Amino lipid, and lipid nanoparticles and use thereof

Abstract

The present invention provides an amino lipid, and lipid nanoparticles (LNPs) and a use thereof, the amino lipid having a structure represented by general formula (I), or an isomer, pharmaceutically acceptable salt, prodrug or solvate of the amino lipid. The present invention further provides LNPs containing the amino lipid. According to the present invention, the amino lipid having a structure represented by general formula (I) is used as an ionizable lipid compound, and the LNPs are obtained by means of self-assembly of the ionizable lipid compound, a steroid, a neutral lipid, and a polymer-bonded lipid. The LNPs can further improve the translation expression level of a nucleic acid load in cells, improve the effect of a nucleic acid-LNP preparation, and enable the nucleic acid-LNP preparation to provide a theoretical basis for personalized treatment.

Description

[Corrected 15.08.2025 according to Regulation 26] Amino lipids, lipid nanoparticles and their applications [Corrected 15.08.2025 according to Rule 26] Technical field

[0001] [Corrected 15.08.2025 according to Rule 26] The present invention relates to the field of biochemical technology, and in particular to an amino lipid, lipid nanoparticles and applications thereof. [Corrected 15.08.2025 according to Rule 26] Background Art

[0002] [Corrected 15.08.2025 according to Rule 26] Lipid nanoparticles (LNPs) are a new type of nucleic acid biomolecule delivery technology. LNPs are usually composed of four components: (1) ionizable lipids, which can self-assemble with mRNA into virus-sized particles and release mRNA from endosomes into the cytoplasm; (2) polymer-bound lipids, which can increase the half-life of LNPs in the blood; (3) steroids, which can increase the stability of nanoparticles; and (4) neutral phospholipids, which help form the lipid bilayer structure.

[0003] [Corrected 15.08.2025 according to Rule 26] LNPs can protect mRNA from degradation by RNases and from recognition by TLRs, thereby preventing overactivation of the innate immune system. The choice of ionizable lipids has the greatest impact on LNPs, as they both promote cellular uptake and aid drug escape from endosomes. This influences the encapsulation efficiency, in vivo delivery efficiency, and cytotoxicity of nucleic acid drugs.

[0004] [Corrected 15.08.2025 according to Rule 26] Currently, Moderna and BioNtech & Pfizer have vaccines against the COVID-19 novel coronavirus. Both vaccines use LNP technology to deliver mRNA drugs, thereby achieving the prevention of COVID-19 novel coronavirus, demonstrating the huge application potential of LNP in the field of mRNA drugs and vaccines. However, LNP still has disadvantages such as low encapsulation efficiency, low endosomal escape rate, low expression level, and low safety. The development direction of LNP delivery systems mainly focuses on ionizable lipids and formulations.

[0005] [Corrected 15.08.2025 according to Rule 26] In view of this, the development of new ionizable lipid compounds is of great significance for the development of nucleic acid drugs. [Corrected 15.08.2025 according to Rule 26] Summary of the invention

[0006] [Corrected 15.08.2025 according to Rule 26] In a first aspect, the present invention provides an amino lipid having a structure represented by general formula (I), or an isomer, pharmaceutically acceptable salt, prodrug or solvate thereof:

[0007] [Corrected 15.08.2025 according to Rule 26] wherein G is selected from H, OR, CN, -C(=O)OR', -OC(=O)R', -C(=O)NR'R", -NR'C(=O)R", NR'R", or a cyclic alkyl structure containing at least one heteroatom; the substitutable carbon atoms or heteroatoms in the cyclic alkyl structure are unsubstituted or substituted with one or more hydroxyl groups, C1-C4 alkyl groups, C2-C4 alkenyl groups, C3-C8 cycloalkyl groups or C3-C8 cycloalkenyl groups;

[0008] [Corrected 15.08.2025 according to Rule 26] M1, M2, M3, M4 are the same as or different from each other and are independently selected from C1-C 24 Alkylene, C3-C 24 Cycloalkylene, C2-C 24 Alkenylene or C3-C 24 cycloalkenylene;

[0009] [Corrected 15.08.2025 in accordance with Rule 26]R 1 、R 2 are the same as or different from each other and are independently selected from H, C1-C 24 Alkyl, C3-C 24 Cycloalkyl, C2-C 24 Alkenyl or C3-C 24 cycloalkenyl;

[0010] [Corrected 15.08.2025 under Rule 26] L1, L2, L3, L4 are the same as or different from each other and are independently selected from -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=O)NR-, -NRC(=O)-, -S(=O)-, -OS(=O)2-, -S(=O)2O-, -O-, -S- or -SS-;

[0011] [Corrected 15.08.2025 according to Rule 26] R, R', R" are the same as or different from each other and are independently selected from H, Cl-C 10 Alkyl, C3-C 10 Cycloalkyl, C3-C 10 Alkenyl or C3-C 10 Cycloalkenyl, C1-C 10 Alkyl, C3-C10 Cycloalkyl, C3-C 10 an alkenyl group, or a cyclic alkyl group containing at least one heteroatom, wherein the cyclic alkyl group is unsubstituted or substituted by one or more C1-C4 alkyl groups, C2-C4 alkenyl groups, C3-C8 cycloalkyl groups, or C3-C8 cycloalkenyl groups;

[0012] [Corrected 15.08.2025 according to Rule 26] M5 is independently selected from a single bond, C1-C 16 Alkylene, C2-C 16 Alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene;

[0013] [Corrected 15.08.2025 according to Rule 26] In a second aspect, the present invention provides a method for preparing the aforementioned amino lipid. The synthesis is primarily comprised of three steps: the first step is a ring-opening reaction; the second step is a condensation reaction; and the third step is a substitution reaction.

[0014] [Corrected 15.08.2025 according to Rule 26] In some embodiments of the present invention, the method for preparing the amino lipid comprises the following steps:

[0015] [Corrected 15.08.2025 according to Rule 26] S1: Ring-opening reaction of epoxy compound and carboxylic acid to prepare intermediate 1R 1 -L1-M1-OH;

[0016] [Corrected 15.08.2025 according to Rule 26] S2: Intermediate 1 and the carboxylic acid compound raw material undergo condensation reaction in the presence of a condensing agent to prepare intermediate 2R 1 -L1-M1-L2-M2-leaving group;

[0017] [Corrected 15.08.2025 according to Rule 26] S3: Intermediate 2 undergoes one or more substitution reactions with the amino compound starting material to prepare the target product;

[0018] [Corrected 15.08.2025 in accordance with Rule 26] or include the following steps:

[0019] [Corrected 15.08.2025 according to Rule 26] S1': The diol is protected with TBS and then oxidized. The oxidation product reacts with the Grignard reagent to prepare the intermediate 1'HO-M1-OTBS;

[0020] [Corrected 15.08.2025 according to Rule 26] S2': Intermediate 1' and a carboxylic acid compound undergo condensation reaction in the presence of a condensing agent to prepare intermediate 2'TBSO-M1-L2-M2-leaving group;

[0021] [Corrected 15.08.2025 according to Rule 26] S3': The intermediate 2'TBSO-M1-L2-M2-leaving group is deprotected from TBS and condensed with a carboxylic acid compound in the presence of a condensing agent to produce intermediate 2R 1 -L1-M1-L2-M2-leaving group;

[0022] [Corrected 15.08.2025 according to Rule 26] S4': Intermediate 2 undergoes one or more substitution reactions with the amino compound starting material to prepare the target product.

[0023] [Corrected 15.08.2025 according to Rule 26] In a third aspect, the present invention provides a lipid nanoparticle comprising any one of the above-mentioned amino lipids.

[0024] [Corrected 15.08.2025 in accordance with Rule 26] According to the present invention, there is provided a lipid nanoparticle, said lipid nanoparticle further comprising a steroid, a neutral lipid and / or a polymer-bound lipid;

[0025] [Corrected 15.08.2025 according to Rule 26] The polymer-bound lipid has the chemical formula PYL, wherein P is a hydrophilic polymer portion, Y is an optional linker, and L is a lipid portion.

[0026] [Corrected 15.08.2025 according to Rule 26] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned lipid nanoparticles and a pharmaceutically acceptable carrier.

[0027] [Corrected 15.08.2025 according to Rule 26] In a fifth aspect, the present invention also provides a method for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases using the above-mentioned amino lipids or lipid nanoparticles or pharmaceutical compositions.

[0028] [Corrected 15.08.2025 according to Rule 26] The present invention also provides a method for gene therapy, gene vaccination, antisense therapy, nucleic acid transfer or treatment by interfering RNA using the above-mentioned amino lipids or lipid nanoparticles or pharmaceutical compositions.

[0029] [Corrected 15.08.2025 according to Rule 26] The present invention also provides the use of the above-mentioned amino lipids or lipid nanoparticles in the preparation of drugs for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases.

[0030] [Corrected 15.08.2025 according to Rule 26] The present invention also provides the use of the above-mentioned amino lipids or lipid nanoparticles in a medicament for gene therapy, gene vaccination, antisense therapy, nucleic acid transfer or treatment by interfering RNA.

[0031] [Corrected 15.08.2025 in accordance with Rule 26] In a sixth aspect, the present invention also provides a method for delivering a pharmaceutical agent to a subject, comprising: administering to the subject the pharmaceutical agent formulated in the above-mentioned lipid nanoparticles.

[0032] [Corrected 15.08.2025 according to Rule 26] The amino lipids of the present invention are combined with steroids, neutral lipids and polymers to obtain LNPs through self-assembly. The LNPs can further increase the translation expression level of the payload - nucleic acid - in cells, improve the effect of the nucleic acid-LNP preparation, and provide a basis for the theoretical treatment of personalized nucleic acid-LNP preparations. [Corrected 15.08.2025 in accordance with Rule 26]

[0033] [Corrected 15.08.2025 according to Rule 26] Figure 1 is the 1H-NMR spectrum of E12LA6B6O3 in Example 2;

[0034] [Corrected 15.08.2025 according to Rule 26] Figure 2 is a tumor size monitoring curve of tumor-bearing mice after intramuscular injection of OVA mRNA vaccine in Example 39;

[0035] [Corrected 15.08.2025 according to Rule 26] Figure 3 is a survival curve of tumor-bearing mice after intramuscular injection of OVA mRNA vaccine in Example 39. [Corrected 15.08.2025 according to Rule 26] Specific implementation method

[0036] [Corrected 15.08.2025 in accordance with Rule 26] Definition

[0037] [Corrected 15.08.2025 pursuant to Rule 26] For clarity and ease of reading, the following scientific background information and definitions are provided. Any technical features mentioned herein or disclosed herein may be part of, or read upon, any embodiment of the present invention. Additional definitions and explanations may be provided in the context of the present invention.

[0038] [Corrected 15.08.2025 as per Rule 26] Unless otherwise defined, or unless the specific context requires otherwise, all technical terms used herein have the same meanings as commonly understood by a person skilled in the relevant technical field.

[0039] [Corrected 15.08.2025 as per Rule 26] Unless the context indicates or requires otherwise, the words “comprises”, “comprising” and “containing” and similar expressions shall be construed in this description and claims in an open and inclusive sense to mean “including but not limited to”.

