Ionizable lipids, stereoisomers or pharmaceutically acceptable salts thereof, methods of making and uses

Abstract

The application provides an ionizable lipid, a stereoisomer or a pharmaceutically acceptable salt thereof, and a structure thereof is shown as formula (I). The application also provides a preparation method and application of the ionizable lipid. The ionizable lipid provided in the application can cooperate with each other through hydrogen bonds, electrostatics and / or hydrophobic interactions, and can stabilize and protect nucleic acid molecules. In cells, the ionizable lipid can promote endocytosis of nucleic acids and realize endosome escape, thereby effectively promoting expression of the nucleic acids. The ionizable lipid provided in the application has good biocompatibility, can efficiently deliver nucleic acid molecules, and has a wide clinical application prospect, and has important application value in the fields of gene therapy, cytokine therapy and nucleic acid vaccine development.

Description

Technical Field

[0001] The present invention relates to the technical field of drug delivery carriers, and in particular to an ionizable lipid, a stereoisomer or a pharmaceutically acceptable salt thereof, a preparation method and an application. Background Art

[0002] In recent years, the rapid development of gene therapy and vaccine development has driven innovation in delivery systems, particularly the widespread use of ionizable lipids (ILs) for nucleic acid delivery. ILs are key components of lipid nanoparticle (LNP) delivery systems. Their unique pH-responsiveness allows them to remain relatively neutral under physiological conditions, thereby reducing nonspecific binding to serum proteins and lowering systemic toxicity. In acidic environments, they acquire a positive charge, forming stable complexes with negatively charged nucleic acids and promoting endosomal escape, resulting in efficient nucleic acid delivery. Currently, LNPs have been widely used to deliver messenger RNA (mRNA) vaccines and small interfering RNA (siRNA) drugs, such as the COVID-19 mRNA vaccine and the FDA-approved siRNA drug Onpattro (Patisiran). Despite the successful application of LNPs and their proven clinical value, the availability of ILs suitable for broad gene therapy remains limited and faces numerous challenges, primarily safety, transfection efficiency, and delivery system stability. Currently approved ILs for clinical use, such as ALC-0315 (used in Pfizer-BioNTech's mRNA vaccine) and SM-102 (used in Moderna's mRNA vaccine), often exhibit adverse effects such as cytotoxicity and inflammatory responses when used at high doses. This limits the dosage and frequency of administration in clinical applications, further impacting therapeutic efficacy. Therefore, the development of novel ILs with excellent safety, high transfection efficiency, and stable assembly capabilities is crucial for further expanding the application of LNPs in clinical gene therapy.

[0003] Currently, the main safety challenge for ILs stems from nucleic acid and lipid leakage caused by insufficient assembly stability. In traditional LNPs, the lipid-nucleic acid bond is not completely stable. This instability, particularly in the dynamic in vivo environment, can lead to the release of some mRNA into peripheral tissues before it effectively enters cells, influenced by blood flow, enzymatic degradation, and other physiological factors. For ILs, their involvement in nucleic acid stabilization primarily relies on electrostatic interactions following protonation and the hydrophobic interaction of hydrophobic groups within their structure. However, the pKa values ​​of ILs have not been adequately designed to adapt to the complex in vivo environment. For example, MC3, with a pKa of approximately 6.7, facilitates endosomal escape, but its ionization in the bloodstream (pH 7.4) is insufficient, leading to premature nucleic acid release or particle aggregation. Studies have shown that after one hour of incubation in serum, SM-102-LNPs exhibit an mRNA leakage rate as high as 30%, significantly impairing target tissue delivery efficiency. Once mRNA or lipid molecules are free, they can be easily mistaken by the immune system as foreign pathogens, triggering inflammatory responses and immune activation, thus compromising therapeutic safety. This "leakage effect" caused by assembly instability not only weakens the effective concentration of nucleic acids in target cells, but may also induce systemic inflammatory responses and increase the risk of adverse reactions in patients. More seriously, this risk will further accumulate during long-term or high-dose administration, limiting the treatment dose and frequency, thereby affecting the overall therapeutic effect. Therefore, in the design and synthesis of ionizable lipids, improving assembly stability is the key to improving safety and ensuring the reliability of clinical applications, and this has also become one of the difficult problems that the academic and industrial communities are currently paying attention to and trying to overcome.

[0004] On the other hand, transfection efficiency has always been another important bottleneck that needs to be broken through in the research and development of nucleic acid delivery systems. Ideal ILs should be able to achieve efficient transfection of nucleic acid molecules such as mRNA and siRNA while ensuring high safety, ensuring their long-term expression in target cells. However, due to the limitations of molecular structure and assembly mode, some current ILs have insufficient binding force with nucleic acids, resulting in low endosome escape efficiency, which limits the effective delivery and expression of nucleic acids in cells. Low transfection efficiency not only means that a higher dose of nucleic acid is required to achieve the desired therapeutic effect, but it may also aggravate the inflammatory response and immune activation caused by nucleic acid leakage, thereby further reducing the safety and effectiveness of the overall treatment. At the same time, high transfection efficiency also has the advantages of reducing the amount of nucleic acid used and achieving long-term expression, which is of great significance for reducing treatment costs and reducing side effects. Therefore, how to balance assembly stability and transfection efficiency when designing new ionizable lipids has become a technical problem that needs to be solved urgently. Summary of the Invention

[0005] The present invention provides an ionizable lipid, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. The ionizable lipid comprises an α-acyloxyamide structure and an ionizable structure, and further includes at least two (e.g., three, four, etc.) lipid tails, at least one of which comprises a long-chain alkyl hydrophobic structure, such as a C12-C30 structure. As a nucleic acid drug carrier, the ionizable lipid of the present invention not only stabilizes the nucleic acid drug through electrostatic and / or hydrophobic interactions but also forms stable hydrogen-bonding interactions with nucleic acid molecules through the excellent hydrogen-bonding ability of the α-acyloxyamide structure, thereby enhancing the binding force between the nucleic acid and the ionizable lipid. In particular, the lipid tail can further enhance the hydrophobic interaction and interact with the nucleic acid molecule, as well as with other lipid molecules in the lipid nanoparticle. This significantly improves the binding stability between the lipid and the nucleic acid, greatly enhancing the encapsulation efficiency and transfection efficiency of the nucleic acid drug, while also exhibiting low cytotoxicity and ensuring high safety.

[0006] Specifically, one aspect of the present invention provides an ionizable lipid represented by formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof,

[0007] (I)

[0008] in,

[0009] R1 is selected from a C1-C18 alkyl group containing a tertiary amine structure, a C1-C18 heteroalkyl group containing a tertiary amine structure, or a C3-C20 heterocycle containing a tertiary amine structure;

[0010] R2 and R3 are the same as or different from each other and are each independently selected from a hydrogen atom, a C1-C18 saturated or unsaturated straight-chain alkyl group / heteroalkyl group, a C8-C30 saturated or unsaturated heteroalkyl group or a branched alkyl group containing an acyloxy group;

[0011] R4 is independently selected from hydrogen, C1-C18 saturated or unsaturated straight-chain alkyl, C8-C30 saturated or unsaturated heteroalkyl or branched alkyl containing an acyloxy group, C1-C20 alkyl containing a tertiary amine structure, C2-C50 heteroalkyl containing a tertiary amine structure, C3-C20 heterocycle containing a tertiary amine structure, and C5-C20 cycloalkyl;

[0012] And at least one of R1 and R4 is an ionizable structure;

[0013] R5 is hydrogen or a C1-C5 straight or branched chain alkyl group;

[0014] L1 and L2 are the same as or different from each other and are each independently selected from a methylene group or a chemical bond selected from O(C=O), (C=O)O, S(C=O), (C=O)S or (C=O)NH;

[0015] L3 is a methylene group or a chemical bond selected from O(C=O), (C=O)O, S(C=O), (C=O)S or (C=O)NH;

[0016] M1, M2 and M3 are the same as or different from each other and are each independently selected from a C1-C5 saturated straight-chain alkylmethylene group, a branched-chain alkylmethylene group or a branched-chain alkylmethine group.

[0017] The present application also provides a method for preparing the ionizable lipid represented by the above formula (1), its stereoisomers or pharmaceutically acceptable salts, which comprises mixing and reacting the carboxylic acid compound represented by the following formula (II), the ketone compound represented by the following formula (III) and the isocyanate compound represented by the following formula (IV) in an organic solvent to obtain the ionizable lipid represented by the formula (I).

[0018] (II), (III), (IV).

[0019] In the above method, the use of a ketone compound as one of the reactive monomers facilitates the introduction of multiple asymmetric lipid tails (e.g., tri- or quadru-tails). In contrast, the use of other monomers, such as aldehydes, requires the addition of other complex monomers to achieve the introduction of multiple asymmetric tails. Furthermore, this preparation method also facilitates (e.g., requiring fewer steps) the introduction of ionizable structures at both the carboxyl and isocyanate ends. Ionizable lipids with ionizable structures at both the carboxyl and isocyanate ends can form more stable hydrogen bonding interactions with nucleic acid molecules, further enhancing the binding force between nucleic acids and ionizable lipid carriers, improving encapsulation efficiency and transfection efficiency, and reducing cytotoxicity. Therefore, the preparation method of the present invention is characterized by simple operation, short reaction time, low equipment requirements, mild reaction conditions, good selectivity, high yield, readily available raw materials, and low cost. It overcomes the technical issues of traditional methods, which require more synthetic steps, complex processes, difficult structural optimization, and high production costs. It also makes it easier to tailor the number of lipid tails and the location and number of ionizable structures, thereby preparing ionizable lipids that meet diverse functional requirements.

[0020] In addition, the present invention also relates to the use of ionizable lipids in the preparation of lipid nanoparticles and a method for preparing lipid nanoparticles; and the use of lipid nanoparticles in the preparation of drugs, preferably, the drugs are drugs for gene therapy, gene vaccination, cytokine products, interfering RNA therapy and nucleic acid transfer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0022] Figure 1 The results of particle size analysis and morphology analysis of lipid nanoparticles containing the ionizable lipids of the present invention are shown.

[0023] Figure 2 Shown are the results of in vitro transfection of lipid nanoparticles containing different ionizable lipids.

[0024] Figure 3 Shown are the results of shelf stability tests of lipid nanoparticles containing different ionizable lipids.

[0025] Figure 4 Shown are the results of in vivo transfection of lipid nanoparticles containing different ionizable lipids in mice.

[0026] Figure 5 Shown are the sustained expression of lipid nanoparticles containing different ionizable lipids in mice.

[0027] Figure 6 The results of the immune efficacy of the new coronavirus mRNA vaccine constructed with different ionizable lipids are shown.

[0028] Figure 7 The results of the immune efficacy of tumor mRNA vaccines constructed with different ionizable lipids are shown. DETAILED DESCRIPTION

[0029] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0030] Given the requirements for high delivery efficiency, good biocompatibility, and low cytotoxicity required of ionizable lipids as nucleic acid carriers, this application provides a class of ionizable lipids having α-acyloxyamides, stereoisomers, or pharmaceutically acceptable salts thereof, as described in Formula (I). These lipid molecules contain three key structural units: 1) an α-acyloxyamide structure; 2) an ionizable structure; and 3) at least two (e.g., three, four, etc.) lipid tails, at least one of which contains a C12-C30 long-chain alkyl hydrophobic structure. As nucleic acid carriers, ionizable lipids containing these three structural units can not only stabilize nucleic acids (such as mRNA, DNA, and siRNA) through electrostatic and hydrophobic interactions, but can also form stable hydrogen bond interactions with nucleic acid molecules through the excellent hydrogen bonding ability of the α-acyloxyamide structure, thereby enhancing the binding force between the nucleic acid and the ionizable lipid carrier, improving the encapsulation efficiency and transfection efficiency of nucleic acid drugs, and exhibiting low cytotoxicity. Therefore, the ionizable lipids provided in this application are used as delivery vehicles for nucleic acid drugs, which can solve the problems of high toxicity and poor biocompatibility of existing ionizable lipids used as delivery vehicles for nucleic acid drugs. Specifically, this application provides an ionizable lipid as described in formula (I), its stereoisomers, or pharmaceutically acceptable salts.

[0031] (I)

[0032] Wherein, R1 is selected from C1~C18 alkyl containing a tertiary amine structure, C1~C18 heteroalkyl containing a tertiary amine structure, and C3~C20 heterocycle containing a tertiary amine structure;

[0033] R2 and R3 are the same as or different from each other and are each independently selected from a hydrogen atom, a C1-C18 saturated or unsaturated straight-chain alkyl group / heteroalkyl group, a C8-C30 saturated or unsaturated heteroalkyl group or a branched alkyl group containing an acyloxy group;

[0034] R4 is independently selected from hydrogen, C1-C18 saturated or unsaturated straight-chain alkyl, C8-C30 saturated or unsaturated heteroalkyl or branched alkyl containing an acyloxy group, C1-C20 alkyl containing a tertiary amine structure, C2-C50 heteroalkyl containing a tertiary amine structure, C3-C20 heterocycle containing a tertiary amine structure, and C5-C20 cycloalkyl;

[0035] and at least one of R1 and R4 is an ionizable structure;

[0036] R5 is hydrogen or a C1-C5 straight or branched chain alkyl group;

[0037] L1 and L2 are the same as or different from each other and are each independently selected from a methylene group or a chemical bond selected from O(C=O), (C=O)O, S(C=O), (C=O)S or (C=O)NH;

[0038] L3 is a methylene group or a chemical bond selected from O(C=O), (C=O)O, S(C=O), (C=O)S or (C=O)NH;

[0039] M1, M2 and M3 are the same as or different from each other and are each independently selected from a hydrogen atom, a C1-C5 saturated straight-chain alkylmethylene group, a branched-chain alkylmethylene group or a branched-chain alkylmethine group.

