J=16.0Hz, J=1.6Hz, 1H), 6.11 (q, J=6.8Hz, 1H), 5.80 (dd, J=9.2Hz, J=1.2Hz, 1H), 4.55(s,1H),4.15(t,J=6.4Hz,2H),3.13-3.08(m,2H),2.15-2.08(m,9H),1.72-1.68(m,8H),1.53-1.37(m,4H).

[0101] Example 8:

[0102]

[0103] 100 mg of 1-(2-aminoethyl)pyrrolidine and two stoichiometric amounts of intermediate F were added to a 5 mL reaction flask equipped with a magnetic inlet (the flask cap had a tetrafluoroethylene liner). The reaction was carried out at 90 °C for 48 h. After the reaction was complete, the product was separated by thin-layer chromatography to obtain the ionizable lipid 3-5-CA.

[0104] The proton NMR spectrum of the obtained product is as follows Figure 7 As shown, the proton NMR data are as follows:

[0105] 1 H NMR (400MHz, CDCl3): 4.71-4.68 (m, 2H), 4.05 (t, J = 7.2Hz, 4H), 3.12-3.02 (m, 8H), 2.65-2.62 (m, 8H), 2.46-2 .42(m,4H),2.14-2.03(m,18H),1.80-1.78(m,4H),1.66-1.60(m,16H),1.51-1.48(m,4H),1.40-1.36(m,4H).

[0106] Example 9:

[0107]

[0108] In a 50 mL reaction tube, 5 mmol of 2-octyldodecyl alcohol, 15 mmol of N,N'-carbonyldiimidazole, 10 mmol of TEA, 20 mL of DCM, and a magnetic stir bar were added sequentially. The reaction tube was placed in a heating mantle at 40 °C and reacted for 24 h. The reaction progress was monitored using thin-layer chromatography (TLC). Once the reaction was complete, the reaction mixture was transferred to a separatory funnel, and DCM (2 x 100 mL) and saturated saline (2 x 100 mL) were added for extraction. The mixture was washed with 1 M HCl (2 x 20 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. Product G was obtained and could be used for the next reaction without further purification.

[0109] Example 10:

[0110]

[0111] In a 50 mL reaction tube equipped with a magnetic stir bar, 5 mmol of intermediate product G, 10 mmol of 5-amino-1-pentanol, and 20 mL of DCM were added sequentially. The reaction tube was placed in a heating mantle at 40 °C and reacted for 24 h. The reaction progress was monitored using TLC. Once the reaction was complete, the reaction mixture was transferred to a separatory funnel, and DCM (2 x 100 mL) and saturated saline (2 x 100 mL) were added for extraction. The mixture was washed with 1 M HCl (2 x 20 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography to obtain the target product H in 76% yield.

[0112] The proton NMR spectrum of the obtained product is as follows Figure 8 As shown, the proton NMR data are as follows:

[0113] 1 H NMR (400MHz, CDCl3): 4.73 (s, 1H), 3.93-3.92 (m, 2H), 3.62 (t, J = 6.4Hz, 2H), 3.19 -3.15(m,2H),1.61-1.36(m,8H),1.30-1.24(m,32H),0.88-0.85(t,J=6.4Hz,6H).

[0114] Example 11:

[0115]

[0116] In a three-necked flask equipped with a magnetic stir bar, 5 mmol of intermediate product H, 7.5 mmol of TEA, and 20 mL of DCM were added sequentially. The flask was pre-cooled in an ice bath for 30 min. 6.25 mmol of acryloyl chloride (premixed in 10 mL of DCM) was slowly added dropwise using a constant-pressure funnel. After the acryloyl chloride addition was complete, the ice bath was removed. The reaction solution was allowed to react at room temperature for 24 h. The reaction progress was monitored using TLC. Once the reaction was complete, the solution was diluted with DCM (2 x 50 mL) and washed with 1 M HCl (2 x 20 mL). The organic layer was collected, dried over anhydrous magnesium sulfate, and filtered. The organic solvent was then removed using a rotary evaporator under reduced pressure. The product was separated by thin-layer chromatography to obtain the target product I in 88% yield.