[0040] [Corrected 15.08.2025 pursuant to Rule 26] The expressions "one embodiment," "an embodiment," "a specific embodiment," etc., mean that a particular characteristic, property, or feature, or a particular group or combination of characteristics, properties, or features as described in conjunction with the corresponding expression, is present in at least one embodiment of the present invention. The appearances of these expressions in various places throughout the description are not necessarily all referring to the same embodiment. Furthermore, the particular characteristics, properties, or features may be combined in any suitable manner in one or more embodiments.

[0041] [Corrected 15.08.2025 in accordance with Rule 26] Unless the context clearly requires otherwise, the singular forms “a”, “an” and “the” are to be construed as including the plural.

[0042] [Corrected 15.08.2025 in accordance with Rule 26] The expression “neutral” when applied to a compound, such as a lipid or steroid, or to a group or moiety, means that it is neither cationic nor anionic, for example a compound which does not have ionizable functional groups under physiological conditions, such as hydrocarbons; or that it is both cationic and anionic under typical physiological conditions, i.e. a zwitterion, such as the typical natural phosphatidylcholines.

[0043] [Corrected 15.08.2025 according to Rule 26] As used herein, "lipid" refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally divided into at least three categories: (1) "simple lipids," including fats, oils, and waxes; (2) "complex lipids," including phospholipids and glycolipids; and (3) "derivative lipids," such as steroids. With respect to glycolipids, in certain embodiments, the LNP comprises a glycolipid (e.g., monosialoganglioside GM1).

[0044] [Corrected 15.08.2025 pursuant to Rule 26] In this context, the prefix "poly" refers to a plurality of atoms or groups in a compound having their own properties. However, the absence of the prefix should not be construed as excluding the plurality. For example, a polycationic compound is also a cationic compound and may be referred to as a cationic compound.

[0045] [Corrected 15.08.2025 under Rule 26] The term "nucleic acid" refers to any compound comprising or consisting of DNA or RNA. The term may be applied to oligonucleotides or oligonucleotides.

[0046] [Corrected 15 August 2025 in accordance with Regulation 26] Immune System: The immune system protects organisms from infection. If a pathogen breaches an organism's physical barriers and enters, the innate immune system provides an immediate but nonspecific response. If a pathogen evades this innate response, vertebrates have a second layer of protection: the adaptive immune system. Here, the immune system adjusts its response during infection to improve its ability to recognize the pathogen. This improved response then persists as immunological memory after the pathogen has been eliminated, allowing the adaptive immune system to mount a faster and more robust attack each time the pathogen is encountered. Thus, the immune system comprises both the innate and adaptive immune systems. Each of these two components consists of so-called humoral and cellular components.

[0047] [Corrected 15 August 2025 in accordance with Regulation 26] Adaptive Immune System: The adaptive immune system consists of highly specialized systemic cells and processes that eliminate or prevent pathogenic growth. The adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens (generate immunity) and mount a stronger attack each time the pathogen is encountered. This system is highly adaptable due to somatic hypermutation (a process in which the frequency of somatic cell mutations increases) and V(D)J recombination (irreversible genetic recombination of antigen receptor gene segments). This mechanism allows a small number of genes to generate a large number of different antigen receptors, which are then uniquely expressed on each individual lymphocyte. Because gene rearrangement causes irreversible changes in the DNA of each cell, all of that cell's progeny (offspring) will inherit genes encoding the same receptor specificity, including memory B cells and memory T cells, which are key to long-lived specific immunity. The immune network theory is a theory of how the adaptive immune system works, based on the interactions between T cells, B cells, and the variable regions of the receptors of molecules with variable regions produced by T and B cells.

[0048] [Corrected 15.08.2025 in accordance with Regulation 26] The term “vaccine” is generally understood to mean a prophylactic or therapeutic material that provides at least one antigen or antigenic function that can stimulate the body’s adaptive immune system to provide an adaptive immune response.

[0049] [Corrected 15.08.2025 in accordance with Rule 26] The term “antigen” generally refers to a substance that can be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, for example by the formation of antibodies and / or antigen-specific T cells as part of the adaptive immune response.

[0050] [Corrected 15.08.2025 according to rule 26] The term "artificial mRNA" (sequence) is generally to be understood as an mRNA molecule that is not naturally occurring. In other words, an artificial mRNA molecule is to be understood as a non-natural mRNA molecule. Such an mRNA molecule may be non-natural due to its individual sequence (which is not naturally occurring) and / or due to other modifications, such as structural modifications of non-naturally occurring nucleotides. Generally, artificial mRNA molecules can be designed and / or generated by genetic engineering methods to correspond to a desired artificial nucleotide sequence (heterologous sequence). In this context, an artificial sequence is generally a non-naturally occurring sequence, i.e. it differs from the wild-type sequence by at least one nucleotide. The term "wild type" is to be understood as a sequence that occurs in nature.

[0051] [Corrected 15.08.2025 as per Rule 26] The term "pharmaceutically acceptable salt" refers to a form of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound.

[0052] [Corrected 15.08.2025 in accordance with Rule 26] The compounds of the present invention may exist in multiple crystalline forms, i.e., different lattice arrangements of the same elemental composition of the compound. Polymorphs typically have different X-ray diffraction spectra, infrared spectra, melting points, density, hardness, crystal form, optical and electrical properties, stability, and solubility. Different factors such as recrystallization solvent, crystallization rate, and storage temperature may result in a recrystallized product dominated by a single crystalline form. It is understood that the amino lipids described herein include all such crystalline forms.

[0053] [Corrected 15.08.2025 in accordance with Rule 26] The compounds of the present invention may have chiral centers and / or axial chirality and may occur as racemates, racemic mixtures, single enantiomers, diastereomeric compounds, single diastereomers, and cis-trans isomers. Each chiral center or axial chirality will independently produce two optical isomers, and all possible optical isomers and diastereomeric mixtures, as well as pure or partially purified compounds, are within the scope of the present invention. The term "isomer" as used herein includes all such isomeric forms of the compound.

[0054] [Corrected 15.08.2025 in accordance with Rule 26] The compounds of the present invention may also exist in various hydrates or solvates. Solvates contain stoichiometric or non-stoichiometric amounts of solvents and are selectively formed during crystallization with pharmaceutically acceptable solvents such as water or other solvents such as ethanol. Hydrates are formed when the solvent is water, and solvates are formed when the solvent is other solvents such as ethanol.

[0055] [Corrected 15 August 2025 in accordance with Rule 26] The term "prodrug," also known as precursor drug, drug precursor, or prodrug, refers to a compound that, after chemical structural modification, is inactive or minimally active in vitro but releases the active drug in vivo through enzymatic or non-enzymatic conversion, exerting its pharmacological effect. There are two main categories of prodrugs: carrier prodrugs, or simply carrier prodrugs, and bioprodrugs. Carrier prodrugs are characterized by a covalent bond between an active compound and a transporter. The carrier is removed in vivo through simple hydrolysis, allowing the active compound to exert its pharmacological effect. Carrier prodrugs are often less active or inactive compared to the parent compound. The carrier structure is often lipophilic, harmless to the organism, and capable of timely release of the active compound. Unlike carrier prodrugs, bioprodrugs do not require a temporary binding of the active substance to a carrier; instead, their activity is achieved through changes in their own molecular structure. Bioprodrugs are inherently inactive; it is their metabolites in the body that are active.

[0056] [Corrected 15.08.2025 according to Rule 26] The present invention unexpectedly discovered through research that the use of the novel amino lipids and / or lipid nanoparticles provided by the present invention can effectively solve the shortcomings of LNPs in the prior art, such as low encapsulation efficiency, low endosomal escape rate, low expression level, and low safety, thereby promoting the development of LNPs in the field of mRNA drugs and vaccines.

[0057] [Corrected 15.08.2025 according to Regulation 26] Amino lipids

[0058] [Corrected 15.08.2025 according to Rule 26] The amino lipid is preferably cationizable, i.e. when the pH is lowered below the pKa of the ionizable group of the lipid, the amino lipid will be protonated and, when positively charged, the lipid will be able to bind to the negatively charged nucleic acid.

[0059] [Corrected 15.08.2025 according to Rule 26] In one aspect, the present invention provides an amino lipid having a structure represented by general formula (I), or an isomer, pharmaceutically acceptable salt, prodrug or solvate thereof:

[0060] [Corrected 15.08.2025 according to Rule 26] wherein G is selected from H, OR, CN, -C(=O)OR', -OC(=O)R', -C(=O)NR'R", -NR'C(=O)R", NR'R" or a cyclic alkyl structure (preferably a 3-10 membered cycloalkyl, more preferably a 4-6 membered cycloalkyl) containing at least one heteroatom (preferably, the heteroatom is N or O, preferably 1 or 2 heteroatoms selected from N or O); the substitutable carbon atoms or heteroatoms in the cyclic alkyl structure are unsubstituted or substituted by one or more C1-C4 alkyl, C2-C4 alkenyl, C3-C8 cycloalkyl or C3-C8 cycloalkenyl groups;

[0061] [Corrected 15.08.2025 according to Rule 26] M1, M2, M3, M4 are the same as or different from each other and are independently selected from C1-C 24 Alkylene, C3-C 24 Cycloalkylene, C2-C 24 Alkenylene or C3-C 24 cycloalkenylene;

[0062] [Corrected 15.08.2025 in accordance with Rule 26]R 1 、R 2 are the same as or different from each other and are independently selected from H, C1-C 24 Alkyl, C3-C 24 Cycloalkyl, C2-C 24 Alkenyl or C3-C 24 cycloalkenyl;

[0063] [Corrected 15.08.2025 under Rule 26] L1, L2, L3, L4 are the same as or different from each other and are independently selected from -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=O)NR-, -NRC(=O)-, -S(=O)-, -OS(=O)2-, -S(=O)2O-, -O-, -S- or -SS-;

[0064] [Corrected 15.08.2025 according to Rule 26] R, R', R" are the same as or different from each other and are independently selected from H, Cl-C 10 Alkyl (preferably C1-C6 alkyl, more preferably C1-C4 alkyl), C3-C 10 Cycloalkyl, C3-C 10 Alkenyl, C3-C 10 Cycloalkenyl, C1-C 10 Alkyl, C3-C 10 Cycloalkyl, C3-C 10Alkenyl, or a cyclic alkyl group containing at least one heteroatom, wherein the cyclic alkyl group is unsubstituted or substituted by one or more C1-C4 alkyl groups, C2-C4 alkenyl groups, C3-C8 cycloalkyl groups, or C3-C8 cycloalkenyl groups; preferably, the terminal end is connected with -N(C 1-6 Alkyl)2C1-C 10 or a C1-C2 alkyl group or a C1-C2 alkyl group having a 3-8 membered cyclic alkyl group (preferably a 4-6 membered cyclic alkyl group) with 1-2 ring nitrogen atoms connected to the terminal end. 10 Alkyl, the C3-C 10 The cycloalkyl group and the 3- to 8-membered cyclic alkyl group (preferably a 4- to 6-membered cyclic alkyl group) are optionally substituted with a C1-C6 alkyl group.