[0040] Herein, the terms "ionizable structure" and "ionizable group" are used interchangeably to refer to structures / groups capable of undergoing protonation and deprotonation reactions at specific pH values. This ionizable structure allows ionizable lipids to be protonated in acidic environments (pH 5-6.5), such as endosomes / lysosomes, while remaining unprotonated at physiological pH (approximately pH 7.4), thereby exerting their desired functions. Exemplary ionizable structures include, but are not limited to, alkyl / heteroalkyl / heterocyclic groups containing tertiary amine structures. In some embodiments, R1 is selected from a C1-C18 alkyl group containing a tertiary amine structure, a C1-C18 heteroalkyl group containing a tertiary amine structure, or a C3-C20 heterocyclic group containing a tertiary amine structure, wherein the heteroatom includes -OH or N. Preferably, R1 is selected from a C2-C10 alkyl group containing a tertiary amine structure, a C4-C10 heteroalkyl group containing a tertiary amine structure, or a C4-C10 heterocyclic group containing a tertiary amine structure.

[0041] In some embodiments, R2 and R3 are the same or different and are each independently selected from a hydrogen atom, a C1-C18 saturated or unsaturated straight-chain alkyl group / heteroalkyl group, a C8-C30 saturated or unsaturated heteroalkyl group or a branched alkyl group containing an acyloxy group. The heteroatoms in the above-mentioned heteroalkyl group include O and S. Preferably, R2 and R3 are the same or different and are each independently selected from a hydrogen atom, a C6-C15 saturated or unsaturated straight-chain alkyl group / heteroalkyl group, a C8-C20 saturated or unsaturated heteroalkyl group or a branched alkyl group containing an acyloxy group.

[0042] In some embodiments, R4 is selected from hydrogen or a C1-C18 saturated or unsaturated straight-chain alkyl group, a C8-C30 saturated or unsaturated heteroalkyl group or branched alkyl group containing an acyloxy group, a C1-C20 alkyl group containing a tertiary amine structure, a C2-C50 heteroalkyl group containing a tertiary amine structure, a C3-C20 heterocycle containing a tertiary amine structure, or a C5-C20 cycloalkyl group, wherein the heteroatom includes O.

[0043] Preferably, R4 is selected from hydrogen, a C6-C15 saturated or unsaturated straight-chain alkyl group, a C8-C24 saturated or unsaturated heteroalkyl group or branched alkyl group containing an acyloxy group, a C3-C10 alkyl group containing a tertiary amine structure, a C3-C30 heteroalkyl group containing a tertiary amine structure, a C4-C10 heterocycle containing a tertiary amine structure, and a C6-C18 cycloalkyl group, wherein the heteroatom includes O.

[0044] Preferably, R4 is an ionizable structure.

[0045] In some embodiments, R5 is hydrogen.

[0046] In some embodiments, R5 is a C1, C2, C3, C4 or C5 linear or branched alkyl group.

[0047] In some embodiments, L1 and L2 are each independently methylene.

[0048] In some embodiments, L1 and L2 are each independently O(C=O), (C=O)O, S(C=O), (C=O)S, or (C=O)NH.

[0049] In some embodiments, L3 is methylene.

[0050] In some embodiments, L3 is O(C=O), (C=O)O, S(C=O), (C=O)S, or (C=O)NH.

[0051] In some embodiments, M1, M2, and M3 are each independently a hydrogen atom or a C1-C5 saturated linear alkyl methylene group.

[0052] In some embodiments, M1, M2, and M3 are each independently a C1-C5 branched alkyl methylene group or a branched alkyl methine group.

[0053] In some preferred embodiments, R1 is selected from any one of the following structural formulas:

[0054]

[0055] Furthermore, in some preferred embodiments, R2 and R3 are the same as or different from each other and are independently selected from any one of the following structural formulas:

[0056]

[0057] Furthermore, in some preferred embodiments, R4 is selected from hydrogen or any one of the following structural formulas:

[0058]

[0059] In some specific embodiments, the ionizable lipid of formula (I), its stereoisomers or pharmaceutically acceptable salts are selected from the following structural formulas:

[0060] ,

[0061] ,

[0062] ,

[0063] ,

[0064] ,

[0065] .

[0066] The ionizable lipid structure of the present invention contains tertiary amine groups, ensuring that the ionizable lipid has pH sensitivity and tunable positive charge. Furthermore, in some embodiments, the number of lipid tails and the branching structure of the ionizable lipid can be adjusted as needed. In addition, in some embodiments, the lipid tail also contains ester bonds, disulfide bonds, etc., which can ensure the degradation ability of the lipid molecules and ensure its efficient drug release ability as a delivery vehicle. In addition, the presence of amide bonds, etc., provides a structure for hydrogen bonding with nucleic acids, ensuring the internal stability of the lipid nanoparticles. Furthermore, in some embodiments, the lipid tail contains alkyl groups (e.g., heteroalkyl groups) and alkenyl groups, etc., and by adjusting their chain length, the hydrophobicity of the ionizable lipid and the strength of its interaction with cell membranes can be easily controlled.

[0067] Through rational structural design, the present invention obtains the above-mentioned series of ionizable lipids with α-acyloxyamides. This type of ionizable lipid molecular materials with α-acyloxyamides can bind to biological macromolecules such as nucleic acids (including mRNA, DNA and siRNA) and form complexes, thereby achieving efficient intracellular delivery of biological macromolecule drugs.

[0068] A second aspect of the present invention provides a method for preparing an ionizable lipid, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, comprising:

[0069] The carboxylic acid compound represented by formula (II), the ketone compound represented by formula (III) and the isocyanate compound represented by formula (IV) are mixed and reacted in an organic solvent to obtain an ionizable lipid represented by formula (I);

[0070] (II), (III), (IV),

[0071] (I),

[0072] in,

[0073] R1 is selected from a C1-C18 alkyl group containing a tertiary amine structure, a C1-C18 heteroalkyl group containing a tertiary amine structure, or a C3-C20 heterocycle containing a tertiary amine structure;

[0074] R2 and R3 are the same as or different from each other and are each independently selected from a hydrogen atom, a C1-C18 saturated or unsaturated straight-chain alkyl group / heteroalkyl group, a C8-C30 saturated or unsaturated heteroalkyl group or a branched alkyl group containing an acyloxy group;

[0075] R4 is independently selected from hydrogen, C1-C18 saturated or unsaturated straight-chain alkyl, C8-C30 saturated or unsaturated heteroalkyl or branched alkyl containing an acyloxy group, C1-C20 alkyl containing a tertiary amine structure, C2-C50 heteroalkyl containing a tertiary amine structure, C3-C20 heterocycle containing a tertiary amine structure, and C5-C20 cycloalkyl;

[0076] And at least one of R1 and R4 is an ionizable structure;

[0077] R5 is hydrogen or a C1-C5 straight or branched chain alkyl group;

[0078] L1 and L2 are the same as or different from each other and are each independently selected from a methylene group or a chemical bond selected from O(C=O), (C=O)O, S(C=O), (C=O)S or (C=O)NH;

[0079] L3 is a methylene group or a chemical bond selected from O(C=O), (C=O)O, S(C=O), (C=O)S or (C=O)NH;

[0080] M1, M2 and M3 are the same as or different from each other and are each independently selected from a C1-C5 saturated straight-chain alkylmethylene group, a branched-chain alkylmethylene group or a branched-chain alkylmethine group.

[0081] In some embodiments, the carboxylic acid compound represented by formula (II) is selected from any one of the following structural formulas (wherein CAR1, CAR2, etc. are numbers of the structural formulas, not parts of the structural formulas):

[0082]

[0083] In some embodiments, the ketone compound represented by formula (III) is selected from any one of the following structural formulas (wherein KET1, KET2, etc. are numbers of the structural formulas, not parts of the structural formulas):

[0084]

[0085] In some embodiments, the isocyanate compound represented by formula (IV) is selected from any one of the following structural formulas:

[0086]

[0087] In the method for preparing ionizable lipids, the carboxylic acid compound, the ketone compound, and the isocyanate compound are sequentially added to an organic solvent. Preferably, the organic solvent is selected from one or more of dichloromethane, chloroform, tetrahydrofuran, dimethyl sulfoxide, and N,N-dimethylformamide. Specifically, the organic solvent is selected from dichloromethane, chloroform, tetrahydrofuran, dimethyl sulfoxide, or N,N-dimethylformamide. In some embodiments, the organic solvent is chloroform.

[0088] The molar ratio of the ketone compound having the structure of formula (III) to the carboxylic acid compound having the structure of formula (II) is (0.5-5):1, for example, the molar ratio of the ketone compound having the structure of formula (III) to the carboxylic acid compound having the structure of formula (II) is (1-3):1, for example, 2:1, 1.5:1, etc. The molar ratio of the isocyanate compound having the structure of formula (IV) to the carboxyl group having the structure of formula (II) is (0.5-5):1, for example, the molar ratio of the isocyanate compound having the structure of formula (IV) to the carboxyl group having the structure of formula (II) is (1.1-3.5):1, for example, 2:1, 1.5:1, etc.

[0089] In the above process, the reaction temperature is 10-60° C. For example, the reaction temperature is 25-55° C., 35-50° C., or 40-45° C. The reaction time is 2-100 h, for example, 2-96 h. Preferably, the reaction time is 24-72 h, for example, 30-65 h, 35-60 h, 40-55 h, or 45-50 h.

[0090] According to the above-mentioned method for preparing ionizable lipids, the use of a ketone compound as one of the reactive monomers facilitates the introduction of multiple asymmetric lipid tails (e.g., tri- or quadru-tails). In contrast, the use of other monomers, such as aldehydes, requires additional monomers with complex structures to achieve the introduction of multiple asymmetric tails. Furthermore, this preparation method also makes it easier (e.g., requiring fewer steps) to introduce ionizable structures at both the carboxyl and isocyanide ends. Therefore, the preparation method of the present invention features simple operation, short reaction time, minimal equipment requirements, mild reaction conditions, good selectivity, high yield, readily available raw materials, and low cost. It overcomes the technical issues of traditional methods, which require more synthetic steps, are complex, difficult to optimize, and have high production costs.

[0091] In another aspect, the present application provides a lipid nanoparticle comprising the aforementioned ionizable lipid, its stereoisomer or pharmaceutically acceptable salt, or the ionizable lipid, its stereoisomer or pharmaceutically acceptable salt prepared by the above method.

[0092] Furthermore, the preparation method of the lipid nanoparticles includes:

[0093] dissolving ionizable lipids, sterols, helper lipids, and polyethylene glycol lipid derivatives in an organic solvent to obtain an organic phase;

[0094] The nucleic acid drug is dissolved in the aqueous phase, then mixed with the organic phase using a microfluidic device or a pipette, and then dialyzed to obtain lipid nanoparticles.

[0095] During the preparation of lipid nanoparticles, the molar ratio of the ionizable lipid, sterol, helper lipid, and polyethylene glycol lipid derivative is (20-70): (20-50): (2-30): (0.1-20). In some embodiments, the molar ratio of the ionizable lipid, sterol, helper lipid, and polyethylene glycol lipid derivative is (30-60): (30-40): (5-20): (1-5), for example (40-55): (35-45): (8-15): (1.5-4).

[0096] In the present application, in some specific embodiments, the sterol is selected from one or more of cholesterol, sitosterol, stigmasterol and cholesterol derivatives. In some embodiments, the sterol is cholesterol and / or a cholesterol derivative.

[0097] In the present application, in some specific embodiments, the auxiliary lipid is selected from one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and ceramide. In some embodiments, the auxiliary lipid is selected from one of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and ceramide. Preferably, the auxiliary lipid is, for example, dipalmitoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and / or dioleoylphosphatidylethanolamine (DOPE).

[0098] In the present application, in some specific embodiments, the polyethylene glycol lipid derivative is selected from diglyceride-PEG (DAG-PEG), dioleoyl-PEG (DAA-PEG), dimyristoyl-PEG (DMG-PEG), distearoyl-PEG (DSPE-PEG), octyl-PEG (C8-PEG), dodecyloxy-PEG (DOG-PEG), ceramide-PEG, distearoyl-rac-glycerol-polyethylene glycol (DSG-PEG) and / or dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG).

[0099] In the present application, in some specific embodiments, the nucleic acid drug is selected from one or more of mRNA, circular RNA, siRNA, microRNA or antisense nucleic acid.

[0100] In the present application, in some specific embodiments, the organic solvent is selected from one or more of ethanol, tetrahydrofuran, dimethyl sulfoxide and N,N-dimethylformamide. In some embodiments, the organic solvent comprises ethanol.

[0101] In the present application, in some specific embodiments, the aqueous phase is selected from one of water, glucose solution, phosphate buffer, carbonate buffer, borate buffer, acetate buffer or Tris-HCl-glucose buffer.

[0102] In the present application, the aqueous phase obtained in the above process is finally added to the organic phase at a ratio of (1-5) (volume) of aqueous phase to (volume) of organic phase (e.g., 2:1, 3:1, or 4:1), and the lipid nanoparticles are obtained after dialysis. In this process, the dialysis is performed according to methods well known to those skilled in the art and is not particularly limited in this application.

[0103] Furthermore, the present application also provides the use of lipid nanoparticles in the preparation of drugs, wherein the lipid nanoparticles are the lipid nanoparticles described in the above scheme.

[0104] In the present application, the drug is a drug for gene therapy, gene vaccination, cytokine production, interfering RNA therapy and nucleic acid transfer. In some embodiments, the nucleic acid drug is an mRNA vaccine.

[0105] Furthermore, the present application also provides a pharmaceutical composition comprising the lipid nanoparticles as described in the present application, their stereoisomers or pharmaceutically acceptable salts, and a pharmaceutically acceptable carrier, wherein the lipid nanoparticles contain nucleic acid drugs. Preferably, the nucleic acid drugs include DNA and RNA drugs for gene therapy, gene vaccination, cytokine products, or interfering RNA therapy, preferably one or more of mRNA, circular RNA, siRNA, microRNA, or antisense nucleic acid. Preferably, the pharmaceutical composition is a vaccine, such as an mRNA vaccine, such as a preventive vaccine or a therapeutic vaccine, such as an anti-tumor vaccine, or, for example, a new crown vaccine.