[0117] The proton NMR spectrum of the obtained product is as follows Figure 9 As shown, the proton NMR data are as follows:

[0118] 1H NMR (400MHz, CDCl3): 6.38 (dd, J=15.6Hz, J=1.6Hz, 1H), 6.10 (q, J=6.8Hz, 1H), 5.80 (dd, J=8.8Hz, J=1.6Hz, 1H), 4.66 (s, 1H), 4.15 (t, J=6.4Hz, 2H),3.95-3.93(m,2H),3.20-3.15(m,2H),1.72-1.67(m,2H),1.57-1.5 2(m,2H),1.44-1.38(m,2H),1.31-1.25(m,32H),0.87(t,J=6.4Hz,6H).

[0119] Example 12:

[0120]

[0121] 100 mg of 1-(2-aminoethyl)piperidine and two stoichiometric amounts of intermediate I were added to a 5 mL reaction flask equipped with a magnetic inlet (the flask cap had a tetrafluoroethylene liner). The reaction was carried out at 90 °C for 48 h. After the reaction was complete, the product was separated by thin-layer chromatography to obtain the ionizable lipid 8-5-C8C10.

[0122] The proton NMR spectrum of the obtained product is as follows Figure 10 As shown, the proton NMR data are as follows:

[0123] 1 H NMR (400MHz, CDCl3): 4.81 (s, 2H), 4.15-3.93 (m, 8H), 3.18-3.16 (m, 4H), 2.79 (t, J = 7.2Hz, 4H), 2.6 0-2.58(m,2H),2.41-2.27(m,10H),1.67-1.51(m,12H),1.42-1.25(m,72H),0.88(t,J=6.4Hz,12H).

[0124] Example 13:

[0125]

[0126] 100 mg of N,N-diethylethylenediamine and two stoichiometric amounts of intermediate I were added to a 5 mL reaction flask equipped with a magnetic inlet (the flask cap had a tetrafluoroethylene liner). The reaction was carried out at 90 °C for 48 h. After the reaction was complete, the product was separated by thin-layer chromatography to obtain the ionizable lipid 14-5-C8C10.

[0127] The proton NMR spectrum of the obtained product is as follows Figure 11 As shown, the proton NMR data are as follows:

[0128] 1 H NMR(400MHz, CDCl3):4.74(s,2H),4.07-3.94(m,8H),3.20-3.14(m,4H),2.91-2.78(m,4H),2.56-2.42(m, 10H),2.04(s,4H),1.68-1.51(m,10H),1.42-1.22(m,68H),1.02(t,J=7.2Hz,4H),0.88(t,J=6.8Hz,12H).

[0129] The structure of the multi-tailed ionizable lipid synthesized in this invention is as follows (for the synthesis methods of other multi-tailed ionizable lipids, please refer to Examples 1-13):

[0130]

[0131]

[0132]

[0133]

[0134]

[0135] Example 14:

[0136] In the 293T cell line, the efficiency of LNP containing ionizable lipids in delivering self-amplified RNA (repRNA-GFP-Luc) encoding green fluorescent protein (GFP) and firefly luciferase (Luc) was verified. The following ionizable lipids were used: 3-6-C8, 7-6-C8, 8-6-C8, 10-6-C8, 11-6-C8, 12-6-C8, 13-6-C8, 14-6-C8, 16-6-C8, 3-5-C2C6, 7-5-C2C6, 8-5-C2C6, 10-5-C2C6, 11-5-C2C6, 12-5-C2C6, 13-5-C2C6, 14-5-C2C6, 16-5-C2C6, 3-5-CA, 7-5-CA, 8-5-CA, 10-5-CA, 11-5-CA, 12-5-CA, 13-5-CA, 14-5-CA, and 16-5-CA. The following proteins were used as delivery materials to express repRNA-GFP-Luc in cells: 3-5-C6C8, 7-5-C6C8, 8-5-C6C8, 10-5-C6C8, 11-5-C6C8, 12-5-C6C8, 13-5-C6C8, 14-5-C6C8, 16-5-C6C8, 3-5-C8C10, 7-5-C8C10, 8-5-C8C10, 10-5-C8C10, 11-8C10, 12-5-C8C10, 13-5-C8C10, 14-5-C8C10, 16-5-C8C10, and the commercially available materials ALC-0315 and SM-102.

[0137] Specific steps:

[0138] 1. Cell Culture

[0139] The day before the experiment, the cultured 293T cells were seeded in a 96-well cell culture plate. When the cell density grew to about 70-80%, the cell transfection experiment was carried out.