[0065] [Corrected 15.08.2025 according to Rule 26] M5 is selected from a single bond, C1-C 16 Alkylene, C2-C 16 C3-C8 cycloalkylene or C3-C8 cycloalkenylene.

[0066] [Corrected 15.08.2025 in accordance with Rule 26] It should be noted that, unless expressly disallowed, the alkyl, alkenyl, alkylene, and alkenylene groups containing multiple carbon atoms referred to in the present invention may be straight-chain or branched. Furthermore, the alkyl, alkenyl, alkylene, and alkenylene groups, as well as cycloalkyl, cycloalkenyl, cycloalkylene, and cycloalkenylene groups referred to in the present invention may be unsubstituted or substituted, and the substituents may be any suitable substituents, i.e., any straight-chain or branched alkyl, aryl, heteroalkyl, or heteroaromatic structure, which may optionally contain other functional groups, such as ester or amide groups.

[0067] [Corrected 15.08.2025 according to Rule 26] In some preferred embodiments, G is selected from H, OR, NR'R" or a cyclic alkyl structure containing at least one heteroatom; wherein the heteroatom is O or N; wherein R, R', R" are the same as or different from each other and are each independently selected from H, C1-C8 alkyl, C3-C8 cycloalkyl, C3-C8 alkenyl or C3-C8 cycloalkenyl.

[0068] [Corrected 15.08.2025 according to Rule 26] In a further preferred embodiment, G is selected from H, OH or NR'R", wherein R' and R" are the same or different and are each independently selected from H or C1-C4 alkyl; that is, NR'R" may be NH2, NHCH3, NHC2H5, NHC3H7, NHC4H9, N(CH3)2, CH3-N-C2H5, CH3-N-C3H7, CH3-N-C4H9, C2H5-N-C2H5, C2H5-N-C3H7, C2H5-N-C4H9, C3H7-N-C3H7, C3H7-N-C4H9 or C4H9-N-C4H9.

[0069] [Corrected 15.08.2025 according to Rule 26] Alternatively, G is selected from substituted or unsubstituted oxa-five-membered cycloalkyl, aza-five-membered cycloalkyl, aza-six-membered cycloalkyl, diaza-six-membered cycloalkyl or aza-oxa-six-membered cycloalkyl. Preferably, the oxa-five-membered cycloalkyl is Aza five-membered cycloalkyl is Aza six-membered cycloalkyl is The diaza six-membered cycloalkyl group is Azaoxa six-membered cycloalkyl group is The * position is connected to M5.

[0070] [Corrected 15.08.2025 according to Rule 26] In some embodiments, when G is a substituted diaza six-membered cycloalkyl group, the substituent position is a nitrogen atom that is not connected to M5.

[0071] [Corrected 15.08.2025 according to Rule 26] In some preferred embodiments, G is selected from H, OR, CN, -C(=O)OR', -OC(=O)R', -C(=O)NR'R", -NR'C(=O)R", NR'R" or a cyclic alkyl structure containing 1 or 2 ring N atoms (preferably a 3-10 membered cycloalkyl, more preferably a 4-6 membered cycloalkyl); the substitutable carbon atoms or heteroatoms in the cyclic alkyl structure are unsubstituted or substituted by one or more C1-C4 alkyl, C2-C4 alkenyl, C3-C8 cycloalkyl or C3-C8 cycloalkenyl groups. R, R', R" are the same or different and are each independently selected from H, C1-C6 alkyl, C3-C8 cycloalkyl or C3-C8 cycloalkenyl groups. 10 Cycloalkyl, terminally connected with -N(C 1-6 Alkyl)2C1-C 10 Alkyl or a C1-C ... 10 Alkyl, the C3-C 10 The cycloalkyl group and the 4- to 6-membered cyclic alkyl group are optionally substituted with a C1-C6 alkyl group.

[0072] [Corrected 15.08.2025 according to Rule 26] In some embodiments, M5 is selected from a single bond, C2-C 16 Alkylene, C2-C 16 C4-C8 cycloalkylene or C3-C8 cycloalkenylene.

[0073] [Corrected 15.08.2025 according to Rule 26] Further preferably, M5 is selected from a single bond, C2-C 16More preferably, M5 is selected from a single bond, a C2-C6 alkylene group or a C4-C6 cycloalkylene group; or is selected from a C3-C5 alkylene group.

[0074] [Corrected 15.08.2025 according to Rule 26] In a preferred embodiment, in the general formula (I), M5 and G are connected to form One selected from A1 to A38:

[0075] [Corrected 15.08.2025 according to Rule 26] Further preferably, the Selected from one of A1-A18, A22-A24, A28-A38.

[0076] [Corrected 15.08.2025 in accordance with Rule 26] More preferably, the Selected from one of A15, A16, A17, A23, A29, A30, A33, A37, A38, A42.

[0077] [Corrected 15.08.2025 according to Rule 26] In some embodiments of the present invention, in the general formula (I), M5 and G are connected to form Select one from A39 to A52:

[0078] [Corrected 15.08.2025 according to Rule 26] In some embodiments of the present invention, L1, L2, L3, L4 are the same as or different from each other and are independently selected from -C(=O)O-, -OC(=O)-, -C(=O)NR- or -NRC(=O)-.

[0079] [Corrected 15.08.2025 according to Rule 26] wherein, when L1, L2, L3, L4 are independently selected from -C(=O)NR- or -NRC(=O)-, wherein R is independently selected from H or C1-C 10 Preferably, R is H.

[0080] [Corrected 15.08.2025 according to Rule 26] In a preferred embodiment of the present invention, L1 is the same as L4 and is -C(=O)O- or -OC(=O)-.

[0081] [Corrected 15.08.2025 according to Rule 26] In some embodiments of the present invention, M1, M2, M3, and M4 are the same or different from each other, and M1 and M4 are each independently selected from a C4-C 22 Alkylene, branched C4-C 22Cycloalkylene, C4-C 22 Alkenylene or branched C4-C 22 Cycloalkenylene, M2 and M3 are each independently selected from C4-C 22 Alkylene, C4-C 22 Cycloalkylene, C4-C 22 Alkenylene or C4-C 22 Cycloalkenylene.

[0082] [Corrected 15.08.2025 according to Rule 26] Further preferably, M1, M2, M3, M4 are the same as or different from each other, and M1 and M4 are each independently selected from a C4-C 22 Alkylene or branched C4-C 22 Alkenylene, preferably a C6-C 16 Alkylene; M2 and M3 are each independently selected from C4-C 22 Alkylene or C4-C 22 Alkenylene, preferably C3-C8 alkylene.

[0083] [Corrected 15.08.2025 according to Rule 26] More preferably, in some embodiments of the present invention, M2 is the same as M3 and is C4-C 22 Alkylene.

[0084] [Corrected 15.08.2025 according to Rule 26] In some embodiments of the present invention, M1 is the same as M4 and is a C4-C 22 Alkylene.

[0085] [Corrected 15.08.2025 according to Rule 26] In some embodiments of the present invention, R 1 、R 2 are the same as or different from each other and are each independently selected from C4-C 22 Alkyl or C4-C 22 Alkenyl, preferably C5-C 12 alkyl.

[0086] [Corrected 15.08.2025 according to Rule 26] In a preferred embodiment, R 1 -L1-M1-L2-M2- fragment is R 1 -C(=O)O-M1-OC(=O)-M2-,R 2 -L4-M4-L3-M3-Fragment R 2 -C(=O)O-M4-OC(=O)-M3-. Further preferably, M1, M2, M3, and M4 are the same or different from each other, and M1 and M4 are each independently selected from a C4-C 22Alkylene or branched C4-C 22 Alkenylene; M2 and M3 are each independently selected from C4-C 22 Alkylene or C4-C 22 Alkenylene; R 1 、R 2 are the same as or different from each other and are each independently selected from C4-C 22 Alkyl or C4-C 22 Alkenyl.

[0087] [Corrected 15.08.2025 according to Rule 26] Those skilled in the art can combine the above preferred embodiments for different groups according to common sense to obtain more preferred embodiments of the compounds of the present invention.

[0088] [Corrected 15.08.2025 according to Rule 26] In a preferred embodiment of the present invention, the structure of formula (I) is selected from one of the following structures:

[0089] [Corrected 15.08.2025 according to Rule 26] In a preferred embodiment of the present invention, the structure of formula (I) is selected from one of the following structures:

[0090] [Corrected 15.08.2025 according to Rule 26] Those skilled in the art can combine the above preferred embodiments for different groups according to common sense to obtain more preferred embodiments of the compounds of the present invention.

[0091] [Corrected 15.08.2025 according to Rule 26] Method for preparing amino lipids

[0092] [Corrected 15.08.2025 according to Rule 26] The present invention provides a method for preparing the above-mentioned amino lipid, comprising the following steps:

[0093] [Corrected 15.08.2025 according to Rule 26] S1: Ring-opening reaction of epoxy compound and carboxylic acid to prepare intermediate 1R 1 -L1-M1-OH;

[0094] [Corrected 15.08.2025 according to Rule 26] S2: Intermediate 1 and the carboxylic acid compound raw material undergo condensation reaction in the presence of a condensing agent to prepare intermediate 2R 1 -L1-M1-L2-M2-leaving group;

[0095] [Corrected 15.08.2025 according to Rule 26] S3: Intermediate 2 undergoes one or more substitution reactions with the amino compound starting material to prepare the target product;

[0096] [Corrected 15.08.2025 in accordance with Rule 26] or include the following steps:

[0097] [Corrected 15.08.2025 according to Rule 26] S1': The diol is protected with TBS and then oxidized. The oxidation product reacts with the Grignard reagent to prepare the intermediate 1'HO-M1-OTBS;

[0098] [Corrected 15.08.2025 according to Rule 26] S2': Intermediate 1' and a carboxylic acid compound undergo condensation reaction in the presence of a condensing agent to prepare intermediate 2'TBSO-M1-L2-M2-leaving group;

[0099] [Corrected 15.08.2025 according to Rule 26] S3': The intermediate 2'TBSO-M1-L2-M2-leaving group is deprotected from TBS and condensed with a carboxylic acid compound in the presence of a condensing agent to produce intermediate 2R 1 -L1-M1-L2-M2-leaving group;

[0100] [Corrected 15.08.2025 according to Rule 26] S4': Intermediate 2 undergoes one or more substitution reactions with the amino compound starting material to prepare the target product.

[0101] [Corrected 15.08.2025 according to Rule 26] Preferably, the carboxylic acid in step S2' is a H-L2-M2-leaving group.

[0102] [Corrected 15.08.2025 according to Rule 26] Preferably, the carboxylic acid in step S3' is R 1 -L1-H.

[0103] [Corrected 15.08.2025 according to Rule 26] Preferably, the leaving group is a halogen.

[0104] [Corrected 15.08.2025 according to Rule 26] Preferably, each intermediate and target product prepared are purified by column chromatography.