[0106] Compared with the prior art, the advantages or beneficial effects of the present invention are, for example:

[0107] The ionizable lipids provided by the present invention utilize a triple-functional design comprising an α-acyloxyamide structure, an ionizable head, and a long-chain hydrophobic tail. The α-acyloxyamide structure exhibits excellent hydrogen bonding ability, forming stable hydrogen-bonding interactions with nucleic acid molecules (such as mRNA, DNA, and siRNA), thereby enhancing the binding force between the nucleic acid and the lipid carrier. The ionizable head, upon protonation, undergoes strong electrostatic interactions with the nucleic acid. This interaction helps securely encapsulate the negatively charged nucleic acid within the lipid nanoparticle, preventing premature degradation or shedding in the body. Furthermore, the specific long-chain hydrophobic lipid tail structure of the present invention not only interacts with the nucleic acid molecules through hydrophobic interactions but also interacts with other lipid molecules within the lipid nanoparticle to form a stable nucleic acid carrier. These triple interactions—hydrogen bonding, electrostatic interactions, and hydrophobic interactions—synergistically enhance the stability of the lipid-nucleic acid complex, significantly increasing the encapsulation efficiency of nucleic acid drugs and improving the overall encapsulation efficiency of the carrier. This characteristic gives the lipid compound significant advantages in the delivery and protection of nucleic acid drugs such as mRNA.

[0108] More importantly, in view of the unique structure of the ionizable lipid of the present invention, it can not only significantly improve the encapsulation efficiency of nucleic acid drugs, but also show lower cytotoxicity and good biocompatibility, and is suitable as a delivery vehicle for nucleic acid drugs. This is of great significance for the clinical transformation of nucleic acid drugs, especially in the fields of gene therapy, mRNA vaccines, etc., and can effectively improve the delivery efficiency of drugs, and reduce the potential toxicity to normal cells. After being internalized by the cell, the ionizable group can be charged to enhance the endosomal escape efficiency of nucleic acid, and enhance the transfection of nucleic acid. Therefore, using the ionizable lipid of the present invention as a delivery vehicle for nucleic acid drugs, it is not only possible to improve delivery efficiency, improve drug loading performance, but also possible to reduce side effects while enhancing therapeutic effects, and there is a wide prospect for the clinical application of future nucleic acid drugs.

[0109] In another aspect, the present invention provides an innovative synthetic method that successfully synthesizes the ionizable lipids of the present invention through a one-pot combined reaction using carboxylic acid compounds, ketone monomers, and isocyanates as reaction raw materials. Compared to other preparation methods (such as protection / deprotection, solvent exchange, or methods using aldehyde monomers / compounds as one of the starting materials), the preparation method of the present invention not only simplifies the traditional multi-step synthesis process but also efficiently obtains the desired target lipid compound under mild reaction conditions. Furthermore, compared to using other monomers (such as aldehyde monomers), using ketone monomers as starting materials facilitates the control of the number of lipid tails (e.g., the preparation of multi-tailed (e.g., tri- and tetra-tailed) lipids) and the manipulation of the ionizable structure (e.g., the facile introduction of ionizable structures at both the carboxyl and isocyanate ends). A significant advantage of this method is its mild reaction conditions, which not only avoids potential damage to the product caused by extreme conditions such as high temperature and high pressure, but also significantly reduces the formation of byproducts, thereby improving product purity and recovery. Furthermore, the one-pot synthesis process makes the entire reaction process simpler and faster, amenable to large-scale production, and exhibits excellent reproducibility and stability. This feature makes this method not only suitable for small-scale laboratory research, but also can be expanded to larger-scale production applications, greatly improving the feasibility of its industrial application.

[0110] In addition, the flexible adjustment of the raw material structure makes it possible to synthesize lipid structures in a high-throughput manner. By adjusting the different raw material components and reaction conditions, ionizable lipids of various structures can be efficiently synthesized (for example, the number of lipid tails and / or the number and position of ionizable structures can be easily adjusted as needed) to meet the needs of different drug delivery systems. Based on this synthesis strategy, the ionizable lipids prepared by the present invention have excellent biocompatibility and can form stable complexes with a variety of drugs (especially nucleic acid drugs), ensuring the efficiency of drug delivery in vivo. More importantly, the lipids have good biocompatibility in vivo and can avoid excessive cytotoxic reactions, thus laying the foundation for the safe and efficient delivery of drugs. Therefore, the ionizable lipids synthesized by the method of the present invention are very suitable for use in fields such as gene therapy and vaccine delivery, and can achieve safe, efficient and stable intracellular delivery of a variety of drugs, with broad application prospects. Example

[0111] The present invention will be described in more detail with reference to the following examples. It should be understood that these examples are provided by way of illustration and are not intended to limit the present invention. In addition, the experimental methods in the following examples are conventional methods in the art unless otherwise specified. The raw materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified. Example 1

[0112] This Example 1 provides a method for preparing an ionizable lipid 6-2-8 having an α-acyloxyamide structure, wherein 6-2-8 is represented by chemical formula (V):

[0113] (V)

[0114] The specific preparation process of ionizable lipid 6-2-8 is as follows:

[0115] Dihydroxyacetone (900.8 mg, 10 mmol), linoleic acid (6450.35 mg, 23 mmol), 4-dimethylaminopyridine (244.34 mg, 2 mmol), N,N-diisopropylethylamine (2972.52 mg, 23 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4409.1 mg, 23 mmol) were dissolved in dichloromethane and stirred at room temperature for 24 h. The compound was purified by flash column chromatography to obtain the compound 2-oxopropane-1,3-diyl (9Z, 9'Z, 12Z, 12'Z)-bis(octadec-9,12-dioate) (1475 mg, 24% yield). The above reaction process is as follows:

[0116]

[0117] The main data of the H NMR spectrum of the compound 2-oxopropane-1,3-diyl (9Z, 9'Z, 12Z, 12'Z)-bis(octadecane-9,12-dioate) are:

[0118] 1 H NMR (300 MHz, Chloroform-d) δ 5.48 – 5.27 (m, 8H), 4.77 (s, 4H), 2.79 (t, J = 5.9 Hz, 4H), 2.44 (t, J = 7.5 Hz, 4H), 2.07 (q, J = 6.5 Hz, 8H), 1.67 (q, J = 7.2 Hz, 4H), 1.36 – 1.21 (m, 28H), 0.96 – 0.85 (m, 6H).

[0119] 2-Oxopropane-1,3-diyl (9Z, 9'Z, 12Z, 12'Z)-bis(octadecane-9,12-dioate) (61.50 mg, 0.1 mmol), 1-methyl-4-piperidinylacetic acid (15.72 mg, 0.1 mmol) and tert-butyl isocyanate (9.14 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55 ° C for 72 h. The compound was purified by flash column chromatography to obtain compound 6-2-8 (52.4 mg, yield 61%). The above reaction process is shown in Formula 1:

[0120]

[0121] Formula 1

[0122] The main data of the H NMR spectrum of ionizable lipid 6-2-8 are:

[0123] 1 H NMR (500 MHz, Chloroform-d) δ 5.40 – 5.27 (m, 8H), 4.78 (d, J =11.8 Hz, 2H), 4.49 (d, J = 11.8 Hz, 2H), 2.80 – 2.66 (m,4H), 2.47 (s, 4H), 2.36 (d, J = 6.6 Hz, 4H), 2.29 (q, J = 6.9, 6.4 Hz, 5H), 2.00 (q, J = 6.7 Hz, 8H), 1.89 (s, 1H) , 1.68 – 1.51 (m, 8H), 1.37 – 1.20 (m, 37H), 0.88 (t, J =7.0 Hz, 6H). Example 2

[0124] This Example 2 provides a method for preparing an ionizable lipid 4-2-8 having an α-acyloxyamide structure, wherein 4-2-8 is represented by chemical formula (VI):

[0125] (VI);

[0126] The specific preparation process of ionizable lipid 4-2-8 is as follows:

[0127] 2-Oxopropane-1,3-diyl (9Z, 9'Z, 12Z, 12'Z)-bis(octadecane-9,12-dioate) (61.50 mg, 0.1 mmol), 1-methylpiperidine-4-carboxylic acid (14.31 mg, 0.1 mmol), and tert-butyl isocyanide (9.14 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 hours. The compound was purified by flash column chromatography to obtain compound 4-2-8 (53.09 mg, 63% yield). The above reaction process is shown in Equation 2:

[0128]

[0129] Formula 2

[0130] The main data of the H NMR spectrum of ionizable lipid 4-2-8 are:

[0131] 1 H NMR (500 MHz, Chloroform-d) δ 5.40 – 5.27 (m, 8H), 4.78 (d, J =11.8 Hz, 2H), 4.49 (d, J = 11.8 Hz, 2H), 2.80 – 2.66 (m,4H), 2.47 (s, 4H),2.42 (s, 1H), 2.36 (d, J = 6.6 Hz, 4H), 2.29 (q, J = 6.9, 6.4 Hz, 3H), 2.00(q, J = 6.7 Hz, 8H), 1.68 – 1.51 (m, 8H), 1.37 – 1.20 (m, 37H), 0.88 (t, J =7.0 Hz, 6H). Example 3

[0132] This Example 3 provides a method for preparing an ionizable lipid 1-3-11 having an α-acyloxyamide structure, wherein 1-3-11 is represented by chemical formula (VII):

[0133] (VII)

[0134] The specific preparation process of ionizable lipid 1-3-11 is as follows:

[0135] Dihydroxyacetone (900.8 mg, 10 mmol), oleic acid (6496.58 mg, 23 mmol), 4-dimethylaminopyridine (244.34 mg, 2 mmol), N,N-diisopropylethylamine (2972.52 mg, 23 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4409.1 mg, 23 mmol) were dissolved in dichloromethane and stirred at room temperature for 24 h. The compound was purified by flash column chromatography to obtain 2-oxopropane-1,3-diyl dioleate (2535 mg, 41% yield). The above reaction process is shown below;

[0136]

[0137] The main data of the H NMR spectrum of 2-oxopropane-1,3-diyl dioleate are:

[0138] 1 H NMR (500 MHz, Chloroform-d) δ 5.34 (qd, J = 3.7, 1.5 Hz, 4H), 4.75(s, 4H), 2.42 (t, J = 7.6 Hz, 4H), 2.01 (q, J = 6.0 Hz, 8H), 1.65 (q, J = 7.3Hz, 4H), 1.30 (dd, J = 20.5, 9.7 Hz, 40H), 0.88 (t, J = 7.0 Hz, 6H).

[0139] 3-Diethylaminopropylamine (1302.3 mg, 10 mmol) was dissolved in ethyl formate (3704 mg, 50 mmol) and stirred at 60°C for 24 hours. After the reaction, the solvent ethyl formate and the by-product ethanol were removed, and triethylamine (5059 mg, 50 mmol) was added. After dissolution in dichloromethane, the mixture was heated to reflux at 55°C. Triphosgene (1038.55 mg, 3.5 mmol) was slowly added dropwise. After the addition was complete, the reaction was continued for 2 hours. The compound was purified by flash column chromatography to obtain N,N-diethyl-3-isocyanatopropan-1-amine (743.19 mg, 53% yield). The reaction process is shown below.

[0140]

[0141] The main data of the H NMR spectrum of N, N-diethyl-3-isocyanatopropan-1-amine are:

[0142] 1H NMR (500 MHz, Chloroform-d) δ 5.35 (s, 2H), 2.80 (s, 2H), 2.57 (s,4H), 2.41 (d, J = 12.5 Hz, 1H), 2.35 (s, 1H), 1.01 (s, 6H).

[0143] 2-Oxopropane-1,3-diyl dioleate (66.71 mg, 0.1 mmol), 4-dimethylaminobutyric acid (13.12 mg, 0.1 mmol) and N,N-diethyl-3-isocyanatopropan-1-amine (15.43 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 h. The compound was purified by flash column chromatography to obtain compound 1-3-11 (55.20 mg, 62% yield). The above reaction is shown in Formula 3:

[0144]

[0145] Formula 3

[0146] The main data of the H NMR spectrum of ionizable lipid 1-3-11 are:

[0147] 1 H NMR (500 MHz, Chloroform-d) δ 5.38 – 5.29 (m, 4H), 4.76 (d, J =12.5 Hz, 2H), 4.62 (d, J = 12.5 Hz, 2H), 3.22 (s, 2H), 2.57 (s, 4H), 2.48 (s,2H), 2.37 (s, 2H), 2.31 (s, 4H), 2.26 (s, 6H), 2.02 (s, 8H), 1.90 (s, 2H),1.66 (s, 2H) ,1.59 (d, J = 1.1 Hz, 4H), 1.34 – 1.26 (m, 42H), 1.01 (s, 6H), 0.90 (s, 6H). Example 4

[0148] This Example 4 provides a method for preparing an ionizable lipid 6-3-9 having an α-acyloxyamide structure, wherein 6-3-9 is represented by chemical formula (VIII):

[0149] (VIII);

[0150] The specific preparation process of ionizable lipid 6-3-9 is as follows:

[0151] 2-Oxopropane-1,3-diyl dioleate (66.71 mg, 0.1 mmol), 1-methyl-4-piperidinyl acetic acid (15.72 mg, 0.1 mmol) and ethyl isocyanoacetate (12.44 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 h. The compound was purified by flash column chromatography to obtain compound 6-3-9 (59.58 mg, 67% yield). The above reaction process is shown in Formula 4:

[0152]

[0153] Formula 4

[0154] The main data of the H NMR spectrum of ionizable lipid 6-3-9 are:

[0155] 1 H NMR (500 MHz, Chloroform-d) δ 5.38 – 5.29 (m, 4H), 4.76 (d, J =11.8 Hz, 2H), 4.49 (d, J = 11.8 Hz, 2H), 4.23 (q, J = 7.1 Hz, 2H), 4.05 (d, J= 4.9 Hz, 2H), 2.36 (d, J = 6.6 Hz, 4H), 2.29 (h, J = 8.1 Hz, 5H), 2.00 (q, J= 6.7 Hz, 16H), 1.89 (s, 1H) , 1.61 – 1.54 (m, 4H), 1.37 – 1.20 (m, 43H),0.88 (t, J = 7.0 Hz, 6H). Example 5

[0156] This Example 5 provides a method for preparing an ionizable lipid 6-3-8 having an α-acyloxyamide structure, wherein 6-3-8 is represented by the chemical formula (IX):

[0157] (IX)