[0140] 2. Preparation of lipid nanoparticles LNP-repRNA-GFP-Luc for cell transfection

[0141] Using ionizable lipids 3-6-C8, 7-6-C8, 8-6-C8, 10-6-C8, 11-6-C8, 12-6-C8, 13-6-C8, 14-6-C8, 16-6-C8, 3-5-C2C6, 7-5-C2C6, 8-5-C2C6, 10-5-C2C6, 11-5-C2C6, 12-5-C2C6, 13-5-C2C6, 14-5-C2C6, 16-5-C2C6, 3-5 -CA, 7-5-CA, 8-5-CA, 10-5-CA, 11-5-CA, 12-5-CA, 13-5-CA, 14-5-CA, 16-5-CA, 3-5-C6C8, 7-5-C6C8 , 8-5-C6C8, 10-5-C6C8, 11-5-C6C8, 12-5-C6C8, 13-5-C6C8, 14-5-C6C8, 16-5-C6C8, 3-5-C8C10, 7-5 -C8C10, 8-5-C8C10, 10-5-C8C10, 11-8C10, 12-5-C8C10, 13-5-C8C10, 14-5-C8C10, and 16-5-C8C10, along with distearate phosphatidylcholine (DSPC), cholesterol, and DSPE-PEG, were dissolved in anhydrous ethanol at concentrations of 10 mg / mL, 3 mg / mL, 6 mg / mL, and 1 mg / mL, respectively. Ionized lipids:Cholesterol:DSPC:DSPE-PEG = 40:48:10:2 were uniformly mixed. Simultaneously, an appropriate amount of repRNA-GFP-Luc was dissolved in sodium acetate buffer (the volume of the sodium acetate buffer was twice the total volume of the lipid mixture, pH = 5.2–5.3). The mRNA buffer and lipid mixture were then rapidly mixed and incubated at room temperature for 15 min to assemble stable LNPs (each well contained 150 ng of repRNA-GFP-Luc). The LNPs were diluted with twice the volume of sterile PBS and added to 96-well cell culture plates for transfection. The nitrogen-to-phosphorus ratio of ionized lipids to mRNA was 24:1, which is the molar ratio between protonated amino groups and phosphate groups on the mRNA (the same applies below).

[0142] Positive control group: LNPs were assembled using commercially available lipids ALC-0315 and SM-102 according to published preparation methods. The specific procedures were as follows: ALC-0315 or SM-102, DSPC, Cholesterol, ALC-0159, or DMG-PEG were added. 2000Dissolve the ALC-0315 in anhydrous ethanol at concentrations of 5 mg / mL, 1.5 mg / mL, 3 mg / mL, and 1 mg / mL, respectively, in a ratio of 46.3:42.7:9.4:1.6 or SM-102:Cholesterol:DSPC:DMG-PEG. 2000 The mRNA, GFP, and Luc molecules were mixed uniformly in a molar ratio of 50:38.5:10:1.5. Simultaneously, an appropriate amount of repRNA-GFP-Luc was dissolved in sodium citrate buffer (the volume of the sodium citrate buffer was three times the total volume of the lipid mixture, pH 4.0). The mRNA buffer and lipid mixture were then rapidly mixed and incubated at room temperature for 15 min to assemble stable LNPs (each well contained 150 ng of repRNA-GFP-Luc). The mixture was diluted with twice the volume of sterile PBS and added to 96-well cell culture plates for transfection. The nitrogen-phosphorus ratio of ALC-0315, SM-102, and mRNA was 6:1.

[0143] Negative control group: 293T cells were cultured normally and then supplemented with unloaded repRNA-GFP-Luc.

[0144] 3. Cell transfection efficiency analysis

[0145] Thirty-six hours after cell transfection, the expression of green fluorescent protein was detected using a fluorescence microscope. The culture medium in the 96-well cell culture plates was aspirated, cell lysis buffer was added, and cells were lysed on ice for 30 minutes. After centrifugation, the supernatant was collected and transferred to a white 96-well detection plate. Firefly luciferase substrate was added, and the firefly luciferase content (chemiluminescence) was detected using a microplate reader. The relative luciferase activity results are as follows: Figure 12 As shown in the figure. The results show that the ionizable lipids synthesized in this invention can greatly enhance the transfection efficiency of self-amplified RNA. The RNA expression efficiency is highest when the tail of the ionizable lipid is 6-C8, 5-C6C8, and 5-C8C10, while the RNA expression efficiency is weaker when the tail is 5-C2C6 and 5-CA. Lipids represented by 14-6-C8, 8-5-C8C10, and 14-5-C8C10 have better transfection efficiency than commercial lipids ALC-0315 and SM-102, with an efficiency increase of approximately 2 to 3 times, verifying the rationality and high efficiency of the overall chemical structure of the ionizable lipids designed in this invention.