[0105] [Corrected 15.08.2025 according to Rule 26] In some embodiments, the preparation method comprises the following steps:

[0106] [Corrected 15.08.2025 according to Rule 26] Ring-opening reaction: Carboxylic acid compound (1.0 eq), epoxy compound raw material (0.6-3.0 eq), ferric chloride (0.5-10 mol%), pyridine (0.2-20 mol%) were mixed and stirred at room temperature overnight. After the reaction, intermediate 1 was purified by column chromatography in a yield of 56%-99.0%.

[0107] [Corrected 15.08.2025 according to Rule 26] Condensation reaction: Intermediate 1 (1.0 eq), halogenated carboxylic acid (0.6-3.0 eq), EDCI-HCl (1.0-6.0 eq), DMAP (0.05-0.5 eq), DIPEA (1.0-8.0 eq), and DCM were mixed in sequence, stirred at room temperature overnight, and purified by column chromatography to obtain intermediate 2 in a yield of 46.0%-96.4%.

[0108] [Corrected 15.08.2025 according to Rule 26] Substitution reaction: Intermediate 2 (1.0-5.0 eq), potassium carbonate (1.0-5.0 eq), an ammonia compound (1.0 eq), sodium iodide (1.0-3.0 eq) and acetonitrile were mixed in sequence, stirred at 20-100°C overnight, and purified by column chromatography to obtain amino lipids with a yield of 32.0% to 92.1%.

[0109] [Corrected 15.08.2025 according to Rule 26] In some embodiments, the preparation method comprises the following steps:

[0110] [Corrected 15.08.2025 according to Rule 26] Addition reaction: After the diol is protected by TBS, it undergoes oxidation reaction with PCC. 0.9-1.2 eq of oxidation product is stirred in anhydrous tetrahydrofuran at -20°C for 2-20 minutes, 1.2-1.5 eq of Grignard reagent is added, and the mixture is stirred at -20°C to 0°C for 1-6 hours. The intermediate 1 is purified by column chromatography;

[0111] [Corrected 15.08.2025 according to Rule 26] Condensation reaction 1: Intermediate 1' (1.0 eq), halogenated carboxylic acid (0.6-3.0 eq), EDCI-HCl (1.0-6.0 eq), DMAP (0.05-0.5 eq), DIPEA (1.0-8.0 eq), and DCM were mixed in sequence, stirred at room temperature overnight, and purified by column chromatography to obtain intermediate 2';

[0112] [Corrected 15.08.2025 according to Rule 26] Condensation reaction 2: Intermediate 2' (1.0 eq) was deprotected from TBS with ammonium fluoride (5.0-20.0 eq), mixed with carboxylic acid (0.6-3.0 eq), EDCI-HCl (1.0-6.0 eq), DMAP (0.05-0.5 eq), DIPEA (1.0-8.0 eq), and DCM, and stirred at room temperature overnight. The mixture was purified by column chromatography to obtain intermediate 2;

[0113] [Corrected 15.08.2025 according to Rule 26] Substitution reaction: Intermediate 2 (1.0-5.0 eq), potassium carbonate (1.0-5.0 eq), an ammonia compound (1.0 eq), sodium iodide (1.0-3.0 eq) and acetonitrile were mixed in sequence, stirred at 20-100°C overnight, and purified by column chromatography to obtain amino lipid.

[0114] [Corrected 15.08.2025 in accordance with Regulation 26] Lipid nanoparticles

[0115] [Corrected 15.08.2025 according to Rule 26] The present invention provides a lipid nanoparticle comprising any one of the above-mentioned amino lipids.

[0116] [Corrected 15.08.2025 according to Rule 26] It will be understood that all the options and preferences mentioned above for amino lipids themselves also apply to the lipid nanoparticles comprising amino lipids of the present invention.

[0117] [Corrected 15.08.2025 according to Rule 26] In some embodiments of the present invention, the lipid nanoparticles further comprise a steroid, a neutral lipid and / or a polymer-bound lipid.

[0118] [Corrected 15.08.2025 in accordance with Regulation 26] Steroids

[0119] [Corrected 15.08.2025 under Rule 26] "Steroid" is an organic compound that has four rings arranged in a specific molecular configuration. It contains the following carbon skeleton:

[0120] [Corrected 15.08.2025 in accordance with Rule 26] Steroids and neutral steroids include naturally occurring steroids and their analogs (such as the amphiphilic lipid cholesterol hemisuccinate (CHEMS), which consists of succinate esterified to the β-hydroxy group of cholesterol as a cholesterol derivative). Neutral steroids can be steroids that do not have atoms or groups that are ionizable under physiological conditions, or can be zwitterionic steroids. In a preferred embodiment, the neutral steroid does not contain atoms or groups that are ionizable under physiological conditions. In some preferred embodiments, the steroid or steroid analog is cholesterol. The terms "steroid" and "neutral steroid" are used interchangeably herein.

[0121] [Corrected 15.08.2025 according to Rule 26] Neutral lipids

[0122] [Corrected 15.08.2025 according to Rule 26] The "neutral lipids" of the present invention, also known as "helper lipids," are preferably phospholipids or neutral phospholipids. As used herein, "neutral phospholipids" are amphiphilic compounds consisting of a molecule typically having two hydrophobic fatty acid "tails" and a hydrophilic "head" containing a phosphate group. The phosphate group can be modified with simple organic molecules such as choline, ethanolamine, or serine. Phospholipids are abundant in nature. "Phospholipids" or "neutral phospholipids" herein include both natural phospholipids and synthetic phospholipids.

[0123] [Corrected 15.08.2025 according to Rule 26] Polymer-bound lipids

[0124] [Corrected 15.08.2025 in accordance with Rule 26] The term "polymer-bound lipid" refers to a molecule comprising both a lipid portion and a polymer portion. Preferably, the polymer-bound lipid is a pegylated lipid or PEG-lipid. The term "pegylated lipid" or "PEG-lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include PEG-DMG, among others.

[0125] [Corrected 15.08.2025 according to Rule 26] In a specific embodiment, the polymer-bound lipid has the chemical formula PYL, wherein P is a hydrophilic polymer moiety, Y is an optional linker, and L is a lipid moiety.

[0126] [Corrected 15.08.2025 according to Rule 26] Specifically, the hydrophilic polymer portion P can be polyethylene glycol PEG. In a specific embodiment, the average molecular weight of the PEG portion is between 1 kDa and 3 kDa, for example, between 1.5 kDa and 2.5 kDa, between 1.7 kDa and 2.3 kDa, between 1.8 kDa and 2.2 kDa, between 1.9 kDa and 2.1 kDa, or 2 kDa. Thus, the PEG can be the PEG commonly known as "PEG 2000".

[0127] [Corrected 15.08.2025 according to Rule 26] In another embodiment, the hydrophilic polymer portion P in the polymer-bound lipid may also be a substantially hydrophilic polymer different from the above-mentioned hydrophilic polymer portion, i.e., the hydrophilic polymer portion P in the polymer-bound lipid may be based on poly(propylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), poly-N-(2-hydroxypropyl)methacrylamide, HESylation process (according to PMID 24681396), PASylation method (i.e., proline-alanine-serine), XTEN method known in the art (i.e., PEG-based peptides), polysarcosine or poly(vinyl acetate).

[0128] [Corrected 15.08.2025 in accordance with Rule 26] In particular, the optional linker Y may be any useful spacer structure, for example selected from those spacers commonly found useful in PEGylated lipids, such as, but not limited to, succinimide, amine, ether, ester, anhydride, aldehyde, ketone, amide, carbamate linkers or combinations thereof.

[0129] [Corrected 15.08.2025 in accordance with Rule 26] Specifically, the lipid moiety L may be derived from a phospholipid, a sphingolipid, or a ceramide. As used herein, the term "derived from a phospholipid or a ceramide" includes free radicals of phospholipids and ceramides. Examples are polymer-bound lipids containing a phosphatidylethanolamine or phosphatidylglycerol moiety.

[0130] [Corrected 15.08.2025 according to Rule 26] In a preferred embodiment, the polymer-bound lipid is a pegylated lipid. The pegylated lipid includes, but is not limited to, the following pegylated lipids: pegylated diacylglycerol lipids (PEG-DAG); pegylated ceramide lipids (PEG-Cer); pegylated phosphatidylethanolamine lipids (PEG-PE); pegylated succinate diacylglycerol lipids (PEG-S-DAG); pegylated dialkoxypropylcarbamate lipids; 1,2-dimyristyl-rac-glycero-3-methoxypolyethylene glycol ("PEG-DMG" or "DMG-PEG").

[0131] [Corrected 15.08.2025 according to Rule 26] In a more preferred embodiment, the polymer-bound lipid is DMG-PEG2000.

[0132] [Corrected 15.08.2025 according to rule 26] Preferably, as used in the art, "DMG-PEG 2000" is considered to be a mixture of 1,2-DMG PEG2000 and 1,3-DMG PEG2000 in a ratio of about 97:3.

[0133] [Corrected 15.08.2025 according to Rule 26] In some embodiments of the present invention, the molar ratio of the amino lipid, steroid, neutral lipid, and polymer-bound lipid in the lipid nanoparticles is 30-70:30-65:0-30:0.2-5. More preferably, it is 30-60:35-60:0-20:0.3-3.

[0134] [Corrected 15.08.2025 according to Rule 26] In a preferred embodiment of the present invention, in the lipid nanoparticles, the amino lipid is the aforementioned preferred amino lipid, the steroid is cholesterol, the neutral lipid is a phospholipid, and the polymer-bound lipid is a pegylated lipid; the molar ratio of the amino lipid, cholesterol, phospholipid and pegylated lipid is 40-50:40-45:10-15:0.5-2.

[0135] [Corrected 15.08.2025 according to Rule 26] In a preferred embodiment, in the lipid nanoparticles, the PEGylated lipid is DMG-PEG2000.

[0136] [Corrected 15.08.2025 pursuant to Rule 26] The lipid nanoparticles of the present invention are not limited to any particular morphology and should be construed to include any morphology resulting from the combination of an amino lipid and, optionally, one or more other lipids, for example, in an aqueous environment and / or in the presence of a nucleic acid compound. For example, liposomes, lipid complexes, lipoplexes, etc. are within the scope of lipid nanoparticles.

[0137] [Corrected 15.08.2025 according to Rule 26] The lipid nanoparticles of the present invention can be combined with at least one pharmaceutically acceptable carrier or excipient to obtain a pharmaceutical composition. Thus, the composition can be a dry composition, such as a powder or granules, or a solid unit, such as a lyophilized form or tablet. Alternatively, the composition can be in liquid form, and each excipient can be added independently in a dissolved or dispersed (e.g., suspended or emulsified) form. In a preferred embodiment, the composition is formulated as a sterile solid composition, such as a powder or lyophilized form, for reconstitution with an aqueous liquid carrier. Such a formulation is also preferred for compositions comprising biologically active components as described in further detail below.