[0158] The specific preparation process of ionizable lipid 6-3-8 is as follows:

[0159] 2-Oxopropane-1,3-diyl dioleate (66.71 mg, 0.1 mmol), 1-methyl-4-piperidinyl acetic acid (15.72 mg, 0.1 mmol) and tert-butyl isocyanate (9.14 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 h. The compound was purified by flash column chromatography to obtain compound 6-3-8 (61.01 mg, 71% yield). The above reaction process is shown in Formula 5:

[0160]

[0161] Formula 5

[0162] The main data of the H-NMR spectrum of ionizable lipid 6-3-8 are:

[0163] 1 H NMR (500 MHz, Chloroform-d) δ 5.38 – 5.29 (m, 4H), 4.76 (d, J =11.8 Hz, 2H), 4.49 (d, J = 11.8 Hz, 2H), 2.36 (d, J = 6.6 Hz, 4H), 2.29 (h, J= 8.1 Hz, 5H), 2.00 (q, J = 6.7 Hz, 16H), 1.89 (s, 1H), 1.61 – 1.54 (m, 4H), 1.37 – 1.20 (m, 49H), 0.88 (t, J = 7.0 Hz, 6H). Example 6

[0164] This Example 6 provides a method for preparing an ionizable lipid 4-3-9 having an α-acyloxyamide structure, wherein 4-3-9 is represented by the chemical formula (X):

[0165] (X)

[0166] The specific preparation process of ionizable lipid 4-3-9 is as follows:

[0167] 2-Oxopropane-1,3-diyl dioleate (66.71 mg, 0.1 mmol), 1-methylpiperidine-4-carboxylic acid (14.31 mg, 0.1 mmol) and ethyl isocyanoacetate (12.44 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 h. The compound was purified by flash column chromatography to obtain compound 4-3-9 (57.77 mg, 66% yield). The above reaction process is shown in Formula 6:

[0168]

[0169] Formula 6

[0170] The main data of the H NMR spectrum of ionizable lipid 4-3-9 are:

[0171] 1 H NMR (500 MHz, Chloroform-d) δ 5.38 – 5.29 (m, 4H), 4.76 (d, J =11.8 Hz, 2H), 4.49 (d, J = 11.8 Hz, 2H), 4.23 (q, J = 7.1 Hz, 2H), 4.05 (d, J= 4.9 Hz, 2H), 2.42 (s, 1H), 2.36 (d, J = 6.6 Hz, 4H), 2.29 (h, J = 8.1 Hz, 3H), 2.00 (q, J = 6.7 Hz, 16H), 1.61 – 1.54 (m, 4H), 1.37 – 1.20 (m, 43H),0.88 (t, J = 7.0 Hz, 6H). Example 7

[0172] This Example 7 provides a method for preparing an ionizable lipid 3-12-9 having an α-acyloxyamide structure, wherein 3-12-9 is represented by the chemical formula (XI):

[0173] (XI)

[0174] The specific preparation process of ionizable lipid 3-12-9 is as follows:

[0175] 9-Heptadecanol (25647 mg, 100 mmol), pimelic acid (17618 mg, 110 mmol), 4-dimethylaminopyridine (244.3 mg, 20 mmol), N,N-diisopropylethylamine (15510 mg, 120 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (23004 mg, 120 mmol) were dissolved in dichloromethane and stirred at room temperature for 24 hours. The compound was purified by flash column chromatography to obtain 7-(heptadecan-9-yloxy)-7-oxoheptanoic acid (27745 mg, 70% yield). The reaction process is shown below:

[0176]

[0177] The main data of the H NMR spectrum of compound 7-(heptadodec-9-yloxy)-7-oxoheptanoic acid are:

[0178] 1 H NMR (500 MHz, Chloroform-d) δ 4.86 (p, J = 6.3 Hz, 1H), 2.35 (t, J= 7.5 Hz, 2H), 2.29 (t, J = 7.5 Hz, 2H), 1.65 (dtd, J = 15.2, 7.6, 4.7 Hz,4H), 1.50 (d, J = 5.4 Hz, 4H), 1.39 (ddd, J = 15.4, 9.1, 3.4 Hz, 2H), 1.27 (d, J = 17.9 Hz, 24H), 0.87 (t, J = 6.9 Hz, 6H).

[0179] Dihydroxyacetone (2252 mg, 25 mmol), 7-(heptadodec-9-yloxy)-7-oxoheptanoic acid (22904.55 mg, 57.5 mmol), 4-dimethylaminopyridine (610.85 mg, 5 mmol), N,N-diisopropylethylamine (7431.87 mg, 57.5 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (11022.75 mg, 57.5 mmol) were dissolved in dichloromethane and stirred at room temperature for 24 hours. The compound was purified by flash column chromatography to obtain 7,7'-di(heptadodec-9-yl) O'1,O1-(2-oxopropane-1,3-diyl) di(pimelate) (6381 mg, 30% yield). The reaction process is shown below:

[0180]

[0181] The main data of the H NMR spectrum of compound 7,7'-di(heptadodec-9-yl) O'1,O1-(2-oxopropane-1,3-diyl) di(pimelate) are:

[0182] 1 H NMR (500 MHz, Chloroform-d) δ 4.88 (s, 2H), 4.77 (s, 4H), 2.32 (s,4H), 2.20 (s, 4H), 1.60 (s, 4H), 1.57 (s, 4H), 1.55 (d, J = 12.3 Hz, 8H), 1.39 – 1.35 (m, 12H), 1.34 – 1.26 (m, 40H), 0.90 (s, 12H).

[0183] Ethyl 4-bromobutyrate (1950.5 mg, 10 mmol), N-methyl-2-hydroxyethylamine (826.2 mg, 11 mmol), and N,N-diisopropylethylamine (1421.75 mg, 11 mmol) were dissolved in ethanol and reacted at 40°C for 18 h. The product was purified by flash column chromatography to obtain ethyl 4-((2-hydroxyethyl)(methyl)amino)butyrate.

[0184] Ethyl 4-((2-hydroxyethyl)(methyl)amino)butanoate was dissolved in 5 volumes of 10M hydrochloric acid solution and reacted at 110°C for at least 4 hours. TLC confirmed complete reaction. HCl was removed using a rotary evaporator to obtain 4-((2-hydroxyethyl)(methyl)amino)butanoic acid (1128 mg, 70% yield). The reaction process is shown below:

[0185]

[0186] The main data of the H NMR spectrum of compound 4-((2-hydroxyethyl)(methyl)amino)butyric acid are:

[0187] 1 H NMR (500 MHz, Chloroform-d) δ 3.64 (d, J = 5.1 Hz, 2H), 2.70 (t, J= 5.0 Hz, 1H), 2.59 (d, J = 1.6 Hz, 4H), 2.32 (s, 2H), 2.28 (s, 3H), 1.80 (s,2H).

[0188] 7,7'-Di(heptadodec-9-yl) O'1,O1-(2-oxopropane-1,3-diyl) di(pimelate) (85.13 mg, 0.1 mmol), 4-((2-hydroxyethyl)(methyl)amino)butanoic acid (16.13 mg, 0.1 mmol), and ethyl isocyanoacetate (12.44 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 hours. The compound was purified by flash column chromatography to obtain compound 3-12-9 (67.54 mg, 60% yield). The reaction process is shown in Equation 7:

[0189]

[0190] The main data of the H NMR spectrum of ionizable lipid 3-12-9 are:

[0191] 1H NMR (500 MHz, Chloroform-d) δ 4.85 (s, 2H), 4.78 (d, J = 12.5 Hz,2H), 4.63 (d, J = 12.3 Hz, 2H), 4.16 (s, 2H), 3.92 (s, 2H), 3.64 (d, J = 5.1Hz, 2H), 2.70 (t, J = 5.0 Hz, 1H), 2.59 (s, 2H), 2.56 (s, 2H), 2.37 (s, 2H), 2.30 (d, J = 12.8 Hz, 7H), 2.25 – 2.15 (m, 4H), 1.88 (s, 2H), 1.68 (d, J =12.5 Hz, 4H), 1.60 (s, 4H), 1.58 – 1.52 (m, 8H), 1.39 – 1.34 (m, 12H), 1.34 –1.24 (m, 43H), 0.90 (s, 12H). Example 8

[0192] This Example 8 provides a method for preparing an ionizable lipid 6-12-12 having an α-acyloxyamide structure, wherein 6-12-12 is represented by the chemical formula (XII):

[0193] (XII);

[0194] The specific preparation process of ionizable lipid 6-12-12 is as follows:

[0195] 7,7'-Di(heptadodec-9-yl) O'1,O1-(2-oxopropane-1,3-diyl) di(pimelate) (85.13 mg, 0.1 mmol), 4-((2-hydroxyethyl)(methyl)amino)butanoic acid (16.13 mg, 0.1 mmol), and 2-morpholinoethyl isocyanide (15.42 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 hours. The compound was purified by flash column chromatography to obtain compound 6-12-12 (73.52 mg, 64% yield). The reaction process is shown in Equation 8:

[0196]

[0197] Formula 8

[0198] The main data of the H NMR spectrum of ionizable lipid 6-12-12 are:

[0199] 1H NMR (500 MHz, Chloroform-d) δ 4.85 (s, 2H), 4.78 (d, J = 12.5 Hz,2H), 4.49 (d, J = 12.3 Hz, 2H) , 3.61 (s, 4H), 3.43 – 3.30 (m, 2H), 2.78 (s,2H), 2.56 (s, 2H), 2.46 (s, 4H), 2.32 (d, J = 15.0 Hz, 8H), 2.30 – 2.15 (m,9H), 1.89 (s, 1H), 1.71 – 1.65 (m, 6H), 1.62 – 1.52 (m, 12H), 1.39 – 1.26 (m,52H), 0.90 (s, 12H). Example 9

[0200] This Example 9 provides a method for preparing an ionizable lipid 4-12-9 having an α-acyloxyamide structure, wherein 4-12-9 is represented by the chemical formula (XIII):

[0201] (XIII)

[0202] The specific preparation process of ionizable lipid 4-12-9 is as follows:

[0203] 7,7'-Di(heptadodec-9-yl) O'1,O1-(2-oxopropane-1,3-diyl) di(pimelate) (85.13 mg, 0.1 mmol), 1-methylpiperidine-4-carboxylic acid (14.31 mg, 0.1 mmol), and ethyl isocyanoacetate (12.44 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 hours. The compound was purified by flash column chromatography to obtain compound 4-12-9 (77.53 mg, 70% yield). The reaction process is shown in Equation 9:

[0204]

[0205] Formula 9

[0206] The main data of the H-NMR spectrum of the ionizable lipid 4-12-9 are:

[0207] 1H NMR (500 MHz, Chloroform-d) δ 4.85 (s, 2H), 4.78 (d, J = 12.5 Hz, 2H), 4.49 (d, J = 12.3 Hz, 2H) , 4.16 (s, 2H), 3.92 (s, 2H), 2.84 (s, 2H),2.43 (d, J = 16.7 Hz, 1H), 2.31 (s, 4H), 2.25 – 2.15 (m, 7H), 1.91 (s, 2H),1.73 – 1.65 (m, 6H), 1.60 (s, 4H), 1.58 – 1.52 (m, 8H), 1.39 – 1.35 (m, 12H), 1.34 – 1.24 (m, 45H), 0.90 (s, 12H). Example 10

[0208] This Example 10 provides a method for preparing an ionizable lipid 4-12-12 having an α-acyloxyamide structure, wherein 4-12-12 is represented by the chemical formula (XIV):

[0209] (XIV)

[0210] The specific preparation process of ionizable lipid 4-12-12 is as follows:

[0211] 7,7'-Di(heptadodec-9-yl) O'1,O1-(2-oxopropane-1,3-diyl) di(pimelate) (85.13 mg, 0.1 mmol), 1-methylpiperidine-4-carboxylic acid (14.31 mg, 0.1 mmol), and 2-morpholinoethyl isocyanide (15.42 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 hours. The compound was purified by flash column chromatography to obtain compound 4-12-12 (74.88 mg, 66% yield). The reaction process is shown in Equation 10:

[0212]

[0213] Formula 10

[0214] The main data of the H NMR spectrum of the ionizable lipid 4-12-12 are:

[0215] 1H NMR (500 MHz, Chloroform-d) δ 4.85 (s, 2H), 4.78 (d, J = 12.5 Hz,2H), 4.49 (d, J = 12.3 Hz, 2H) , 3.61 (s, 4H), 3.45 – 3.29 (m, 2H), 2.84 (s,2H), 2.56 (s, 2H), 2.48 – 2.40 (m, 7H), 2.31 (s, 4H), 2.25 – 2.15 (m, 7H),1.91 (s, 2H), 1.73 – 1.65 (m, 6H), 1.62 – 1.52 (m, 12H), 1.39 – 1.35 (m,12H), 1.35 – 1.26 (m, 40H), 0.90 (s, 12H). Example 11

[0216] This Example 11 provides a method for preparing an ionizable lipid 1-2-12 having an α-acyloxyamide structure, wherein 1-2-12 is represented by chemical formula (XV):

[0217] (XV)

[0218] The specific preparation process of ionizable lipid 1-2-12 is as follows:

[0219] 2-Oxopropane-1,3-diyl (9Z, 9'Z, 12Z, 12'Z)-bis(octadecane-9,12-dioate) (61.50 mg, 0.1 mmol), 4-dimethylaminobutyric acid (13.12 mg, 0.1 mmol), and 2-morpholinoethyl isocyanide (15.42 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 hours. The compound was purified by flash column chromatography to obtain compound 1-2-12 (50.52 mg, 57% yield). The above reaction process is shown in Equation 11:

[0220]

[0221] Formula 11

[0222] The main data of the H NMR spectrum of ionizable lipid 1-2-12 are:

[0223] 1H NMR (500 MHz, Chloroform-d) δ 5.40 – 5.27 (m, 8H), 4.78 (d, J =11.8 Hz, 2H), 4.49 (d, J = 11.8 Hz, 2H), 3.61 (s, 4H), 3.41 (d, J = 12.4 Hz,1H), 3.32 (d, J = 12.4 Hz, 1H), 2.78 – 2.66 (m, 5H), 2.63 (d, J = 12.3 Hz,1H), 2.56 (s, 2H), 2.46 (s, 4H), 2.37 (s, 2H), 2.31 (s, 4H), 2.26 (s, 6H),2.08 – 2.01 (m, 8H), 1.90 (s, 2H), 1.59 (d, J = 1.1 Hz, 4H), 1.37 – 1.28 (m, 28H), 0.90 (s, 6H). Example 12

[0224] This Example 12 provides a method for preparing an ionizable lipid 1-3-12 having an α-acyloxyamide structure, wherein 1-3-12 is represented by the chemical formula (XVI):

[0225] (XVI)

[0226] The specific preparation process of ionizable lipid 1-3-12 is as follows:

[0227] 2-Oxopropane-1,3-diyl dioleate (66.71 mg, 0.1 mmol), 4-dimethylaminobutyric acid (13.12 mg, 0.1 mmol), and 2-morpholinoethyl isocyanate (15.42 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 h. The compound was purified by flash column chromatography to obtain compound 1-3-12 (52.53 mg, 59% yield). The above reaction is shown in Formula 12:

[0228]

[0229] Formula 12

[0230] The main data of the H-NMR spectrum of ionizable lipid 1-3-12 are:

[0231] 1H NMR (500 MHz, Chloroform-d) δ 5.38 – 5.29 (m, 4H), 4.79 (d, J =11.8 Hz, 2H), 4.50 (d, J = 11.8 Hz, 2H), 3.61 (s, 4H), 3.43 – 2.59 (m, 4H),2.56 (s, 2H), 2.46 (s, 4H), 2.37 (s, 2H), 2.31 (s, 4H), 2.26 (s, 6H), 2.02(s, 8H), 1.90 (s, 2H), 1.59 (s, 4H), 1.34 – 1.26 (m, 40H), 0.90 (s, 6H). Example 13

[0232] This Example 13 provides a method for preparing an ionizable lipid 1-4-12 having an α-acyloxyamide structure, wherein 1-4-12 is represented by chemical formula (XVII):

[0233] (XVII)

[0234] The specific preparation process of ionizable lipid 1-4-12 is as follows:

[0235] Dihydroxyacetone (1261 mg, 14 mmol), stearic acid (2844.8 mg, 10 mmol), 4-dimethylaminopyridine (244.34 mg, 2 mmol), N,N-diisopropylethylamine (1292.5 mg, 10 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1917 mg, 10 mmol) were dissolved in dichloromethane and stirred at room temperature for 24 h. The compound was purified by flash column chromatography to obtain 3-hydroxy-2-oxopropyl stearate (1104 mg, 31% yield). The above reaction is shown in the following formula:

[0236]

[0237] The main data of the H NMR spectrum of the compound 3-hydroxy-2-oxopropyl stearate are:

[0238] 1 H NMR (500 MHz, Chloroform-d) δ 4.76 (s, 2H), 4.16 (d, J = 5.0 Hz,2H), 2.31 (s, 2H), 1.34 – 1.25 (m, 30H), 0.90 (s, 3H).

[0239] 2,2-Disulfide dipyridine (24234 mg, 110 mmol), n-dodecyl mercaptan (18837 mg, 100 mmol) and 4-dimethylaminopyridine (24434 mg, 200 mmol) were dissolved in dichloromethane and stirred at room temperature for 24 h. The compound was purified by flash column chromatography to obtain compound 2-(undecyldisulfanyl)pyridine (19637 mg, yield 66%).

[0240] The main data of the H NMR spectrum of compound 2-(undecyldisulfanyl)pyridine are:

[0241] 1 H NMR (500 MHz, Chloroform-d) δ 8.45 (dd, J = 5.0, 1.2 Hz, 1H), 7.69 (td, J = 8.0, 1.2 Hz, 1H), 7.30 (dd, J = 8.0, 0.8 Hz, 1H), 7.19 (ddd, J =8.1, 5.0, 1.1 Hz, 1H), 2.87 (s, 2H), 1.39 (s, 2H), 1.32 (d, J = 2.4 Hz, 4H), 1.30 – 1.26 (m, 8H), 1.27 (d, J = 1.5 Hz, 4H), 0.90 (s, 3H).

[0242] The compound 2-(undecyldisulfanyl)pyridine was dissolved in dichloromethane, and 3-mercaptopropionic acid (8491.2 mg, 80 mmol) and 4-dimethylaminopyridine (24434 mg, 200 mmol) were added. The mixture was stirred at room temperature for 24 hours. The compound was purified by flash column chromatography to obtain the compound 3-(dodecyldisulfanyl)propionic acid (16367 mg, 81% yield). The above reaction is shown in the following formula:

[0243]

[0244] The main data of the H NMR spectrum of compound 3-(dodecyldisulfanyl)propionic acid are:

[0245] 1 H NMR (500 MHz, Chloroform-d) δ 3.13 (s, 2H), 2.77 (s, 2H), 2.64 (s,2H), 1.39 (s, 2H), 1.32 (d, J = 2.4 Hz, 4H), 1.30 – 1.25 (m, 16H), 0.90 (s,3H).

[0246] The compound 3-hydroxy-2-oxopropyl stearate (1068.87 mg, 3 mmol), 3-(dodecyldisulfanyl)propionic acid (1010.36 mg, 3.3 mmol), 4-dimethylaminopyridine (73.03 mg, 0.6 mmol), N,N-diisopropylethylamine (426.52 mg, 3.3 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (630.86 mg, 3.3 mmol) were dissolved in dichloromethane and stirred at room temperature for 24 hours. The compound was purified by flash column chromatography to obtain the compound 2-oxopropane-1,3-diylbis(3-dodecyldisulfanyl)propionate (1546.68 mg, 80% yield). The above reaction process is shown in the following formula:

[0247]

[0248] The main data of the H NMR spectrum of the compound 2-oxopropane-1,3-diylbis(3-dodecyldisulfanyl)propionate are:

[0249] 1 H NMR (500 MHz, Chloroform-d) δ 4.70 (d, J = 1.3 Hz, 4H), 3.16 (s,2H), 2.77 (s, 2H), 2.65 (s, 2H), 2.32 (s, 2H), 1.61 (d, J = 17.8 Hz, 4H), 1.39 (s, 2H), 1.32 (d, J = 2.4 Hz, 9H), 1.29 – 1.25 (m, 35H), 0.90 (s, 6H).

[0250] 2-Oxopropane-1,3-diylbis(3-dodecyldisulfanyl)propionate (64.51 mg, 0.1 mmol), 4-dimethylaminobutyric acid (13.12 mg, 0.1 mmol), and 2-morpholinoethyl isocyanate (15.42 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 hours. The compound was purified by flash column chromatography to obtain compound 1-4-12 (63.10 mg, 69% yield). The above reaction process is shown in Formula 13:

[0251]

[0252] Formula 13

[0253] The main data of the H NMR spectrum of ionizable lipid 1-4-12 are:

[0254] 1H NMR (500 MHz, Chloroform-d) δ 4.75 (dd, J = 12.4, 11.0 Hz, 2H), 4.49 (dd, J = 12.4, 5.8 Hz, 2H), 3.62 (d, J = 10.4 Hz, 4H), 3.44 – 3.29 (m,2H), 3.16 (d, J = 3.7 Hz, 2H), 2.83 – 2.71 (m, 2H), 2.71 – 2.66 (m, 4H), 2.56 (d, J = 2.4 Hz, 2H), 2.46 (d, J = 16.1 Hz, 4H), 2.37 (s, 2H), 2.31 (s, 2H),2.26 (s, 6H), 1.90 (s, 2H), 1.66 – 1.58 (m, 4H), 1.39 (s, 2H), 1.36 – 1.25 (m, 44H), 0.90 (s, 6H). Example 14

[0255] This Example 14 provides a method for preparing an ionizable lipid 1-6-12 having an α-acyloxyamide structure, wherein 1-6-12 is represented by the chemical formula (XVIII):

[0256] (XVIII)

[0257] The specific preparation process of ionizable lipid 1-6-12 is as follows:

[0258] Dihydroxyacetone (1261 mg, 14 mmol), oleic acid (2824.7 mg, 10 mmol), 4-dimethylaminopyridine (244.34 mg, 2 mmol), N,N-diisopropylethylamine (1292.5 mg, 10 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1917 mg, 10 mmol) were dissolved in dichloromethane and stirred at room temperature for 24 h. The compound was purified by flash column chromatography to obtain the compound 3-hydroxy-2-oxopropyl oleate (1240 mg, 35% yield). The above reaction is shown in the following formula:

[0259]

[0260] The main data of the H NMR spectrum of the compound 3-hydroxy-2-oxopropyl oleate are:

[0261] 1H NMR (500 MHz, Chloroform-d) δ 5.35 (s, 2H), 4.76 (s, 2H), 4.16 (d,J = 5.0 Hz, 2H), 3.35 (t, J = 4.9 Hz, 2H), 2.31 (s, 2H), 2.02 (s, 4H), 1.34 –1.26 (m, 20H), 0.90 (s, 3H).

[0262] The compound 3-hydroxy-2-oxopropyl oleate (1063.59 mg, 3 mmol), 3-(dodecyldisulfonyl)propionic acid (1010.36 mg, 3.3 mmol), 4-dimethylaminopyridine (73.03 mg, 0.6 mmol), N,N-diisopropylethylamine (426.52 mg, 3.3 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (630.86 mg, 3.3 mmol) were dissolved in dichloromethane and stirred at room temperature for 24 hours. The compound was purified by flash column chromatography to obtain the compound 3-((3-dodecyldisulfonyl)propionyl)oxy)-2-oxopropyl oleate (1137 mg, 59% yield). The above reaction process is shown below:

[0263]

[0264] The main data of the H NMR spectrum of the compound 3-((3-dodecyldisulfonyl)propionyl)oxy)-2-oxopropyl oleate are:

[0265] 1 H NMR (500 MHz, Chloroform-d) δ 5.35 (s, 2H), 4.70 (d, J = 1.3 Hz,4H), 3.16 (s, 2H), 2.77 (s, 2H), 2.65 (s, 2H), 2.32 (s, 2H), 2.02 (s, 4H), 1.61 (d, J = 17.8 Hz, 4H), 1.39 (s, 2H), 1.34 – 1.25 (m, 36H), 0.90 (s, 6H).

[0266] 3-((3-dodecyldisulfonyl)propionyl)oxy)-2-oxopropyl oleate (64.30 mg, 0.1 mmol), 4-dimethylaminobutyric acid (13.12 mg, 0.1 mmol), and 2-morpholinoethyl isocyanide (15.42 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 hours. The compound was purified by flash column chromatography to obtain compound 1-6-12 (64.92 mg, 71% yield). The above reaction process is shown in Formula 14:

[0267]

[0268] Formula 14

[0269] The main data of the H-NMR spectrum of ionizable lipid 1-6-12 are:

[0270] 1 H NMR (500 MHz, Chloroform-d) δ 5.35 (s, 2H), 4.75 (dd, J = 12.4,11.0 Hz, 2H), 4.63 (dd, J = 12.4, 5.8 Hz, 2H), 3.62 (d, J = 10.4 Hz, 4H),3.45 – 3.28 (m, 2H), 3.16 (d, J = 3.7 Hz, 2H), 2.83 – 2.71 (m, 2H), 2.71 –2.61 (m, 4H), 2.56 (d, J = 2.4 Hz, 2H), 2.46 (d, J = 16.1 Hz, 4H), 2.37 (s,2H), 2.31 (s, 2H), 2.26 (s, 6H), 2.02 (s, 4H), 1.90 (s, 2H), 1.66 – 1.58 (m, 4H), 1.39 (s, 2H), 1.36 – 1.24 (m, 36H), 0.90 (s, 6H). Example 15

[0271] This Example 15 provides a method for preparing an ionizable lipid 1-8-12 having an α-acyloxyamide structure, wherein 1-8-12 is represented by the chemical formula (XIX):

[0272] (XIX)

[0273] The specific preparation process of ionizable lipid 1-8-12 is as follows:

[0274] 7-Pentadecanol (22841 mg, 100 mmol), pimelic acid (17618 mg, 110 mmol), 4-dimethylaminopyridine (244.3 mg, 20 mmol), N,N-diisopropylethylamine (15510 mg, 120 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (23004 mg, 120 mmol) were dissolved in dichloromethane and stirred at room temperature for 24 hours. The compound was purified by flash column chromatography to obtain 7-oxo-7-(pentadecan-7-yloxy)heptanoic acid (27030 mg, 73% yield). The reaction process is shown below:

[0275]

[0276] The main data of the H NMR spectrum of the compound 7-oxo-7-(pentadecan-7-yloxy)heptanoic acid are:

[0277] 1 H NMR (500 MHz, Chloroform-d) δ 4.78 (s, 1H), 2.24 (d, J = 1.8 Hz,2H), 2.20 (d, J = 7.5 Hz, 2H), 1.68 (d, J = 12.5 Hz, 2H), 1.60 – 1.52 (m,4H), 1.39 (s, 4H), 1.37 (s, 2H), 1.39 – 1.30 (m, 4H), 1.33 – 1.26 (m, 14H), 0.90 (s, 6H).

[0278] Dihydroxyacetone (1261 mg, 14 mmol), linoleic acid (2804.5 mg, 10 mmol), 4-dimethylaminopyridine (244.34 mg, 2 mmol), N,N-diisopropylethylamine (1292.5 mg, 10 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1917 mg, 10 mmol) were dissolved in dichloromethane and stirred at room temperature for 24 h. The compound was purified by flash column chromatography to obtain the compound 3-hydroxy-2-oxopropyl (9Z, 12Z)-octadec-9,12-dienoate (1233 mg, 35% yield). The above reaction is shown in the following formula:

[0279]

[0280] The main data of the H NMR spectrum of the compound 3-hydroxy-2-oxopropyl (9Z, 12Z)-octadec-9,12-dienoate are:

[0281] 1 H NMR (500 MHz, Chloroform-d) δ 5.37 (s, 2H), 5.33 (s, 2H), 4.76 (s,2H), 4.16 (d, J = 5.0 Hz, 2H), 2.78 – 2.66 (m, 2H), 2.31 (s, 2H), 2.04 (dt, J= 11.2, 1.0 Hz, 4H), 1.35 – 1.28 (m, 12H), 0.90 (s, 3H).