[0146] Example 15:

[0147] The efficiency of LNP delivery encoding green fluorescent protein and firefly luciferase (repRNA-GFP-Luc) with ionizable lipids was validated in the 293T cell line. The auxiliary lipids for LNP were optimized using ionizable lipids 14-6-C8, 8-5-C8C10, and 14-5-C8C10.

[0148] Specific steps:

[0149] 1. Referring to Example 14, the difference is that in Example 15, the ionizable lipids used in the experimental group were: 14-6-C8, 8-5-C8C10, and 14-5-C8C10. The ionizable lipids, dioleoylphosphatidylethanolamine (DOPE), DSPC or dioleoyllecithin (DOPC), Cholesterol, and DSPE-PEG were dissolved in anhydrous ethanol at concentrations of 10 mg / mL, 3 mg / mL, 6 mg / mL, and 1 mg / mL, respectively. The ratio used was ionizable lipid 14-6-C8, 8-5-C8C10 or 14-5-C8C10:Cholesterol:DOPE, DSPC or DOPC:DSPE-PEG = 40:48:10:2.

[0150] 2. Cell transfection efficiency analysis

[0151] Thirty-six hours after transfection, the culture medium in the 96-well cell culture plate was aspirated, cell lysis buffer was added, and the cells were lysed on ice for 30 minutes. After centrifugation, the supernatant was collected and transferred to a white 96-well detection plate. Firefly luciferase substrate was added, and the firefly luciferase content was detected using a microplate reader (chemiluminescence). The relative luciferase activity results are as follows: Figure 13 As shown in the figure. The results show that the chemical structure of the assisting lipid has a significant impact on RNA delivery efficiency. When the assisting lipid is DSPC, the delivery efficiency of the three ionizable lipids is significantly better than that of DOPE or DOPC. Therefore, DSPC is the preferred assisting lipid.

[0152] Example 16:

[0153] The efficiency of LNP containing ionizable lipids in delivering repRNA-GFP-Luc was validated in the 293T cell line. The proportions of each component in the LNP were optimized using ionizable lipids 14-6-C8, 8-5-C8C10, and 14-5-C8C10.

[0154] Specific steps:

[0155] 1. Referring to Example 14, the difference is that in Example 16, the ionizable lipids used in the experimental group were: 14-6-C8, 8-5-C8C10, and 14-5-C8C10. Ionizable lipids, DSPC, Cholesterol, and DSPE-PEG, were dissolved in anhydrous ethanol at concentrations of 10 mg / mL, 3 mg / mL, 6 mg / mL, and 1 mg / mL, respectively. Five different molar ratios were used for mixing: Ratio A: Ionizable lipids:Cholesterol:DSPC:DSPE-PEG = 40:48:10:2; Ratio B: Ionizable lipids:Cholesterol:DSPC:DSPE-PEG = 30:28.5:10:0.75; Ratio C: Ionizable lipids:Cholesterol:DSPC:DSPE-PEG = 50:38.5:10:1.5; Ratio D: Ionizable lipids:Cholesterol:DSPC:DSPE-PEG = 35:46:16:2.5; Ratio E: Ionizable lipids:Cholesterol:DSPC:DSPE-PEG = 46.3:42.7:9.4:1.6

[0156] 2. Cell transfection efficiency analysis

[0157] Thirty-six hours after transfection, the culture medium in the 96-well cell culture plate was aspirated, cell lysis buffer was added, and the cells were lysed on ice for 30 minutes. After centrifugation, the supernatant was collected and transferred to a white 96-well detection plate. Firefly luciferase substrate was added, and the firefly luciferase content was detected using a microplate reader (chemiluminescence). The relative luciferase activity results are as follows: Figure 14 As shown in the figure. The results show that the molar ratio between the components of LNP also affects the RNA delivery efficiency to some extent, and the optimal ratio is A: ionizable lipids: Cholesterol: DSPC: DSPE-PEG = 40:48:10:2.