[0138] [Corrected 15.08.2025 according to Rule 26] As used herein, a "nanoparticle" is a submicron particle having any structure or morphology. Submicron particles may also be referred to as colloids or colloids. Submicron particles may also be referred to as colloids or colloids. Nanoparticles may be classified with respect to the material on which they are based, as well as the structure or morphology, for example as nanocapsules, vesicles, liposomes, lipid nanoparticles, micelles, cross-linked micelles, lipoplexes, polymers, hybrids or hybrid complexes, to mention only a few possible nomenclatures for specific types of nanoparticles. "Lipid nanoparticles" (LNPs) are nanoparticles formed from lipids, typically comprising at least one amphiphilic, membrane-forming lipid and optionally other lipids, and further optionally comprising a payload material such as a nucleic acid compound. As used herein, the expression "lipid nanoparticle" or "LNP" includes any subtype and morphology of nanoparticles formed or co-formed by lipids, such as liposomes and lipoplexes.

[0139] [Corrected 15.08.2025 pursuant to Rule 26] As defined above, lipid nanoparticles include any type of nanoparticle formed from or co-formed with lipids. In particular, lipid nanoparticles may be co-formed with a combination of lipids including at least one amphiphilic, vesicle-forming lipid. Liposomes and lipoplexes are examples of lipid nanoparticles.

[0140] [Corrected 15.08.2025 according to Rule 26] Preferably, in some embodiments of the present invention, the lipid nanoparticles further comprise a biologically active component.

[0141] [Corrected 15.08.2025 as per Rule 26] A biologically active ingredient is any compound or material that has biological activity and, due to that activity, the compound or material is useful in the prevention, management, amelioration, treatment or therapy of a disease or condition in a subject, such as an animal, in particular a human subject.

[0142] [Corrected 15.08.2025 according to Rule 26] In some embodiments, the biologically active component is a nucleic acid compound selected from the group consisting of artificial mRNA, chemically modified or unmodified messenger RNA comprising at least one coding sequence, self-replicating RNA, circular RNA, viral RNA, and replicon RNA; or any combination thereof. Preferably, the biologically active component is mRNA or an mRNA compound.

[0143] [Corrected 15.08.2025 according to Rule 26] In some embodiments, the biologically active component is selected from small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and mixtures thereof.

[0144] [Corrected 15.08.2025 according to Rule 26] In an embodiment of the present invention, the mRNA comprises one or more of a stem-loop, a chain-terminating nucleoside, a polyA sequence, a polyadenylation signal and / or a 5' cap structure.

[0145] [Corrected 15.08.2025 according to Rule 26] In an embodiment of the present invention, the encapsulation efficiency of the bioactive component is at least 50-90%. More preferably, the encapsulation efficiency of the bioactive component is at least 60-80%.

[0146] [Corrected 15.08.2025 according to Rule 26] In an embodiment of the present invention, in the lipid nanoparticles, the weight / weight ratio of the lipid component to the bioactive component is about 10:1 to about 60:1. More preferably, the weight / weight ratio of the lipid component to the bioactive component is about 20:1.

[0147] [Corrected 15.08.2025 according to Rule 26] In an embodiment of the present invention, the N:P ratio in the lipid nanoparticles is about 2:1 to about 30:1. Further preferably, the N:P ratio is about 5.67:1.

[0148] [Corrected 15.08.2025 as per Rule 26] In an embodiment of the present invention, the average size of the lipid nanoparticles is from about 70 nm to about 100 nm.

[0149] [Corrected 15.08.2025 as per Rule 26] In an embodiment of the present invention, the polydispersity index of the lipid nanoparticles is from about 0.10 to about 0.20.

[0150] [Corrected 15.08.2025 as per Rule 26] In an embodiment of the present invention, the lipid nanoparticles have a zeta potential of about -10 mV to about +20 mV.

[0151] [Corrected 15.08.2025 according to Rule 26] In a preferred embodiment, the biologically active component is complexed or associated with one or more lipids (e.g., amino lipids and / or neutral lipids) to form liposomes, lipid nanoparticles (LNPs), lipoplexes and / or nanoliposomes. In this context, the term "complexed" or "associated" refers to a substantially stable combination of the biologically active component and one or more lipids into a larger complex or assembly that is not covalently bound.

[0152] [Corrected 15.08.2025 according to Rule 26] Application

[0153] [Corrected 15.08.2025 according to Rule 26] The present invention provides a method for treating or preventing infectious diseases, cancer, tumor diseases, genetic diseases, allergies, toxicity, and autoimmune diseases using the above-mentioned amino lipids or lipid nanoparticles or pharmaceutical compositions.

[0154] [Corrected 15.08.2025 according to Rule 26] Accordingly, the present invention provides the use of the above-mentioned amino lipids or lipid nanoparticles or pharmaceutical compositions in the preparation of drugs for treating or preventing infectious diseases, cancer, tumor diseases, genetic diseases, allergies, toxicity, and autoimmune diseases.

[0155] [Corrected 15.08.2025 in accordance with Rule 26] Wherein, the infectious diseases include viral, bacterial or protozoal infectious diseases. The viruses include but are not limited to SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus, SARS coronavirus (SARS-CoV), bunyavirus, cytomegalovirus (CMV), dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4), Ebola virus, flavivirus, hepatitis B virus (HBV), herpes simplex virus (HSV), human immunodeficiency virus (HIV), human metapneumovirus (hMPV), human papillomavirus (HPV), human parainfluenza virus (HPIV), influenza virus, extra-enteropathogenic Escherichia coli, Lassa virus (LASV), MERS coronavirus, Mycobacterium tuberculosis, Nipah virus, norovirus, rabies virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, vaccinia virus, yellow fever virus, and Zika virus.

[0156] [Corrected 15.08.2025 in accordance with Rule 26] The cancers mentioned include lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer and prostate cancer.

[0157] [Corrected 15.08.2025 according to Rule 26] The present invention also provides a method for gene therapy, gene vaccination, antisense therapy, nucleic acid transfer or treatment by interfering RNA using the above-mentioned amino lipids or lipid nanoparticles or pharmaceutical compositions.

[0158] [Corrected 15.08.2025 according to Rule 26] Accordingly, the present invention also provides the use of the above-mentioned amino lipids or lipid nanoparticles in the preparation of a medicament for gene therapy, gene vaccination, antisense therapy, nucleic acid transfer or treatment by interfering RNA.

[0159] [Corrected 15.08.2025 according to Rule 26] The present invention also provides a method for delivering a pharmaceutical agent to a subject, comprising: administering to the subject the pharmaceutical agent formulated in the above-mentioned lipid nanoparticles.

[0160] [Corrected 15.08.2025 in accordance with Rule 26] When administering a drug, the route of administration is determined by the drug's dosage form. The drug's dosage form is related to the excipient and / or carrier. Common routes of systemic administration include transdermal, oral, and parenteral routes, including subcutaneous, intravenous, intramuscular, intraarterial, intradermal, and intraperitoneal injections, and / or intranasal administration. Local administration routes generally include intradermal, transdermal, subcutaneous, or intramuscular injections, or intralesional, intracranial, intrapulmonary, intracardial, intratumoral, and sublingual injections. When the drug is in the form of a vaccine, preferred routes of administration are intramuscular and intradermal injections.

[0161] [Corrected 15.08.2025 according to Rule 26] The following examples are used to illustrate the present invention but are not intended to limit the scope of the invention.

[0162] [Corrected 15.08.2025 according to Rule 26] If specific techniques or conditions are not specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments used without manufacturer's indication are conventional products available through regular channels.

[0163] [Corrected 15.08.2025 according to Rule 26] For the purpose of illustration, the general method for preparing the compounds provided by the present invention is shown in the following examples. For a more detailed description of each reaction step, see the Examples section below. Those skilled in the art will appreciate that other synthetic routes can be used to synthesize the compounds of the present invention. Although specific raw materials and reagents are described in the scheme and discussed below, other raw materials and reagents can be easily substituted to provide various derivatives and / or reaction conditions. In addition, in conjunction with the disclosure of the following examples, many compounds prepared by the following methods can be further modified using conventional chemical methods well known to those skilled in the art, and other compounds within the scope of the present invention can be obtained.

[0164] [Corrected 15.08.2025 according to Rule 26] The abbreviations in the text correspond to the following substances: Py pyridine EDCI 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride DMAP p-dimethylaminopyridine DIPEA diisopropylethylamine DCM dichloromethane MeCN acetonitrile DSPC distearoylphosphatidylcholine DMG-PEG2000 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000.

[0165] [Corrected 15.08.2025 according to Rule 26] Preparation of amino lipid compounds

[0166] [Corrected 15.08.2025 according to Rule 26] Example 1 Synthesis of amino lipid E8LA12B6O3

[0167] [Corrected 15.08.2025 according to Rule 26] Step 1: Synthesis of E8LA12:

[0168] [Corrected 15.08.2025 according to Rule 26] FeCl3 (16.2 mg, 2.5 mol%), Py (4 mg, 1.25 mmol%), lauric acid (200.32 g / mol, 800 mg, 4.0 mmol) and 1,2-epoxyoctane (128.22 g / mol, 1.02 g, 8 mmol) were added sequentially to a 25 mL reaction tube. The mixture was stirred at room temperature overnight. After completion of the reaction, 1.2 g of the intermediate E8LA12 (colorless oily liquid) was obtained by column chromatography purification with a yield of 91%.

[0169] [Corrected 15.08.2025 according to Rule 26] Step 2: Synthesis of E8LA12B6:

[0170] [Corrected 15.08.2025 according to Rule 26] To a 25 mL reaction tube, E8LA12 (328.54 g / mol, 1.2 g, 3.65 mmol), 6-bromohexanoic acid (195.06 g / mol, 855 mg, 4.38 mmol), EDCI (191.70 g / mol, 2.8 g, 14.6 mmol), DMAP (45 mg, 0.365 mmol), DIPEA (2.83 g, 21.9 mmol), and DCM (10 mL) were added sequentially and stirred at room temperature overnight. After completion of the reaction, the intermediate E8LA12B6 was isolated and purified by column chromatography to obtain 1.65 g of the intermediate (colorless oily liquid) in an 89% yield.

[0171] [Corrected 15.08.2025 according to Rule 26] Step 3: Synthesis of E8LA12B6O3:

[0172] [Corrected 15.08.2025 according to Rule 26] To a 25 mL reaction tube were added E8LA12B6 (505.58 g / mol, 1.65 g, 3.26 mmol), potassium carbonate (138.21 g / mol, 902 mg, 6.52 mmol), sodium iodide (149.89 g / mol, 489 mg, 3.26 mmol), aminopropanol (75.11 g / mol, 113 mg, 1.5 mmol), and acetonitrile (10 mL). The mixture was stirred at 75°C overnight. After completion of the reaction, 0.94 g of E8LA12B6O3 (colorless oily liquid) was obtained by column chromatography and purification. The yield was 68%. 1H NMR (400MHz, CDCl3): δ5.08-5.03(m,2H),4.22(dd,J1=11.6Hz,J2=3.6Hz,2H),4.02(dd,J1=12.0Hz,J2=6.4Hz,2H),3.82(t,J=5.6Hz,2H),2.96 (t,J=7.6Hz,2H),2.77(t,J=6.0Hz,4H),2.34-2.28(m,8H),1.89-1.88( m,2H),1.72-1.56(m,12H),1.40-1.25(m,56H),0.87(t,J=7.0Hz,12H). 13 C NMR (100MHz, CDCl3): δ173.1,70.7,65.5,59.0,58.5,57.2,34.2,33.9,31.9,31. 8,30.7,30.3,29.6,29.3,29.0,28.0,26.7,25.0,25.3,24.7,22.7,14.1.ESI-MS C 55 H 106 NO9 + [M+H] + Calculated value: 924.7862, measured value: 924.7850.