[0282] Compound 3-hydroxy-2-oxopropyl (9Z, 12Z)-octadec-9,12-dienoate (1057.53 mg, 3 mmol), compound 7-oxo-7-(pentadecan-7-yloxy)heptanoic acid (1222.02 mg, 3.3 mmol), 4-dimethylaminopyridine (73.03 mg, 0.6 mmol), N,N-diisopropylethylamine (426.52 mg, 3.3 mmol) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (630.86 mg, 3.3 mmol) was dissolved in dichloromethane and stirred at room temperature for 24 hours. The compound was purified by flash column chromatography to obtain 1-(3-(((9Z,12Z)-octadec-9,12-dienoyl)oxy)-2-oxopropyl)-7-(pentadec-7-yl)heptanedioate (1248 mg, 59% yield). The reaction process is shown below:

[0283]

[0284] The main data of the H NMR spectrum of the compound 1-(3-(((9Z,12Z)-octadec-9,12-dienoyl)oxy)-2-oxopropyl)7-(pentadecyl-7-yl)heptanedioate are:

[0285] 1H NMR (500 MHz, Chloroform-d) δ 5.37 (s, 2H), 5.33 (s, 2H), 4.78 (s,1H), 4.69 (d, J = 3.3 Hz, 4H), 2.78 – 2.66 (m, 2H), 2.32 (s, 4H), 2.25 – 2.15(m, 2H), 2.08 – 2.01 (m, 4H), 1.68 (d, J = 12.5 Hz, 2H), 1.64 – 1.58 (m, 4H), 1.58 – 1.52 (m, 4H), 1.40 – 1.37 (m, 6H), 1.35 (d, J = 1.3 Hz, 2H), 1.34 –1.26 (m, 28H), 0.90 (s, 9H).

[0286] 1-(3-(((9Z,12Z)-octadec-9,12-dienoyl)oxy)-2-oxopropyl)-7-(pentadec-7-yl) heptanoate (70.51 mg, 0.1 mmol), 4-dimethylaminobutyric acid (13.12 mg, 0.1 mmol), and 2-morpholinoethyl isocyanide (15.42 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 hours. The compound was purified by flash column chromatography to obtain compound 1-8-12 (60.53 mg, 62% yield). The above reaction is shown in Formula 15:

[0287]

[0288] Formula 15

[0289] The main data of the H NMR spectrum of ionizable lipid 1-8-12 are:

[0290] 1H NMR (500 MHz, Chloroform-d) δ 5.37 (s, 2H), 5.33 (s, 2H), 4.78 (s,1H), 4.75 (s, 2H), 4.49 (d, J = 12.3 Hz, 2H), 3.62 (d, J = 10.4 Hz, 4H), 3.45– 3.26 (m, 2H), 2.77 – 2.60 (m, 4H), 2.56 (d, J = 2.4 Hz, 2H), 2.46 (d, J =16.1 Hz, 4H), 2.37 (s, 2H), 2.31 (d, J = 1.5 Hz, 4H), 2.26 (s, 6H), 2.20 (d,J = 7.5 Hz, 2H), 2.08 – 2.01 (m, 4H), 1.90 (s, 2H), 1.68 (d, J = 12.5 Hz, 2H), 1.64 – 1.57 (m, 6H), 1.55 (d, J = 12.5 Hz, 2H), 1.39 (s, 2H), 1.38 –1.26 (m, 34H), 0.90 (s, 9H). Example 16

[0291] This Example 16 provides a method for preparing an ionizable lipid 1-9-12 having an α-acyloxyamide structure, wherein 1-9-12 is represented by the chemical formula (XX):

[0292] (XX)

[0293] The specific preparation process of ionizable lipid 1-9-12 is as follows:

[0294] The compound 3-hydroxy-2-oxopropyl stearate (1068.87 mg, 3 mmol), 7-(heptadodec-9-yloxy)-7-oxoheptanoic acid (1315.48 mg, 3.3 mmol), 4-dimethylaminopyridine (73.03 mg, 0.6 mmol), N,N-diisopropylethylamine (426.52 mg, 3.3 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (630.86 mg, 3.3 mmol) were dissolved in dichloromethane and stirred at room temperature for 24 hours. The compound was purified by flash column chromatography to obtain the compound 1-(heptadodec-9-yl) 7-(2-oxo-3-(stearoyloxy)propyl) heptanoate (1747 mg, 79% yield). The reaction process is shown below:

[0295]

[0296] The main data of the H NMR spectrum of compound 1-(heptadodec-9-yl) 7-(2-oxo-3-(stearoyloxy)propyl) heptanoate are:

[0297] 1 H NMR (500 MHz, Chloroform-d) δ 4.78 (s, 1H), 4.69 (s, 4H), 2.32 (s,4H), 2.20 (s, 2H), 1.68 (d, J = 12.5 Hz, 2H), 1.61 – 1.52 (m, 8H), 1.39 –1.35 (m, 6H), 1.35 – 1.25 (m, 49H), 0.90 (s, 9H).

[0298] 1-(Heptadodec-9-yl)-7-(2-oxo-3-(stearoyloxy)propyl)heptanedioate (73.71 mg, 0.1 mmol), 4-dimethylaminobutyric acid (13.12 mg, 0.1 mmol), and 2-morpholinoethyl isocyanide (15.42 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 hours. The compound was purified by flash column chromatography to obtain compound 1-9-12 (63.53 mg, 63% yield). The above reaction process is shown in Equation 16:

[0299]

[0300] Formula 16

[0301] The main data of the H-NMR spectrum of ionizable lipid 1-9-12 are:

[0302] 1H NMR (500 MHz, Chloroform-d) δ 4.78 (s, 1H), 4.76 (d, J = 12.5 Hz, 2H), 4.62 (d, J = 12.3 Hz, 2H), 3.62 (d, J = 10.4 Hz, 4H), 3.44 – 3.27 (m,2H), 2.72 – 2.54 (m, 4H), 2.46 (d, J = 16.1 Hz, 4H), 2.37 (s, 2H), 2.31 (d, J= 1.5 Hz, 4H), 2.26 (s, 6H), 2.20 (d, J = 7.5 Hz, 2H), 1.90 (s, 2H), 1.68 (d,J = 12.5 Hz, 2H), 1.64 – 1.52 (m, 8H), 1.39 – 1.34 (m, 6H), 1.34 – 1.31 (m,12H), 1.31 – 1.25 (m, 36H), 0.90 (s, 9H). Example 17

[0303] This Example 17 provides a method for preparing an ionizable lipid 1-10-12 having an α-acyloxyamide structure, wherein 1-10-12 is represented by the chemical formula (XXI):

[0304] (XXI)

[0305] The specific preparation process of ionizable lipid 1-10-12 is as follows:

[0306] Compound 3-hydroxy-2-oxopropyl (9Z, 12Z)-octadec-9,12-dienoate (1057.53 mg, 3 mmol), compound 7-(heptadodec-9-yloxy)-7-oxoheptanoic acid (1315.48 mg, 3.3 mmol), 4-dimethylaminopyridine (73.03 mg, 0.6 mmol), N,N-diisopropylethylamine (426.52 mg, 3.3 mmol) The product was dissolved in dichloromethane and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (630.86 mg, 3.3 mmol). The mixture was stirred at room temperature for 24 hours. The compound was purified by flash column chromatography to obtain 1-(heptadodec-9-yl) 7-(3-((9Z,12Z)-octadecyl-9,12-dienoyl)oxy)-2-oxopropyl) heptanoate (1363 mg, 62% yield). The reaction process is shown below:

[0307]

[0308] The main data of the H NMR spectrum of the compound 1-(heptadodec-9-yl) 7-(3-((9Z,12Z)-octadecyl-9,12-dienoyl)oxy)-2-oxopropyl) heptanoate are:

[0309] 1 H NMR (500 MHz, Chloroform-d) δ 5.37 (s, 2H), 5.33 (s, 2H), 4.78 (s,1H), 4.69 (s, 4H), 2.72 (dt, J = 17.4, 0.9 Hz, 1H), 2.32 (s, 4H), 2.20 (s,2H), 2.04 (dt, J = 11.2, 1.0 Hz, 4H), 1.68 (d, J = 12.5 Hz, 2H), 1.62 – 1.54(m, 7H), 1.39 – 1.34 (m, 6H), 1.34 – 1.26 (m, 34H), 0.90 (s, 9H).

[0310] 1-(Heptadodec-9-yl)-7-(3-((9Z,12Z)-octadecyl-9,12-dienoyl)oxy)-2-oxopropyl) heptanoate (73.31 mg, 0.1 mmol), 4-dimethylaminobutyric acid (13.12 mg, 0.1 mmol), and 2-morpholinoethyl isocyanide (15.42 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 hours. The compound was purified by flash column chromatography to obtain compound 1-10-12 (65.29 mg, 65% yield). The reaction process is shown in Equation 17:

[0311]

[0312] Formula 17

[0313] The main data of the H-NMR spectrum of ionizable lipid 1-10-12 are:

[0314] 1H NMR (500 MHz, Chloroform-d) δ 5.35 (d, J = 20.1 Hz, 4H), 4.78 (s,1H), 4.76 (d, J = 12.5 Hz, 2H), 4.62 (d, J = 12.3 Hz, 2H), 3.62 (d, J = 10.4Hz, 4H), 3.44 – 3.30 (m, 2H), 2.78 – 2.60 (m, 4H), 2.56 (d, J = 2.4 Hz, 2H), 2.46 (d, J = 16.1 Hz, 4H), 2.37 (s, 2H), 2.31 (d, J = 1.5 Hz, 4H), 2.26 (s,6H), 2.24 – 2.15 (m, 2H), 2.08 – 2.01 (m, 4H), 1.90 (s, 2H), 1.71 – 1.52 (m,10H), 1.39 – 1.35 (m, 6H), 1.34 – 1.26 (m, 34H), 0.90 (s, 9H). Example 18

[0315] This Example 18 provides a method for preparing an ionizable lipid 1-12-9 having an α-acyloxyamide structure, wherein 1-12-9 is represented by the chemical formula (XXII):

[0316] (XXII)

[0317] The specific preparation process of ionizable lipid 1-12-9 is as follows:

[0318] 7,7'-Di(heptadodec-9-yl) O'1,O1-(2-oxopropane-1,3-diyl) di(pimelate) (85.13 mg, 0.1 mmol), 4-dimethylaminobutyric acid (13.12 mg, 0.1 mmol), and ethyl isocyanoacetate (12.44 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 hours. The compound was purified by flash column chromatography to obtain compound 1-12-9 (72.25 mg, 66% yield). The reaction process is shown in Equation 18:

[0319]

[0320] Formula 18

[0321] The main data of the H NMR spectrum of ionizable lipid 1-12-9 are:

[0322] 1H NMR (500 MHz, Chloroform-d) δ 4.78 (s, 2H), 4.74 (d, J = 12.5 Hz,2H), 4.63 (d, J = 12.3 Hz, 2H), 4.16 (s, 2H), 3.92 (s, 2H), 2.73 – 2.60 (m,2H), 2.37 (s, 2H), 2.31 (s, 4H), 2.26 (s, 6H), 2.24 – 2.15 (m, 4H), 1.90 (s,2H), 1.68 (d, J = 12.5 Hz, 4H), 1.62 – 1.52 (m, 12H), 1.39 – 1.34 (m, 12H),1.34 – 1.24 (m, 43H), 0.90 (s, 12H). Example 19

[0323] This Example 19 provides a method for preparing an ionizable lipid 1-12-12 having an α-acyloxyamide structure, wherein 1-12-12 is represented by the chemical formula (XXIII):

[0324] (XXIII)

[0325] The specific preparation process of ionizable lipid 1-12-12 is as follows:

[0326] 7,7'-Di(heptadodec-9-yl) O'1,O1-(2-oxopropane-1,3-diyl) di(pimelate) (85.13 mg, 0.1 mmol), 4-dimethylaminobutyric acid (13.12 mg, 0.1 mmol), and 2-morpholinoethyl isocyanide (15.42 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 hours. The compound was purified by flash column chromatography to obtain compound 1-12-12 (77.46 mg, 69% yield). The reaction process is shown in Equation 19:

[0327]

[0328] Formula 19

[0329] The main data of the H NMR spectrum of ionizable lipid 1-12-12 are:

[0330] 1H NMR (500 MHz, Chloroform-d) δ 4.78 (s, 2H), 4.76 (d, J = 12.5 Hz,2H), 4.62 (d, J = 12.5 Hz, 2H), 3.61 (s, 4H), 3.44 – 3.29 (m, 2H), 2.73 –2.60 (m, 2H), 2.56 (s, 2H), 2.46 (s, 4H), 2.37 (s, 2H), 2.31 (s, 4H), 2.26(s, 6H), 2.20 (d, J = 7.5 Hz, 4H), 1.90 (s, 2H), 1.71 – 1.52 (m, 16H), 1.40 –1.26 (m, 52H), 0.90 (s, 12H). Example 20

[0331] This Example 20 provides a method for preparing an ionizable lipid 6-12-9 having an α-acyloxyamide structure, wherein 6-12-9 is represented by the chemical formula (XXIV):

[0332] (XXIV)

[0333] The specific preparation process of ionizable lipid 6-12-9 is as follows:

[0334] 7,7'-Di(heptadodec-9-yl) O'1,O1-(2-oxopropane-1,3-diyl) di(pimelate) (85.13 mg, 0.1 mmol), 4-((2-hydroxyethyl)(methyl)amino)butanoic acid (16.12 mg, 0.1 mmol), and ethyl isocyanoacetate (12.44 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 hours. The compound was purified by flash column chromatography to obtain compound 6-12-9 (68.66 mg, 61% yield). The reaction process is shown in Equation 20:

[0335]

[0336] Formula 20

[0337] The main data of the H-NMR spectrum of ionizable lipid 6-12-9 are:

[0338] 1H NMR (500 MHz, Chloroform-d) δ 4.78 (s, 2H), 4.74 (d, J = 12.5 Hz,2H), 4.63 (d, J = 12.3 Hz, 2H), 4.16 (s, 2H), 3.92 (s, 2H), 2.78 (s, 2H),2.32 (d, J = 15.0 Hz, 6H), 2.29 – 2.15 (m, 9H), 1.89 (s, 1H), 1.71 – 1.65 (m,6H), 1.62 – 1.52 (m, 12H), 1.39 – 1.24 (m, 57H), 0.90 (s, 12H).