[0158] Example 17:

[0159] The efficiency of LNP containing ionizable lipids in delivering repRNA-GFP-Luc was validated in the 293T cell line. The nitrogen-phosphorus ratio of the LNP was optimized using ionizable lipids 14-6-C8, 8-5-C8C10, and 14-5-C8C10.

[0160] Specific steps:

[0161] 1. Referring to Example 14, the difference is that in Example 17, the ionizable lipids used in the experimental group were: 14-6-C8, 8-5-C8C10, and 14-5-C8C10. The ionizable lipids, DSPC, Cholesterol, and DSPE-PEG, were dissolved in anhydrous ethanol at concentrations of 10 mg / mL, 3 mg / mL, 6 mg / mL, and 1 mg / mL, respectively. The ratio used was 14-6-C8, 8-5-C8C10, or 14-5-C8C10:Cholesterol:DSPC:DSPE-PEG = 40:48:10:2. The nitrogen-to-phosphorus ratio of the LNPs was 12:1, 18:1, 24:1, and 32:1, respectively.

[0162] 2. Cell transfection efficiency analysis

[0163] Thirty-six hours after transfection, the culture medium in the 96-well cell culture plate was aspirated, cell lysis buffer was added, and the cells were lysed on ice for 30 minutes. After centrifugation, the supernatant was collected and transferred to a white 96-well detection plate. Firefly luciferase substrate was added, and the firefly luciferase content was detected using a microplate reader (chemiluminescence). The relative luciferase activity results are as follows: Figure 15 As shown in the figure. The results indicate that the repRNA-GFP-Luc transfection efficiency is optimal when the nitrogen-phosphorus ratio is 18:1.

[0164] Example 18:

[0165] The efficiency of LNP delivery of repRNA-GFP-Luc containing ionizable lipids was validated in the 293T cell line. The buffer formulation of LNP was optimized using ionizable lipids 14-6-C8, 8-5-C8C10, and 14-5-C8C10.

[0166] Specific steps:

[0167] 1. Referring to Example 14, the difference is that in Example 18, the ionizable lipids used in the experimental group were: 14-6-C8, 8-5-C8C10, and 14-5-C8C10. The ionizable lipids, DSPC, Cholesterol, and DSPE-PEG, were dissolved in anhydrous ethanol at concentrations of 10 mg / mL, 3 mg / mL, 6 mg / mL, and 1 mg / mL, respectively. The ratio used was 14-6-C8, 8-5-C8C10 or 14-5-C8C10:Cholesterol:DSPC:DSPE-PEG = 40:48:10:2. The premixed RNA solution was sodium acetate or sodium citrate buffer, and the nitrogen-to-phosphorus ratio for preparing LNPs was 18:1.

[0168] 2. Cell transfection efficiency analysis

[0169] Thirty-six hours after transfection, the culture medium in the 96-well cell culture plate was aspirated, cell lysis buffer was added, and the cells were lysed on ice for 30 minutes. After centrifugation, the supernatant was collected and transferred to a white 96-well detection plate. Firefly luciferase substrate was added, and the firefly luciferase content was detected using a microplate reader (chemiluminescence). The relative luciferase activity results are as follows: Figure 16 As shown in the figure. The results indicate that when sodium acetate buffer is used as the buffer for preparing nanoparticles, the RNA delivery efficiency is better than that of sodium citrate buffer. Therefore, sodium acetate buffer is preferred.

[0170] Example 19:

[0171] LNPs containing ionizable lipids 14-6-C8, 8-5-C8C10, 10-5-C8C10, 14-5-C8C10 and commercial lipids SM-102 and ALC-0315 were used to deliver self-amplified RNA (repRNA-Luc) encoding firefly luciferase in Balb / c mice. The expression of reporter gene luciferase was detected using an in vivo imaging system (IVIS) on days 2, 5, 7, 10, 12, and 15 after intramuscular injection.