[0173] [Corrected 15.08.2025 according to Rule 26] Example 2 Synthesis of amino lipid E12LA6B6O3

[0174] [Corrected 15.08.2025 according to Rule 26] Step 1: Synthesis of E12LA6:

[0175] [Corrected 15.08.2025 according to Rule 26] FeCl3 (16.2 mg, 2.5 mol%), Py (4 mg, 1.25 mmol%), n-hexanoic acid (116.1600 g / mol, 464 mg, 4.0 mmol) and 1,2-epoxydodecane (184.18 g / mol, 1.47 g, 8 mmol) were added sequentially to a 25 mL reaction tube. The mixture was stirred at room temperature overnight. After completion of the reaction, 1.02 g of the intermediate E12LA6 (colorless oily liquid) was obtained by column chromatography for purification in a yield of 85%.

[0176] [Corrected 15.08.2025 according to Rule 26] Step 2: Synthesis of E12LA6B6:

[0177] [Corrected 15.08.2025 according to Rule 26] To a 25 mL reaction tube, E12LA6 (300.48 g / mol, 901 mg, 3.0 mmol), 6-bromohexanoic acid (195.06 g / mol, 703 mg, 3.6 mmol), EDCI (191.70 g / mol, 2.3 g, 12.0 mmol), DMAP (37 mg, 0.3 mmol), DIPEA (2.33 g, 18 mmol), and DCM (10 mL) were added sequentially and stirred at room temperature overnight. After completion of the reaction, the intermediate E12LA6B6 was isolated and purified by column chromatography to obtain 1.08 g of the intermediate (colorless oily liquid) in a 76% yield.

[0178] [Corrected 15.08.2025 according to Rule 26] Step 3: Synthesis of E12LA6B6O3:

[0179] [Corrected 15.08.2025 in accordance with Article 26] To a 25 mL reaction tube, E12LA6B6 (477.52 g / mol, 956 mg, 2.0 mmol), potassium carbonate (138.21 g / mol, 221 mg, 1.6 mmol), sodium iodide (149.89 g / mol, 120 mg, 0.8 mmol), aminopropanol (75.11 g / mol, 60 mg, 0.8 mmol), and acetonitrile (5 mL) were added sequentially and stirred at 75°C overnight. After completion of the reaction, the product was separated and purified by column chromatography to obtain 458 mg of E12LA6B6O3 (a colorless oily liquid), in a 66% yield. 1 H NMR (400 MHz, CDCl3): δ 5.08-5.06 (m, 2H), 4.22 (dd, J1 = 12.0 Hz, J2 = 3.6 Hz, 2H), 4.02 (dd, J1 = 11.6 Hz, J2 = 6.4 Hz, 2H), 3.79 (t, J = 5.2 Hz, 2H), 2.73-2.70 (m, 2H), 2.52-2.48 (m, 4H), 2.33-2.28 (m, 8H), 1.74-1.50 (m, 14H), 1.42-1.25 (m, 48H), 0.91-0.86 (m, 12H). (As shown in Figure 1) ESI-MS C 51 H 98 NO9 + [M+H] + Calculated value 868.7236, measured value 868.7234.

[0180] [Corrected 15.08.2025 according to Rule 26] Example 3 Synthesis of amino lipid E8LA8B6O3

[0181] [Corrected 15.08.2025 according to Rule 26] The amino lipid E8LA8B6O3 can be prepared by the same method by replacing lauric acid in Example 1 with caprylic acid, and its structural formula is as follows:

[0182] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ5.08-5.02(m,2H),4.22(dd,J1=12.0Hz,J2=3.6Hz,2H),4.01(dd,J1=11.6Hz,J2=6.0Hz,2H),3.81(t,J=5.2Hz,2H),2.98( t,J=6.0Hz,2H),2.79(t,J=7.6Hz,4H),2.34-2.27(m,8H),1.92-1.86(m, 2H),1.73-1.56(m,12H),1.40-1.26(m,40H),0.88-0.85(m,12H).ESI-MS C 47 H 90 NO9 + [M+H] + Calculated value 812.6610, measured value 812.6598.

[0183] [Corrected 15.08.2025 according to Rule 26] Example 4 Synthesis of amino lipid E8LA10B6O3

[0184] [Corrected 15.08.2025 according to Rule 26] By replacing lauric acid in Example 1 with capric acid, the amino lipid E8LA10B6O3 can be prepared according to the same method, and its structural formula is as follows:

[0185] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.85-0.89(m,12H),1.25-1.40(m,48H),1.56-1.72(m,12H),1.88-1.90(m,2H),2.28-2.33(m,8 H),2.77-2.79(m,4H),2.96-2.98(m,2H),3.79-3.80(m,2H),4.01-4.05(m,2H),4.21-4.24(m,2H),5.03-5.08(m,2H). ESI-MSC 51 H 98 NO9 + [M+H] + Calculated value 868.7, measured value 868.7.

[0186] [Corrected 15.08.2025 according to Rule 26] Example 5 Synthesis of amino lipid E10LA6B6O3

[0187] [Corrected 15.08.2025 according to Rule 26] The amino lipid E10LA6B6O3 can be prepared by replacing 1,2-epoxydodecane in Example 2 with 1,2-epoxydecane according to the same method, and its structural formula is as follows:

[0188] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ5.08-5.02(m,2H),4.22(dd,J1=12.0Hz,J2=3.2Hz,2H),4.01(dd,J1=11.6Hz,J2=6.4Hz,2H),3.79(t,J=5.2Hz,2H),2.82( t,J=5.6Hz,2H),2.62(t,J=8.0Hz,4H),2.36-2.27(m,8H),1.81-1.76(m, 2H),1.68-1.53(m,12H),1.37-1.24(m,40H),0.90-0.84(m,12H).ESI-MS C 47 H 90 NO9 + [M+H] + Calculated value 812.6610, measured value 812.6634.

[0189] [Corrected 15.08.2025 according to Rule 26] Example 6 Synthesis of amino lipid E10LA8B6O3

[0190] [Corrected 15.08.2025 according to Rule 26] The amino lipid E10LA8B6O3 can be prepared by the same method by replacing 1,2-epoxyoctane with 1,2-epoxydecane and lauric acid with n-octanoic acid in Example 1. Its structural formula is as follows:

[0191] [Corrected 15.08.2025 in accordance with Article 26] 1H NMR (400MHz, CDCl3): δ0.84-0.89(m,12H),1.26-1.41(m,48H),1.56-1.73(m,12H),1.89-1.91(m,2H),2.26-2.32(m,8 H),2.78-2.80(m,4H),2.96-2.98(m,2H),3.80-3.81(m,2H),4.01-4.04(m,2H),4.20-4.24(m,2H),5.03-5.07(m,2H). ESI-MSC 51 H 98 NO9 + [M+H] + Calculated value 868.7, measured value 868.7.

[0192] [Corrected 15.08.2025 according to Rule 26] Example 7 Synthesis of amino lipid E10LA10B6O3

[0193] [Corrected 15.08.2025 according to Rule 26] The amino lipid E10LA10B6O3 can be prepared by the same method by replacing 1,2-epoxyoctane with 1,2-epoxydecane and lauric acid with capric acid in Example 1. Its structural formula is as follows:

[0194] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.86-0.92(m,12H),1.25-1.40(m,56H),1.56-1.71(m,12H),1.87-1.89(m,2H),2.27-2.32(m,8 H),2.77-2.80(m,4H),2.97-2.99(m,2H),3.79-3.80,(m,2H),4.00-4.04(m,2H),4.21-4.25(m,2H),5.02-5.07(m,2H). ESI-MSC 55 H 106 NO9 + [M+H] + Calculated value 924.8, measured value 924.9.

[0195] [Corrected 15.08.2025 according to Rule 26] Example 8 Synthesis of amino lipid E12LA8B6O3

[0196] [Corrected 15.08.2025 according to Rule 26] The amino lipid E12LA8B6O3 can be prepared by the same method by replacing n-hexanoic acid with n-octanoic acid in Example 2, and its structural formula is as follows:

[0197] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ5.07-5.04(m,2H),4.23(dd,J1=12.0Hz,J2=3.6Hz,2H),4.01(dd,J1=12.0Hz,J2=6.4Hz,2H),3.80(t,J=5.2Hz,2H),2.86( t,J=5.6Hz,2H),2.66(t,J=7.6Hz,4H),2.33-2.28(m,8H),1.83-1.80(m, 2H),1.69-1.56(m,12H),1.38-1.25(m,56H),0.89-0.85(m,12H).ESI-MS C 55 H 106 NO9 + [M+H] + Calculated value: 924.7862, measured value: 924.7891.

[0198] [Corrected 15.08.2025 according to Rule 26] Example 9 Synthesis of Compound E12LA12B6O3

[0199] [Corrected 15.08.2025 according to Rule 26] Step 1: Synthesis of compound E12LA12B6O3-2

[0200] [Corrected 15.08.2025 according to Rule 26] Ferric chloride (16.2 mg, 0.1 mmol, 0.025 eq), pyridine (4 mg, 0.05 mmol, 0.0125 eq), lauric acid (800 mg, 4.0 mmol, 1.0 eq), and 1,2-epoxydodecane (884 mg, 4.8 mmol, 1.2 eq) were added sequentially to a 25 mL reaction tube and stirred at room temperature overnight. After completion of the reaction, the reaction solution was concentrated and purified using a flash column chromatography system (n-heptane:ethyl acetate = 50:1 to 10:1) to obtain compound E12LA12B6O3-2 (1.2 g, 86.0%). ESI-MS C 24 H 49 O3 + [M+H] + Calculated value 385.4, measured value 385.4.

[0201] [Corrected 15.08.2025 according to Rule 26] Step 2: Synthesis of compound E12LA12B6O3-3

[0202] [Corrected 15.08.2025 according to Rule 26] In a 25 mL reaction tube, E12LA12B6O3-2 (1.2 g, 3.65 mmol, 1.0 eq), 6-bromohexanoic acid (973 mg, 4.38 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.8 g, 14.6 mmol, 4.0 eq), 4-dimethylaminopyridine (45 mg, 0.365 mmol, 0.1 eq) and N,N-diisopropylethylamine (2.83 g, 21.9 mmol, 6.0 eq) and 20 mL of dichloromethane were added in sequence. The reaction was stirred at room temperature overnight. After the reaction, the reaction solution was concentrated and purified using a flash column chromatography system (n-heptane:ethyl acetate = 100:1 to 20:1) to obtain compound E12LA12B6O3-3 (1.65 g, 92.0%). ESI-MS C 30 H 58 BrO4 + [M+H] + Calculated value 561.4, measured value 561.4.