[0339] Reference Example 1

[0340] Reference Example 1 provides a method for preparing an ionizable lipid H18A4B4 having an α-acyloxyamide structure. H18A4B4 is shown in Chemical Formula (XXV):

[0341] (XXV)

[0342] The specific preparation process of ionizable lipid H18A4B4 is as follows:

[0343] Compound 6-hydroxyhexanal (3485 mg, 30 mmol), compound 2-hexyl undecanoic acid (8462 mg, 33 mmol), 4-dimethylaminopyridine (730 mg, 6 mmol), N,N-diisopropylethylamine (4265 mg, 33 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6309 mg, 33 mmol) were dissolved in dichloromethane and stirred at room temperature for 24 hours. The compound was purified by flash column chromatography to obtain compound 6-oxooxy 2-hexyldecanoate (6061 mg, yield 57%). The above reaction process is shown in the following formula:

[0344]

[0345] The main data of the H NMR spectrum of compound 6-oxooxy 2-hexyldecanoate are:

[0346] 1H NMR (500 MHz, Chloroform-d) δ 9.57 (s, 1H), 4.15 – 4.05 (m, 2H), 2.57 – 2.48 (m, 2H), 2.29 (s, 1H), 1.67 (s, 2H), 1.61 (s, 2H), 1.54 (s, 1H), 1.51 – 1.44 (m, 2H), 1.42 (d, J = 7.7 Hz, 2H), 1.37 – 1.32 (m, 4H), 1.32 –1.26 (m, 16H), 0.90 (s, 6H).

[0347] 6-Oxooxy 2-hexyldecanoate (35.44 mg, 0.1 mmol), 4-((2-hydroxyethyl)(methyl)amino)butanoic acid (16.12 mg, 0.1 mmol), and (Z)-1-isocyanooctadec-9-ene (30.53 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 hours. The compound was purified by flash column chromatography to obtain compound H18A4B4 (43.35 mg, 59% yield). The reaction process is shown in Equation 22:

[0348]

[0349] Formula 22

[0350] The main data of the H NMR spectrum of the ionizable lipid H18A4B4 are:

[0351] 1 H NMR (500 MHz, Chloroform-d) δ 5.35 (s, 2H), 4.82 (s, 1H), 4.15 –4.05 (m, 2H), 3.20 – 3.10 (m, 2H), 2.74 – 2.59 (m, 2H), 2.38 – 2.27 (m, 2H),2.26 (s, 6H), 2.02 (s, 4H), 1.91 (d, J = 1.6 Hz, 2H), 1.86 (d, J = 0.9 Hz,2H), 1.65 (d, J = 1.6 Hz, 2H), 1.59 – 1.52 (m, 2H), 1.52 – 1.46 (m, 4H), 1.46– 1.38 (m, 4H), 1.38 – 1.33 (m, 5H), 1.33 – 1.26 (m, 38H), 0.90 (s, 9H).

[0352] Reference Example 2

[0353] Reference Example 2 provides a method for preparing an ionizable lipid 2 (sometimes also referred to as "Compound 2") having an α-acyloxyamide structure. The ionizable lipid 2 is shown in Chemical Formula (XXVI):

[0354] (XXVI)

[0355] The specific preparation process of ionizable lipid 2 is as follows:

[0356] 5-Hydroxypentanoic acid (3544 mg, 30 mmol), 2-hexylundecanoic acid (6611 mg, 33 mmol), 4-dimethylaminopyridine (730 mg, 6 mmol), N,N-diisopropylethylamine (4265 mg, 33 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6309 mg, 33 mmol) were dissolved in dichloromethane and stirred at room temperature for 24 hours. The compound was purified by flash column chromatography to obtain 5-((2-butyloctanoyl)oxy)pentanoic acid)pentanoic acid (5140 mg, 57% yield). The above reaction process is shown in the following formula:

[0357]

[0358] The main data of the H NMR spectrum of compound 5-((2-butyloctanoyl)oxy)pentanoic acid)pentanoic acid are:

[0359] 1 H NMR (500 MHz, Chloroform-d) δ 4.11 (d, J = 2.4 Hz, 2H), 2.30 (s,2H), 2.28 (s, 1H), 1.78 – 1.68 (m, 2H), 1.65 – 1.41 (m, 7H), 1.37 – 1.26 (m,13H), 0.90 (d, J = 3.5 Hz, 6H).

[0360] Dihydroxyacetone (2252 mg, 25 mmol), 5-((2-butyloctanoyl)oxy)pentanoic acid)pentanoic acid (17275 mg, 57.5 mmol), 4-dimethylaminopyridine (610.85 mg, 5 mmol), N,N-diisopropylethylamine (7431.87 mg, 57.5 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (11022.75 mg, 57.5 mmol) were dissolved in dichloromethane and stirred at room temperature for 24 hours. The compound was purified by flash column chromatography to obtain ((2-oxopropane-1,3-diyl)bis(oxy))bis(5-oxopentan-5,1-diyl)bis(2-butyloctanoate) (6381 mg, 30% yield). The reaction process is shown below:

[0361]

[0362] The main data of the H NMR spectrum of the compound ((2-oxopropane-1,3-diyl)bis(oxy))bis(5-oxopentan-5,1-diyl)bis(2-butyloctanoate) are:

[0363] 1 H NMR (500 MHz, Chloroform-d) δ 4.69 (d, J = 3.3 Hz, 4H), 4.11 (d, J= 2.4 Hz, 4H), 2.39 – 2.30 (m, 4H), 2.28 (s, 2H), 1.79 – 1.69 (m, 4H), 1.65 (s, 4H), 1.59 – 1.41 (m, 9H), 1.37 – 1.26 (m, 25H), 0.90 (d, J = 3.5 Hz, 12H).

[0364] N-nonylamine (1433 mg, 10 mmol) and ethyl formate (3704 mg, 50 mmol) were heated and stirred at 60°C for 24 hours. After the reaction, the solvent ethyl formate and the by-product ethanol were removed, and triethylamine (5059 mg, 50 mmol) was added. After dissolution in dichloromethane, phosphorus oxychloride (1533.3 mg, 10 mmol) was slowly added dropwise in an ice-water bath. After the addition was complete, the reaction was continued for 2 hours. The compound was purified by flash column chromatography to obtain 1-isocyanamide (1181 mg, 77% yield). The reaction process is shown below.

[0365]

[0366] The main data of the H NMR spectrum of compound 1-isocyanamide are:

[0367] 1H NMR (500 MHz, Chloroform-d) δ 5.32 (s, 2H), 2.25 (s, 2H), 1.36 (s,2H), 1.33 – 1.26 (m, 10H), 0.90 (s, 3H).

[0368] Ethyl 4-bromobutyrate (1950.5 mg, 10 mmol), diethylamine (804 mg, 11 mmol) and N,N-diisopropylethylamine (1421.75 mg, 11 mmol) were dissolved in ethanol and reacted at 40°C for 18 h. The compound 4-(diethylamino)butyric acid ethyl ester was purified by flash column chromatography.

[0369] Dissolve ethyl 4-(diethylamino)butyrate in 5 volumes of 10M hydrochloric acid solution and react at 110°C for at least 4 hours. TLC confirms complete reaction. Remove the HCl on a rotary evaporator to obtain 4-(diethylamino)butyric acid (1115 mg, 70% yield). The reaction process is shown below:

[0370]

[0371] The main data of the H NMR spectrum of compound 4-(diethylamino)butyric acid are:

[0372] 1 H NMR (500 MHz, Chloroform-d) δ 2.57 (s, 6H), 2.31 (s, 2H), 1.77 (s,2H), 1.01 (s, 6H).

[0373] The compound ((2-oxopropane-1,3-diyl)bis(oxy)bis(5-oxypentane-5,1-diyl)bis(2-butyloctanoate) (65.49 mg, 0.1 mmol), 4-(diethylamino)butyric acid (15.23 mg, 0.1 mmol), and 1-isocyanamide (16.86 mg, 0.11 mmol) were dissolved in 1 mL of chloroform and stirred at 55°C for 72 hours. The compound was purified by flash column chromatography to obtain compound 2 (43.35 mg, 59% yield). The above reaction process is shown in Equation 22:

[0374]

[0375] Formula 22

[0376] The main data of the H NMR spectrum of ionizable lipid 2 are:

[0377] 1H NMR (500 MHz, Chloroform-d) δ 4.76 (d, J = 12.5 Hz, 2H), 4.62 (d,J = 12.5 Hz, 2H), 4.11 (d, J = 2.4 Hz, 4H), 3.21 (d, J = 2.8 Hz, 2H), 2.56(dd, J = 9.4, 1.0 Hz, 6H), 2.43 – 2.31 (m, 6H), 2.28 (s, 2H), 1.84 (d, J =4.2 Hz, 2H), 1.79 – 1.69 (m, 4H), 1.65 (s, 4H), 1.59 – 1.40 (m, 10H), 1.38 –1.26 (m, 36H), 1.01 (s, 6H), 0.90 (d, J = 3.5 Hz, 15H).

[0378] Example 21 Preparation of lipid nanoparticles

[0379] The ionizable lipids, auxiliary phospholipids (distearoylphosphatidylcholine (DSPC), Shanghai MacLean Biochemical Technology Co., Ltd.), cholesterol (Beijing Puxitang Biotechnology Co., Ltd.), and PEGylated hydrophilic polymer lipids (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 DMG-PEG2000, Beijing Puxitang Biotechnology Co., Ltd.) prepared above were dissolved in anhydrous ethanol to prepare four lipid premixes. These premixes were thoroughly mixed at a predetermined molar ratio (50:10:38.5:1.5) to form a four-component lipid phase. A certain amount of Luci-mRNA stock solution expressing luciferase was diluted in citric acid-sodium citrate buffer (pH 4) to a mass ratio of 1:40 and a volume ratio of 3:1 to the lipid phase to prepare an aqueous phase. The lipid phase was added to the aqueous phase, mixed thoroughly, vortexed for 5 seconds, and incubated at 20-25°C for 15 minutes to form mRNA-encapsulated lipid nanoparticles. For in vitro testing, the assembled lipid nanoparticles can be used directly. For mouse experiments, the assembled lipid nanoparticles should be dialyzed against deionized water for 4 hours, ultrafiltered, and diluted to a fixed volume before use.

[0380] The obtained lipid nanoparticles were subjected to particle size analysis (using a Malvern particle size analyzer to measure the particle size and polydispersity index PDI) and morphology analysis (using a transmission electron microscope to photograph the morphology of the lipid nanoparticles). The results of lipid nanoparticles containing ionizable lipid 4-3-9 are shown in Figure 1 middle.

[0381] according to Figure 1As can be seen, the diameter of the lipid nanoparticles constructed with 4-3-9 before ultrafiltration was 187 nm as measured by a particle size analyzer, and transmission electron microscopy images showed that the nanoparticles exhibited a spherical, multilayered morphology. After ultrafiltration, the particle size increased slightly to 202 nm, but still maintained a low PDI value, indicating that the lipid nanoparticles constructed with 4-3-9 maintained a uniform size after ultrafiltration and a good assembly effect. Transmission electron microscopy images showed that the nanoparticles still exhibited a spherical, multilayered structure. These results demonstrate that the nanoparticles constructed with the ionizable lipids of the present invention have good shear stress tolerance, thus maintaining their assembled structure and exhibiting good stability during in vivo use.

[0382] Example 22 In vitro transfection studies of ionizable lipids

[0383] This experiment used human embryonic kidney cells (HEK 293T cells) as a cell model, Luci-mRNA (Changchun Jinchuan Technology Co., Ltd.) as a model nucleic acid drug, and commercial lipid SM-102 (Shanghai MacLean Biochemical Technology Co., Ltd.) as a positive control. SM-102 and the previously prepared ionizable lipids were all prepared using the method of Example 21 to prepare lipid nanoparticles encapsulating Luci-mRNA. HEK 293T cells were plated at 1.5×10 cells per well. 4 The cells were seeded at a density of 100 cells / well in a 96-well plate and cultured for 24 h. According to the amount of 0.2 μg mRNA per well, lipid nanoparticles loaded with Luci-mRNA were quantitatively added to each well, and three parallel groups were set up for each group. After incubation for 24 h, the DMEM medium in the well (high glucose DMEM medium, Dalian Meilun Biotechnology Co., Ltd.) was discarded, and 50 μL of firefly luciferase reporter gene cell lysate (Shanghai Biyuntian Biotechnology Co., Ltd.) was added to each well for cell lysis. Subsequently, luciferase substrate was added, and the luminescence intensity was quantitatively detected using a GloMax® 20 / 20 luminometer. The results are shown in Figure 2 .

[0384] according to Figure 2 It can be seen that the ionizable lipids 6-2-8, 1-3-11, 6-3-9, 6-3-8, 4-3-9, 4-12-9, 4-12-12, 1-3-12, 1-8-12, 1-9-12, 1-12-9, 1-12-12, and 6-12-9 all have good luciferase expression effects, and the mRNA transfection efficiency is greater than or equal to that of SM-102.

[0385] In particular, the mRNA transfection efficiencies of nine ionizable lipids, 6-12-9, 4-12-12, 4-3-9, 6-3-9, 1-12-9, 4-12-9, 1-8-12, 6-2-8, and 1-3-11, were 6.12 times, 5.72 times, 4.01 times, 3.84 times, 1.93 times, 1.89 times, 1.73 times, 1.68 times, and 1.54 times that of SM-102, respectively, which were significantly stronger than those of the reference ionizable lipid H18A4B4 and compound-2.