[0172] 1. The specific steps are as described in Example 14, except that in Example 19, the ionizable lipids used in the experimental group were: 14-6-C8, 8-5-C8C10, 10-5-C8C10, and 14-5-C8C10. Ionizable lipids, DSPC, Cholesterol, and DSPE-PEG, were dissolved in anhydrous ethanol at concentrations of 10 mg / mL, 6 mg / mL, 12 mg / mL, and 5 mg / mL. An appropriate amount of repRNA-Luc was dissolved in sodium acetate buffer (the volume of the sodium acetate buffer was twice the total volume of the lipid mixture, pH = 5.3). The buffer containing repRNA-Luc was added to the ethanol solution of the lipid mixture and quickly mixed to assemble LNPs. The mixed solution was incubated at room temperature for 15 min, dialyzed in PBS for 1 h using a dialysis bag (MWCO = 14000MW), and then intramuscularly injected (each injection contained 1.5 μg of repRNA-Luc LNPs). The ratio used was ionizable lipids 14-6-C8, 8-5-C8C10, 10-5-C8C10 or 14-5-C8C10:Cholesterol:DSPC:DSPE-PEG = 40:48:10:2. The premixed RNA solution was sodium acetate buffer, and the nitrogen-to-phosphorus ratio for preparing LNPs was 18:1.

[0173] Positive control group: Commercially available lipids ALC-0315 and SM-102 were used to assemble the corresponding positive control LNPs according to published preparation methods. The specific procedures are as follows: ALC-0315 or SM-102, DSPC, Cholesterol, ALC-0159, or DMG-PEG were added. 2000 Dissolve the mRNA-Luc in anhydrous ethanol at concentrations of 5 mg / mL, 1.5 mg / mL, 3 mg / mL, and 1 mg / mL respectively, and mix them thoroughly according to the molar ratios of ALC-0315:Cholesterol:DSPC:ALC-0159 = 46.3:42.7:9.4:1.6 or SM-102:Cholesterol:DSPC:DMG-PEG2000 = 50:38.5:10:1.5. At the same time, take an appropriate amount of repRNA-Luc and dissolve it in sodium citrate buffer (the volume of sodium citrate buffer should be three times the total volume of the lipid mixture, pH = 4.0). Then, rapidly mix the buffer containing mRNA with the lipid mixture solution and incubate at room temperature for 15 min to assemble LNPs. Then, dialyze the LNPs in PBS for 1 h using a dialysis bag (MWCO = 14000MW) and administer intramuscular injections (each injection contains 1.5 μg of repRNA-Luc LNPs). The nitrogen-phosphorus ratio of ALC-0315, SM-102, and mRNA is 6:1.

[0174] 2. Analysis of in vivo imaging results

[0175] IVIS results indicate that ( Figure 17-18 From the second day after intramuscular injection, the expression intensity of the ionizable lipid 10-5-C8C10 of the present invention is superior to that of commercial lipids SM-102 and ALC-0315, and the decrease in the expression level of 8-5-C8C10 in vivo is slower than that of commercial lipids.

[0176] According to literature reports, the expression value of self-amplified RNA reaches its peak 7-10 days after injection, while that of ordinary mRNA reaches its peak 48 hours after injection. This indicates that the self-amplified RNA delivered by the ionizable lipid of the present invention has a longer expression time and a higher expression level, which can bring more efficient and lasting immune effects in the application of mRNA vaccines.

[0177] Example 20:

[0178] The efficiency of LNPs containing ionizable lipids in delivering circular RNA (circRNA-Luc) encoding firefly luciferase was verified in the 293T cell line. Using ionizable lipids 3-6-C8, 7-6-C8, 8-6-C8, 10-6-C8, 11-6-C8, 12-6-C8, 13-6-C8, 14-6-C8, 16-6-C8, 3-5-C2C6, 7-5-C2C6, 8-5-C2C6, 10-5-C2C6, 11-5-C2C6, 12-5-C2C6, 13-5-C2C6, 14-5-C2C6, 16-5-C2C6, 3-5-CA, 7-5-CA, 8-5-CA, 10-5-CA, 11-5-CA, 12-5-CA, 13-5-CA, 14-5-CA, 16-5- CA, 3-5-C6C8, 7-5-C6C8, 8-5-C6C8, 10-5-C6C8, 11-5-C6C8, 12-5-C6C8, 13-5-C6C8, 14-5-C6C8, 16-5-C6C8, 3-5-C8C10, 7-5-C8C10, 8-5-C8C10, 10-5-C8C10, 11-8C10, 12-5-C8C10, 13-5-C8C10, 14-5-C8C10, 16-5-C8C10, and commercial lipids ALC-0315 and SM-102 were used as delivery materials to express circRNA-Luc in cells.