[0203] [Corrected 15.08.2025 according to Rule 26] Step 3: Synthesis of E12LA12B6O3

[0204] [Corrected 15.08.2025 according to Rule 26] In a 25 mL reaction tube, E12LA12B6O3-3 (1.65 g, 3.36 mmol, 2.2 eq), potassium carbonate (464 mg, 3.36 mmol, 2.2 eq), sodium iodide (150 mg, 1.0 mmol, 0.67 eq), 3-aminopropanol (113 mg, 1.5 mmol), and 10 mL of acetonitrile were added in sequence. The mixture was stirred at 70-80°C overnight. After the reaction, 0.94 g of E12LA12B6O3 (0.94 g, 68%) was obtained by column chromatography. 1 H NMR (400MHz, CDCl3): δ0.87-0.91(m,12H),1.24-1.38(m,68H),1.42-1.58(m,4H),1.55-1.69(m,14H),2.29-2.34(m,8 H),2.38-2.42(m,4H),2.61-2.64(m,2H),3.78-3.81,(m,2H),4.01-4.06(m,2H),4.20-4.24(m,2H),5.04-5.10(m,2H). ESI-MS C 63 H 122 NO9 + [M+H] + Calculated value 1036.9, measured value 1037.0.

[0205] [Corrected 15.08.2025 according to Rule 26] Example 10 Synthesis of amino lipid E8CA5B6O3

[0206] [Corrected 15.08.2025 according to Rule 26] In Example 9, 1,2-epoxydodecane is replaced by 1,2-epoxyoctane, and lauric acid is replaced by 2-pentyl-heptanoic acid. The amino lipid E8CA5B6O3 can be prepared by the same method, and its structural formula is as follows:

[0207] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.85-0.90(m,18H),1.25-1.37(m,48H),1.40-1.70(m,18H),2.25-2.36(m,6H),2.44- 2.48(m,4H),2.66-2.68(m,2H),3.77-3.79,(m,2H),4.00-4.05(m,2H),4.21-4.25(m,2H),5.03-5.08(m,2H). ESI-MS C 55 H 106 NO9 + [M+H] + Calculated value 924.8, measured value 924.8.

[0208] [Corrected 15.08.2025 according to Rule 26] Example 11 Synthesis of amino lipid E12LA6B6O9

[0209] [Corrected 15.08.2025 according to Rule 26] The amino lipid E12LA6B6O9 can be prepared by the same method by replacing 3-aminopropanol in Example 2 with n-pentylamine, and its structural formula is as follows:

[0210] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.85-0.90(m,15H),1.24-1.36(m,54H),1.53-1.74(m,16H),2. 27-2.33(m,8H),2.83(s,6H),3.98-4.03(m,2H),4.21-4.25(m,2H),5.02-5.07(m,2H). ESI-MS C 53 H 102 NO8 + [M+H] +Calculated value 880.8, measured value 880.8

[0211] [Corrected 15.08.2025 according to Rule 26] Example 12 Synthesis of amino lipid E12LA6B6O10

[0212] [Corrected 15.08.2025 according to Rule 26] The amino lipid E12LA6B6O10 can be prepared by the same method by replacing the 3-aminopropanol in Example 2 with trans-p-aminocyclohexanol, and its structural formula is as follows:

[0213] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.84-0.90(m,12H),1.24-1.31(m,54H),1.53-1.65(m,14H),1.99-2.01(m,2 H),2.26-2.31(m,8H),2.42-2.46(m,4H),3.99-4.03(m,2H),4.19-4.22(m,2H),5.03-5.08(m,2H). ESI-MS C 54 H 102 NO9 + [M+H] + Calculated value 908.8, measured value 908.7.

[0214] [Corrected 15.08.2025 according to Rule 26] Example 13 Synthesis of amino lipid E12LA6B6O12

[0215] [Corrected 15.08.2025 according to Rule 26] The amino lipid E12LA6B6O12 can be prepared by the same method by replacing 3-aminopropanol in Example 2 with 3-dimethylaminopropylamine, and its structural formula is as follows:

[0216] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.86-0.94(m,12H),1.26-1.35(m,42H),1.41-1.48(m,6H),1.55-1.72(m,18H),2.29- 2.36(m,8H),2.70-2.76(m,2H),3.37-3.41(m,4H),3.99-4.04(m,2H),4.24-4.34(m,6H),5.00-5.06(m,2H). ESI-MS C 53 H 103 N2O8+ [M+H] + Calculated value 895.8, measured value 895.8.

[0217] [Corrected 15.08.2025 according to Rule 26] Example 14 Synthesis of amino lipid E12LA6B6O13

[0218] [Corrected 15.08.2025 according to Rule 26] The amino lipid E12LA6B6O13 can be prepared by the same method by replacing 3-aminopropanol in Example 2 with 1-(2-aminoethyl)piperidine, and its structural formula is as follows:

[0219] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.82-0.92(m,12H),1.24-1.36(m,50H),1.44-1.69(m,18H),2.27-2.31(m,8H),2.47- 2.50(m,4H),2.57-2.61(m,4H),2.72-2.74(m,2H),3.99-4.04(m,2H),4.19-4.23(m,2H),5.03-5.08(m,2H). ESI-MS C 55 H 105 N2O8 + [M+H] + Calculated value 921.8, measured value 921.8.

[0220] [Corrected 15.08.2025 according to Rule 26] Example 15 Synthesis of amino lipid E12LA6B6O15

[0221] [Corrected 15.08.2025 according to Rule 26] The amino lipid E12LA6B6O15 can be prepared by the same method by replacing 3-aminopropanol in Example 2 with N-(2-aminoethyl)-4-hydroxypiperidine, and its structural formula is as follows:

[0222] [Corrected 15.08.2025 in accordance with Article 26] 1H NMR (400MHz, CDCl3): δ0.86-0.90(m,12H),1.26-1.37(m,50H),1.48-1.67(m,16H),2.28-2.33(m,8H),2. 54-2.61(m,6H),2.84-2.89(m,4H),3.76(s,1H).4.00-4.07(m,2H),4.20-4.24(m,2H),5.04-5.09(m,2H). ESI-MS C 55 H 105 N2O9 + [M+H] + Calculated value 937.8, measured value 937.9.

[0223] [Corrected 15.08.2025 according to Rule 26] Example 16 Synthesis of amino lipid E12LA12B6O30

[0224] [Corrected 15.08.2025 according to Rule 26] The amino lipid E12LA12B6O30 can be prepared by the same method by replacing 3-aminopropanol in Example 2 with β-alanine, and its structural formula is as follows:

[0225] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.86-0.92(m,12H),1.24-1.37(m,68H),1.44-1.60(m,4H),1.54-1.70(m,14H),2.29-2 .44(m,12H),2.61-2.64(m,2H),3.78-3.81,(m,2H),4.01-4.05(m,2H),4.21-4.24(m,2H),5.02-5.08(m,2H). ESI-MS C 64 H 122 NO 10 + [M+H] + Calculated value 1064.9, measured value 1065.0.

[0226] [Corrected 15.08.2025 according to Rule 26] Example 17 Synthesis of amino lipid E12LA6B6O31

[0227] [Corrected 15.08.2025 according to Rule 26] The amino lipid E12LA12B6O31 can be prepared by the same method by replacing 3-aminopropanol in Example 2 with 4-aminobutyronitrile, and its structural formula is as follows:

[0228] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.86-0.91(m,12H),1.25-1.39(m,68H),1.42-1.60(m,4H),1.58-1.72(m,12H),1.78-1.82(m,2H) ),2.31-2.42(m,12H),2.60-2.64(m,2H),3.79-3.82,(m,2H),4.00-4.04(m,2H),4.18-4.22(m,2H),5.02-5.09(m,2H). ESI-MS C 64 H 121 N2O8 + [M+H] + Calculated value 1045.9, measured value 1045.9.

[0229] [Corrected 15.08.2025 according to Rule 26] Example 18 Synthesis of amino lipid K3LA6B6O3

[0230] [Corrected 15.08.2025 according to Rule 26] Step 1: Synthesis of compound K3LA6B6O3-2

[0231] [Corrected 15.08.2025 according to Rule 26] Imidazole (6.54 g, 96 mmol, 1.2 eq) was added to a 250 mL round-bottom flask and the flask was evacuated to be filled with nitrogen. Dichloromethane (60 mL) and N,N-dimethylformamide (30 mL) and 1,3-propylene glycol (6.09 g, 80 mmol, 1.0 eq) were added in sequence and the mixture was stirred at 0°C for 10 minutes. Tert-butyldimethylsilyl chloride (12.06 g, 80 mmol, 1.0 eq) dissolved in dichloromethane (60 mL) was added dropwise to the mixture and the resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (n-heptane:ethyl acetate = 100:1 to 10:1) to obtain compound K3LA6B6O3-2 (10.96 g, 72%). ESI-MS: C9H 23 O2Si + [M+H] + Calculated value 191.1, measured value 191.1.

[0232] [Corrected 15.08.2025 according to Rule 26] Step 2: Synthesis of compound K3LA6B6O3-3

[0233] [Corrected 15.08.2025 according to Rule 26] A solution of pyridinium chlorochromate (6.09 g, 80 mmol, 1.0 eq) and compound K3LA6B6O3-2 (10.96 g, 57.6 mmol, 1.0 eq) in dichloromethane (100 mL) was stirred at room temperature for 5 hours. The reaction mixture was filtered, the filtrate was concentrated, and purified using a flash column chromatography system (n-heptane:ethyl acetate = 100:1 to 20:1) to obtain compound K3LA6B6O3-3 (8.69 g, 80%). ESI-MS: C9H 21 O2Si + [M+H] + Calculated value 189.1, measured value 189.1.

[0234] [Corrected 15.08.2025 according to Rule 26] Step 3: Synthesis of compound K3LA6B6O3-4

[0235] [Corrected 15.08.2025 according to Rule 26] A 500 mL round-bottom flask was ventilated to fill the flask with nitrogen, and compound K3LA6B6O3-3 (8.69 g, 46.14 mmol, 1.0 eq) and anhydrous tetrahydrofuran (150 mL) were added. The mixture was stirred at -20°C for 10 minutes, and 70 mL of decylmagnesium bromide solution (1 mol / L anhydrous tetrahydrofuran solution, 1.5 eq) was added dropwise to the mixture. The mixture was stirred at -20°C to 0°C for 3 hours, and quenched by adding 50 mL of saturated ammonium chloride solution. The mixture was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by flash column chromatography (n-heptane:ethyl acetate = 100:1 to 20:1) to obtain compound K3LA6B6O3-4 (9.93 g, 65%). ESI-MS C 19 H 43 O2Si + [M+H] + Calculated value 331.3, measured value 331.4.

[0236] [Corrected 15.08.2025 according to Rule 26] Step 4: Synthesis of compound K3LA6B6O3-5

[0237] [Corrected 15.08.2025 according to Rule 26] A solution of K3LA6B6O3-4 (9.93 g, 30 mmol), 6-bromohexanoic acid (7.02 g, 36 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (11.50 g, 60 mmol, 2.0 eq), N,N-diisopropylethylamine (15.51 g, 120 mmol, 4.0 eq), 4-dimethylaminopyridine (0.37 g, 3 mmol, 0.1 eq) in dichloromethane (100 mL) was stirred at room temperature overnight. TLC showed that the reaction was complete. The reaction solution was extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (n-heptane:ethyl acetate = 100:1 to 20:1) to obtain compound K3LA6B6O3-5 (12.79 g, 84%). ESI-MS C 25 H 52 BrO3Si + [M+H] + Calculated value 507.3, measured value 507.2.