[0386] Example 23 Cytotoxicity Study of Ionizable Lipids

[0387] The cytotoxicity of Luci-mRNA-encapsulated lipid nanoparticles containing ionizable lipids (6-2-8, 1-3-11, 6-3-9, 6-3-8, 4-3-9, 4-12-12, 4-12-9, 1-3-12, 1-8-12, 1-9-12, 1-12-9, 1-12-12, 6-12-9, H18A4B4, and Compound-2) was assessed using the MTT assay. The MTT assay is a well-known technique in the art and is based on the MTT (3-(4,5)-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay to assess viable cell count (or cell viability). A higher viable cell count or cell viability indicates a lower cytotoxicity of the lipid nanoparticle. Other appropriate methods can also be used to assess cytotoxicity. Relative cell viability is calculated by comparing the absorbance values ​​of the sample wells with those of the control wells. In this experiment, HEK 293T cells were used as the cell model. HEK 293T cells were plated at 8×10 3 Cells were seeded at a density of 100 μg / mL in a 96-well plate, and the culture medium was removed after 24 hours of incubation. Subsequently, 200 μL of culture medium containing lipid nanoparticles at 1, 2, 4, or 8 μg / mL mRNA was added to each well. A control group received 200 μL of fresh culture medium, with six replicates per well for each concentration. After 24 hours of co-incubation, 25 μL of 5 mg / mL MTT in PBS buffer was added to each well, and incubation continued for 4 hours. After 4 hours, the mixture of culture medium and MTT buffer was aspirated, and 150 μL / well of DMSO was added to dissolve the purple formazan crystals in the viable cells. After thorough shaking, the absorbance at 490 nm was measured using a microplate reader.

[0388] The lipid nanoparticles constructed by the ionizable lipids prepared in the present application, such as ionizable lipids 6-2-8, 4-2-8, 1-3-11, 6-3-9, 6-3-8, 4-3-9, 4-12-9, 4-12-12, 1-3-12, 1-8-12, 1-9-12, 1-12-12, 6-12-9, and 1-12-9, have a cell survival rate greater than 90% under conditions of an mRNA concentration of 1 μg / mL or 2 μg / mL, or even under conditions of a higher mRNA concentration (4 μg / mL), indicating that the ionizable lipids of the present invention have good biocompatibility, low cytotoxicity, and high safety.

[0389] Example 24 Study on the shelf stability of lipid nanoparticles

[0390] After ultrafiltration, the lipid nanoparticles constructed with 4-3-9 were diluted to 0.05 mg Luci-mRNA / mL using 10 mM phosphate buffer at pH 7.4 prepared with enzyme-free water. The cells were then placed in an incubator at 25°C. Samples were taken on days 0, 1, 2, 3, 4, 5, 6, and 7. The nanoparticle size was measured using a particle size analyzer, and the transfection effect of the nanoparticles was measured according to the method described in Example 22 to evaluate the stability of the cells. The results are shown in Figure 3 In the figure, the left graph shows the change in diameter of lipid nanoparticles over storage days; the right graph shows the change in luminescence intensity (LU) over time. The smaller the change in LU, the better the stability.

[0391] according to Figure 3 As can be seen, nanoparticles constructed with the ionizable lipid 4-3-9 exhibited no significant size change after seven days at 25°C, and no significant change in fluorescence intensity after seven days of storage at room temperature, indicating that mRNA transfection efficiency remained high. These results demonstrate the robust stability of lipid nanoparticles constructed with the ionizable lipids of the present invention.

[0392] Example 25 In vivo transfection study of ionizable lipids

[0393] To investigate the in vivo mRNA transfection efficiency of lipid nanoparticles prepared with the ionizable lipids of the present invention, this experiment used Luci-mRNA stably expressing luciferase as a model. Following the method described in Example 21, Luci-mRNA-encapsulated lipid nanoparticles were prepared using the ionizable lipids 6-12-9, 4-12-9, 4-12-12, 4-3-9, 6-3-9, 1-12-9, H18A4B4, and Compound 2. Luci-mRNA-encapsulated lipid nanoparticles constructed with SM-102 served as positive controls, while naked mRNA and phosphate buffered saline (PBS) served as negative controls. C57BL / 6 mice were immunized intramuscularly with the prepared lipid nanoparticles. Live animal imaging of the Luci-mRNA-lipid nanoparticles was performed 24 hours after administration to assess the mRNA transfection efficiency of the ionizable lipids. During the experiment, each mouse was injected intramuscularly with 2 μg of Luci-mRNA. At 24 hours after immunization, 200 μL of D-luciferin (15 mg / mL), a luciferase substrate, was injected intraperitoneally. The luciferase protein expression was then read out by in vivo bioluminescence using an in vivo imaging system (IVIS). The results are shown in Figure 4 .in, Figure 4 The upper figure shows the in vivo imaging of mice 24 hours after administration; Figure 4 The figure below shows a bar chart of the in vivo imaging results of firefly luciferase-mRNA delivery after intramuscular injection in C57BL / 6 mice. The vertical axis represents the total luciferin luminescence intensity, which is proportional to the luciferase expression level and, correspondingly, the transfection efficiency.

[0394] according to Figure 4 It can be seen that Luci-mRNA-lipid nanoparticles with ionizable lipid compounds 1-12-9, 4-3-9, 4-12-9, 6-12-9, and SM-102 as lipid components all showed high luciferase expression at the injection site after intramuscular injection. The mRNA transfection efficiency of 4-3-9 and 1-12-9 was significantly better than that of SM-102, at 4.15 times and 1.40 times that of SM-102, respectively. The mRNA transfection efficiency of ionizable lipid compounds 1-12-9, 4-3-9, 4-12-12, 4-12-9, and 6-12-9 was significantly higher than that of the reference material H18A4B4 and compound-2 in vivo.

[0395] This study also investigated the in vivo sustained expression of Luci-mRNA-lipid nanoparticles constructed with ionizable lipid 4-3-9. After intramuscular immunization of animals with the prepared lipid nanoparticles, in vivo imaging of C57BL / 6 mice was performed using bioluminescence to evaluate Luci-mRNA-lipid nanoparticle expression at 24, 48, 72, and 96 hours (h) after administration. Figure 5 The upper figure shows the evaluation of the persistence of mRNA transfection with ionizable lipids. During the experiment, each mouse was injected intramuscularly with 2 μg of Luci-mRNA. At 24, 48, 72, and 96 hours after immunization, 200 μL of D-luciferin (15 mg / mL), a luciferase substrate, was injected intraperitoneally. Luciferase protein expression was then read out by in vivo bioluminescence using an in vivo imaging system (IVIS). Figure 5 (see the figure below).

[0396] according to Figure 5 It can be seen that after intramuscular injection, 4-3-9 has a higher mRNA transfection efficiency than SM-102, and can achieve sustained protein expression, and can still maintain high luciferase expression 96 hours after injection.

[0397] Example 26 Study on the Immune Efficacy of COVID-19 mRNA Vaccines Constructed with Ionizable Lipids

[0398] This example investigated the potential application of ionizable lipids as mRNA vaccine vectors for viral prevention. Lipid nanoparticle mRNA vaccines were constructed using 6-3-9, 4-3-9, 1-12-9, 6-12-9, 4-12-9, and SM-102, along with mRNA encoding the Omicron BA.1 Spike protein. BABL / c mice were divided into several groups (e.g., a PBS control group and various vaccine-immunized groups). For each vaccine-immunized group, each mouse was immunized intramuscularly with 10 μg of mRNA encoding the Omicron BA.1 Spike protein. The first immunization was designated day 0 (d0), and a booster immunization was administered two weeks after the first immunization (d14). Blood samples were collected two weeks after the first immunization (d14) and four weeks after the first immunization (d28) for detection of RBD-specific IgG antibodies. Antibody titers were analyzed four weeks after the first immunization (d28).

[0399] The specific antibody titer in serum was determined using an enzyme-linked immunosorbent assay (ELISA). The main steps are: 100 μL of a 10 μg / mL RBD antigen protein solution was plated onto an ELISA plate and allowed to adsorb overnight. The serum was diluted 100-fold as a starting dilution, followed by two-fold dilutions. 100 μL of the solution was then added to the ELISA plate to bind to the specific antigen. Finally, HRP-labeled secondary antibody and TMB colorimetric solution were added for development (development at 37°C for 15 minutes). The absorbance at 450 nm (after subtracting the background absorbance at 570 nm) was measured using a microplate reader and recorded as DO450 nm. The absorbance data for each dilution were calculated, and the dilution immediately preceding the one with an absorbance less than 0.03 was defined as the specific antibody titer for that serum sample. Figure 6 The antibody titer analysis results at 2 weeks (d14) and 4 weeks (d28) after the first immunization are shown in FIG.

[0400] according to Figure 6 It can be seen that after intramuscular injection, the mRNA vaccines constructed with ionizable lipids 6-3-9, 4-3-9, 1-12-9, 6-12-9 and 4-12-9 had higher RBD antigen-specific IgG antibody levels than the mRNA vaccine constructed with SM-102 on days 14 and 28 after the first immunization. In particular, the mRNA vaccines constructed with synthetic lipids 4-3-9 and 1-12-9 of the present invention had RBD antigen-specific IgG antibody titers of 5.1 times and 6.3 times that of the mRNA vaccine constructed with SM-102 on day 28 after the first immunization, respectively, indicating that they have stronger virus protection efficacy.

[0401] Example 27 Study on the Immune Efficacy of Tumor mRNA Vaccines Constructed with Ionizable Lipids

[0402] This example investigates the potential application of ionizable lipids as anti-tumor mRNA vaccine carriers. Lipid nanoparticle tumor mRNA vaccines were constructed using 6-3-9, 4-3-9, 1-12-9, 6-12-9, and 4-12-9, along with SM-102, and mRNA encoding the model tumor antigen OVA protein. In the B16-OVA model anti-tumor assay, 6- to 8-week-old female C57BL / 6 mice were subcutaneously injected with 5×10 5 B16-OVA tumor cells were injected into the mice, with the day of injection designated as day 0. Mice were divided into several groups (including a PBS control group and various vaccine-immunized groups). On days 5 and 12, each mouse in the vaccine-immunized group received an intramuscular injection of 10 μg of mRNA encoding OVA protein. Tumor volume was measured every two days, and the tumor volume was calculated using the formula V = a × b. 2 ×0.5, where a is the length of the tumor and b is the width of the tumor. The tumor volume was recorded from the 8th day after tumor implantation to the 22nd day, and a tumor growth curve was prepared, as shown in Figure 7As shown in the figure above. The ELISOPT method was used to determine the specific response of spleen cells to antigens and secretion of IFN-γ after the tumor mRNA vaccine was used in the B16-OVA model. The capture antibody was diluted to a working concentration and added to the ELISPOT plate at 100 μL per well. The plate was incubated at 4 degrees overnight. The plate was washed three times with PBS, and then 100 μL of blocking solution was added to each well and placed at room temperature for 2 hours. The blocking solution was discarded and RPMI 1640 medium containing the antigen was prepared. 200 μL of RPMI1640 medium containing antigen (30 μg OVA protein) was added to each well. The spleen cells of the treated mice were separated and inoculated into the well plates at a density of 2×10 per well. 5 cells. Place the well plate in an incubator at 37 degrees with 5% carbon dioxide and culture for 72 hours. Discard the cells and culture medium, and wash twice with deionized water. Add detection antibody diluted to the working concentration, add 100 μL to each well, and incubate at room temperature for 2 hours. Wash the well plate twice with PBST, add 100 μL of streptavidin-HRP to each well, incubate at room temperature for 1 hour, and then wash the well plate 3 times with PSB. Add 100 μL of color development solution to each well until a plate appears. Use deionized water to wash to terminate the reaction. The results are as follows. Figure 7 As shown in the figure below.

[0403] according to Figure 7 It can be seen that after intramuscular injection, the mRNA vaccine effectively inhibited tumor growth compared to the PBS-treated group. In particular, the tumor mRNA vaccines constructed with the ionizable lipids 6-3-9, 4-3-9, 1-12-9, 6-12-9, and 4-12-9 all had smaller average tumor volumes on day 22 after two immunizations than the tumor mRNA vaccine constructed with SM-102. In particular, the tumor mRNA vaccines constructed with the ionizable lipids 4-3-9 and 6-12-9 achieved tumor inhibition rates of 87.8% and 92.2%, respectively, far exceeding the 50.1% tumor inhibition rate of the tumor mRNA vaccine constructed with SM-102. In addition, the ELISPOT results show that compared to the tumor mRNA vaccine constructed with SM-102, the tumor mRNA vaccines constructed with the ionizable lipids 4-3-9 and 1-12-9 can induce stronger antigen recognition and response in spleen cells, activating a stronger cellular immune response.

[0404] The above embodiments are only intended to help understand the method and core concept of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the present invention, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. An ionizable lipid or a pharmaceutically acceptable salt thereof, characterized in that: It is selected from: 、 、 、 、 or 。 2. Use of the ionizable lipid or a pharmaceutically acceptable salt thereof according to claim 1 in preparing lipid nanoparticles, characterized in that: The ionizable lipid promotes the endocytosis of the nucleic acid drug and achieves endosomal escape, thereby effectively promoting the expression of the nucleic acid.

3. A lipid nanoparticle, characterized in that It comprises the ionizable lipid according to claim 1 or a pharmaceutically acceptable salt thereof.

4. A pharmaceutical composition, characterized in that It comprises the lipid nanoparticles according to claim 3 and a pharmaceutically acceptable carrier, wherein the lipid nanoparticles contain nucleic acid drugs.

5. A method for preparing lipid nanoparticles, characterized in that: It includes: dissolving ionizable lipids, sterols, helper lipids, and polyethylene glycol lipid derivatives in an organic solvent to obtain an organic phase; Dissolve the nucleic acid drug in the aqueous phase, mix the organic phase with the aqueous phase, and optionally dialysis, Wherein, the ionizable lipid is the ionizable lipid according to claim 1 or a pharmaceutically acceptable salt thereof.

6. The method according to claim 5, characterized in that in, The molar ratio of the ionizable lipid, the sterol, the auxiliary lipid and the polyethylene glycol lipid derivative is (20-70): (20-50): (2-30): (0.1-20).

7. Use of the lipid nanoparticles according to claim 3 in preparing medicines, characterized in that: The drug is an mRNA drug.

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