[0179] Specific steps:

[0180] 1. Refer to Example 14, except that in Example 20, repRNA-GFP-Luc is replaced with circRNA-Luc.

[0181] 2. Cell transfection efficiency analysis

[0182] 24 hours after cell transfection, the culture medium in the 96-well cell culture plate was aspirated, cell lysis buffer was added, and the cells were lysed on ice for 30 minutes. After centrifugation, the supernatant was collected and transferred to a white 96-well detection plate. Firefly luciferase substrate was added, and the firefly luciferase content (chemiluminescence) was detected using a microplate reader. The relative luciferase activity results are as follows: Figure 19As shown in the figure. The results show that the ionizable lipids synthesized in this invention can enhance the transfection efficiency of circRNA. The RNA expression efficiency is highest when the tail of the ionizable lipid is 6-C8, 5-C6C8, and 5-C8C10, while the RNA expression efficiency is weaker when the tail is 5-C2C6 or 5-CA. Lipids represented by 10-6-C8, 14-6-C8, 16-6-C8, 7-5-C6C8, 13-5-C8C10, and 14-5-C8C10 have better transfection efficiency than commercial lipids ALC-0315 and SM-102, with a 2-3 fold increase in transfection efficiency. This indicates that the ionizable lipids involved in this invention have a wide range of applications and can be used for the delivery of different types of mRNA.

[0183] Example 21:

[0184] circRNA-Luc was delivered in Balb / c mice using LNPs containing ionizable lipids 8-5-C8C10, 10-5-C8C10, or commercial lipids SM-102 and ALC-0315. The results were measured using IVIS at 6 h, 12 h, 1 day, 2 days, 3 days, 4 days, and 5 days after intramuscular injection.

[0185] 1. The specific steps are as described in Examples 14 and 19, except that in Example 21, the ionizable lipids used in the experimental group were 8-5-C8C10 and 10-5-C8C10. Ionizable lipids, DSPC, Cholesterol, and DSPE-PEG, were dissolved in anhydrous ethanol at concentrations of 10 mg / mL, 6 mg / mL, 12 mg / mL, and 5 mg / mL, respectively. An appropriate amount of circRNA-Luc was dissolved in sodium acetate buffer (the volume of the sodium acetate buffer was twice the total volume of the lipid mixture, pH = 5.3). The buffer containing circRNA-Luc was added to the lipid mixture solution and quickly mixed to assemble LNPs. The mixed solution was incubated at room temperature for 15 min, dialyzed in PBS for 1 h using a dialysis bag (MWCO = 14000MW), and then intramuscularly injected (each injection contained 1.5 μg of circRNA-Luc LNPs). The ratio used was ionizable lipids 8-5-C8C10 or 10-5-C8C10:Cholesterol:DSPC:DSPE-PEG = 40:48:10:2. The premixed RNA solution was sodium acetate, and the nitrogen-phosphorus ratio of ionizable lipids to RNA was 18:1.

[0186] Positive control group: Commercially available lipids ALC-0315 and SM-102 were used to assemble LNPs according to publicly available preparation methods. The specific procedures were as follows: ALC-0315 or SM-102, DSPC, Cholesterol, ALC-0159, or DMG-PEG were added. 2000Dissolve circRNA-Luc in anhydrous ethanol at concentrations of 5 mg / mL, 1.5 mg / mL, 3 mg / mL, and 1 mg / mL respectively, and mix them thoroughly according to the molar ratios of ALC-0315:Cholesterol:DSPC:ALC-0159 = 46.3:42.7:9.4:1.6 or SM-102:Cholesterol:DSPC:DMG-PEG2000 = 50:38.5:10:1.5. Simultaneously, dissolve an appropriate amount of circRNA-Luc in sodium citrate buffer (the volume of sodium citrate buffer should be three times the total volume of the lipid mixture, pH = 4.0). Then, rapidly mix the buffer containing circRNA with the lipid mixture solution and incubate at room temperature for 15 min to assemble stable LNPs. After dialyzing in PBS for 1 h using a dialysis bag (MWCO = 14000MW), administer an intramuscular injection (each injection contains 1.5 μg of circRNA-Luc LNPs). The nitrogen-phosphorus ratio of ALC-0315, SM-102, and RNA is 6:1.

[0187] 2. Analysis of in vivo imaging results

[0188] IVIS results indicate that ( Figure 20 The expression peak of LNPs carrying circRNA-Luc was between 12 and 24 h. Among them, the expression intensity of the ionizable lipid 8-5-C8C10 of the present invention was comparable to that of commercial lipids SM-102 and ALC-0315.

[0189] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A multi-tailed ionizable lipid, characterized in that, The structure is as follows: ； ； 。 2. The method for preparing the multi-tailed ionizable lipid according to claim 1, characterized in that, It is obtained by Michael addition reaction of organic amine compounds and branched tail compounds; The structure of the branched tail compound is as follows: ； Wherein, n, x, m1, and m2 are consistent with the compound of claim 1; The organic amine compound is one of the following compounds: 。 3. The preparation method according to claim 2, characterized in that, The branched tail compound is obtained by esterification of chlorinated acrylate with compound 1; The structure of compound 1 is as follows: ； Wherein, n, x, m1, and m2 are consistent with the compound of claim 1.

4. The use of the multi-tailed ionizable lipid of claim 1 in the preparation of drug carriers.

5. The application according to claim 4, characterized in that, The active ingredient of the drug is selected from nucleic acid molecules and protein drugs.

6. The application according to claim 5, characterized in that, The nucleic acid molecules are selected from siRNA, miRNA, mRNA, circRNA, antisense RNA, CRISPR guide RNAs, replicable RNA, cyclic dinucleotides, polyIC, CpGODN, plasmid DNA, and microcircular DNA; the protein drugs are selected from cell colony-stimulating factors, interleukins, lymphotoxins, interferon proteins, tumor necrosis factor, antibodies, and protein antigens.

7. The application according to claim 4, characterized in that, The method for preparing the drug carrier includes the following steps: (a) Mix the multi-tailed ionizable lipids with an ethanol solution of cholesterol or cholesterol derivatives, auxiliary lipids, and polyethylene glycol-modified lipids to prepare a lipid mixture solution; mix the drug with an acidic buffer solution, and then mix it with the lipid mixture solution; incubate at room temperature for 15 min to 1 h, and dilute with PBS or dialyze to obtain the drug carrier. Alternatively (b) dissolve the multi-tailed ionizable lipids and cholesterol or cholesterol derivatives in chloroform, blow dry with nitrogen to evaporate the solvent, add acidic or neutral buffer solution and sonicate for 1-20 min to prepare liposome nanoparticles for later use; mix protamine with the drug, then mix with the liposome nanoparticles, let stand for 5-30 min, add polyethylene glycol modified lipids, and place at 30-65℃ for 5-20 min to obtain the drug carrier.

8. The application according to claim 7, characterized in that, In step (a), the molar ratio of the multi-tailed ionizable lipid to cholesterol or cholesterol derivatives, auxiliary lipids, and polyethylene glycol-modified lipids is 10~100:0~90:0~90:0~90; and the nitrogen-to-phosphorus ratio of the protonable amino group to the nucleic acid drug in the multi-tailed ionizable lipid is 1~100:

1. The auxiliary lipids mentioned in step (a) are selected from at least one of egg yolk lecithin, hydrogenated egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, sphingomyelin, phosphatidylethanolamine, myristoyl phosphatidylcholine, myristoyl phosphatidylglycerol, dipalmitoyl phosphatidylcholine, distearyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, dioleoyl lecithin, dioleoyl phosphatidylcholine, and dilauroyl phosphatidylcholine. The molar ratio of the multi-tailed ionizable lipid to cholesterol or cholesterol derivatives in step (b) is 1:5 to 5:1; the mass ratio of the multi-tailed ionizable lipid to the drug is 1 to 100:

1. The polyethylene glycol-modified lipids described in steps (a) and (b) are selected from at least one of DSPE-PEG, C14-PEG, DMG-PEG, ALC-0159, DSPE-PEG-Maleimide, DSPE-PEG-COOH, and DSPE-PEG-NH2. The acidic buffer solution described in step (a) has a pH of 3 to 7; the acidic buffer solution is sodium acetate or sodium citrate buffer solution. The acidic or neutral buffer solution described in step (b) has a pH of 3 to 7; the acidic or neutral buffer solution is sodium citrate, sodium acetate buffer, or DPEC water.

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