[0238] [Corrected 15.08.2025 according to Rule 26] Step 5: Synthesis of compound K3LA6B6O3-6

[0239] [Corrected 15.08.2025 according to Rule 26] K3LA6B6O3-5 (12.79 g, 25.2 mmol, 1.0 eq), ammonium fluoride (9.33 g, 252 mmol, 10.0 eq), methanol (75 mL), refluxed at 65°C with stirring for 3 hours. TLC showed the reaction was complete. The reaction solution was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (n-heptane:ethyl acetate = 50:1 to 5:1) to obtain compound K3LA6B6O3-6 (8.72 g, 88%). ESI-MS C 19 H 38 BrO3 + [M+H] + Calculated value 393.2, measured value 393.2.

[0240] [Corrected 15.08.2025 according to Rule 26] Step 6: Synthesis of compound K3LA6B6O3-7

[0241] [Corrected 15.08.2025 according to Rule 26] A solution of K3LA6B6O3-6 (8.72 g, 22.2 mmol), hexanoic acid (3.09 g, 26.64 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8.51 g, 44.4 mmol, 2.0 eq), N,N-diisopropylethylamine (11.48 g, 88.8 mmol, 4.0 eq), 4-dimethylaminopyridine (0.27 g, 2.2 mmol, 0.1 eq) in dichloromethane (50 mL) was stirred at room temperature overnight. TLC showed that the reaction was complete. The reaction solution was extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (n-heptane:ethyl acetate = 100:1 to 20:1) to obtain compound K3LA6B6O3-7 (7.86 g, 72%). ESI-MS C 25 H 48 BrO4 + [M+H] + Calculated value 491.3, measured value 491.2.

[0242] [Corrected 15.08.2025 according to Rule 26] Step 7: Synthesis of compound K3LA6B6O3

[0243] [Corrected 15.08.2025 according to Rule 26] A solution of compound K3LA6B6O3-7 (7.86 g, 15.99 mmol, 3.0 eq), 3-aminopropanol (0.40 g, 5.33 mmol, 3.0 eq), potassium carbonate (1.47 g, 10.66 mmol, 2.0 eq), and sodium iodide (0.80 g, 5.33 mmol, 1.0 eq) in acetonitrile (10 mL) was stirred at 75°C overnight. After concentration, the reaction mixture was purified using a flash column chromatography system (dichloromethane:methanol = 100:1 to 20:1) to obtain compound K3LA6B6O3-7 (3.58 g, 75%). 1 H NMR (400MHz, CDCl3): δ0.85-0.90(m,12H),1.24-1.36(m,48H),1.56-1.73(m,18H),2.25-2.29(m,8 H),2.48-2.52(m,4H),2.70-2.72(m,2H),3.76-3.79(m,2H),4.03-4.11(m,4H),4.93-4.96(m,2H). ESI-MS C 53 H 102 NO9 + [M+H] + Calculated value 896.8, measured value 896.9.

[0244] [Corrected 15.08.2025 according to Rule 26] Example 19 Synthesis of amino lipid K4LA6B6O3

[0245] [Corrected 15.08.2025 according to Rule 26] The amino lipid K4LA6B6O3 can be prepared by the same method by replacing 1,3-propanediol with 1,4-butanediol in Example 18, and its structural formula is as follows:

[0246] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.85-0.91(m,12H),1.26-1.41(m,48H),1.56-1.72(m,18H),2.27-2.33(m,8 H),2.81-2.84(m,4H),3.00-3.04(m,2H),3.81-3.84(m,2H),4.04-4.07(m,4H),4.87-4.90(m,2H). ESI-MS C 55 H 106 NO9 + [M+H] + Calculated value 924.8, measured value 924.8.

[0247] [Corrected 15.08.2025 according to Rule 26] Example 20 Synthesis of amino lipid K5LA6B6O3

[0248] [Corrected 15.08.2025 according to Rule 26] The amino lipid K5LA6B6O3 can be prepared by the same method by replacing 1,3-propanediol with 1,5-pentanediol in Example 18, and its structural formula is as follows:

[0249] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.86-0.91(m,12H),1.26-1.41(m,54H),1.50-1.67(m,18H),2.27-2.34(m,8 H),2.91-2.96(m,4H),3.10-3.14(m,2H),3.83-3.85(m,2H),4.03-4.06(m,4H),4.85-4.89(m,2H). ESI-MS C 57 H 110 NO9 + [M+H] + Calculated value 952.8, measured value 952.8.

[0250] [Corrected 15.08.2025 according to Rule 26] Example 21 Synthesis of amino lipid E12LA6B6O3A2

[0251] [Corrected 15.08.2025 according to Rule 26] Step 1: Synthesis of compound E12LA6B6O3

[0252] [Corrected 15.08.2025 according to Rule 26] According to the method of Example 2, E12LA6B6O3 was synthesized.

[0253] [Corrected 15.08.2025 according to Rule 26] Step 2: Synthesis of compound E12LA6B6O3A2

[0254] [Corrected 15.08.2025 according to Rule 26] A dichloromethane solution of E12LA6B6O3 (0.87 g, 1 mmol, 1.0 eq), N,N'-dicyclohexylcarbodiimide (0.41 g, 1 mmol, 2.0 eq), DMAP (0.12 g, 0.1 mmol, 0.1 eq), and 3-(4-phenyl-piperazin-1-yl)-propionic acid (0.26 g, 1.5 mmol, 1.5 eq) was stirred at room temperature for 4 hours. TLC showed that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated and purified using a flash column chromatography system (dichloromethane:methanol = 100:1 to 10:1) to obtain compound E12LA6B6O3A2 (0.80 g, 78%). 1 H NMR (400MHz, CDCl3): δ0.85-0.90(m,12H),1.25-1.32(m,48H),1.36-1.49(m,4H),1.53-1.66(m,12H),2.27- 2.50(m,25H),2.67-2.71(m,2H),4.00-4.04(m,2H),4.08-4.11(m,2H),4.19-4.23(m,2H),5.04-5.09(m,2H). ESI-MS C 59 H 112 N3O 10 + [M+H] + Calculated value 1022.8, measured value 1022.9.

[0255] [Corrected 15.08.2025 according to Rule 26] Example 22 Synthesis of amino lipid E12LA6B6O3A3

[0256] [Corrected 15.08.2025 according to Rule 26] The amino lipid E12LA6B6O3A3 can be prepared by the same method by replacing 3-(4-phenyl-piperazin-1-yl)-propionic acid in Example 21 with 4-(4-methyl-1-piperazinyl)butanoic acid, and its structural formula is as follows:

[0257] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.85-0.90(m,12H),1.25-1.45(m,50H),1.55-1.66(m,12H),1.69-1.83(m,4 H),2.27-2.47(m,27H),4.00-4.04(m,2H),4.07-4.10(m,2H),4.19-4.23(m,2H),5.04-5.10(m,2H). ESI-MS C 60 H 114 N3O 10 + [M+H] + Calculated value 1036.8, measured value 1036.8.

[0258] [Corrected 15.08.2025 according to Rule 26] Example 23 Synthesis of amino lipid E12LA6B6O3A4

[0259] [Corrected 15.08.2025 according to Rule 26] The amino lipid E12LA6B6O3A4 can be prepared by the same method by replacing 3-(4-phenyl-piperazin-1-yl)-propionic acid in Example 21 with 3-(dimethylamino)propionic acid, and its structural formula is as follows:

[0260] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.84-0.89(m,12H),1.24-1.35(m,50H),1.41-1.48(m,6H),1.52-1.65(m,12H),2.23(s,6H),2.26-2.30(m,8H) ,2.35-2.39(m,4H),2.44-2.48(m,4H),2.58-2.62(m,2H),3.99-4.03(m,2H),4.08-4.11(m,2H),4.18-4.22(m,2H),5.03-5.09(m,2H). ESI-MS C 56 H 107 N2O 10 +[M+H] + Calculated value 967.8, measured value 967.8.

[0261] [Corrected 15.08.2025 according to Rule 26] Example 24 Synthesis of amino lipid E12LA6B6O3A5

[0262] [Corrected 15.08.2025 according to Rule 26] The amino lipid E12LA6B6O3A5 can be prepared by the same method by replacing 3-(4-phenyl-piperazin-1-yl)-propionic acid in Example 21 with 5-(dimethylamino)pentanoic acid, and its structural formula is as follows:

[0263] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.84-0.89(m,12H),1.24-1.35(m,48H),1.41-1.75(m,18H),2.21(s,6H),2.22-2.30(m,12H) ,2.35-2.37(m,4H),2.42-2.45(m,2H),3.98-4.03(m,2H),4.05-4.09(m,2H),4.18-4.22(m,2H),5.03-5.09(m,2H). ESI-MS C 58 H 111 N2O 10 + [M+H] + Calculated value 995.8, measured value 995.9.

[0264] [Corrected 15.08.2025 according to Rule 26] Example 25 Synthesis of amino lipid E12LA6B6O3A6

[0265] [Corrected 15.08.2025 according to Rule 26] The amino lipid E12LA6B6O3A3 can be prepared by the same method by replacing 3-(4-phenyl-piperazin-1-yl)-propionic acid in Example 21 with 7-(dimethylamino)heptanoic acid, and its structural formula is as follows:

[0266] [Corrected 15.08.2025 in accordance with Article 26] 1H NMR (400MHz, CDCl3): δ0.84-0.91(m,12H),1.23-1.37(m,50H),1.39-1.49(m,6H),1.52-1.64(m,14H),2.21(s,6H),2.25-2.3 0(m,12H),2.33-2.37(m,4H),2.42-2.45(m,2H),3.98-4.03(m,2H),4.05-4.08(m,2H),4.18-4.22(m,2H),5.03-5.08(m,2H). ESI-MS C 60 H 115 N2O 10 + [M+H] + Calculated value 1023.8, measured value 1023.8.

[0267] [Corrected 15.08.2025 according to Rule 26] Example 26 Synthesis of amino lipid E8LA6B6O3A6

[0268] [Corrected 15.08.2025 according to Rule 26] The amino lipid E8LA6B6O3A6 can be prepared by the same method by replacing E12LA6B6O3 in Example 21 with E8LA6B6O3, and its structural formula is as follows:

[0269] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.86-0.91(m,12H),1.26-1.43(m,36H),1.39-1.49(m,6H),1.56-1 .79(m,12H),2.23-2.48(m,24H),4.00-4.11(m,4H),4.20-4.24(m,2H),5.07-5.11(m,2H). ESI-MS C 52 H 99 N2O 10 + [M+H] + Calculated value 911.7, measured value 911.9.

[0270] [Corrected 15.08.2025 according to Rule 26] Example 27 Synthesis of amino lipid E12LA6B6O3A7

[0271] [Corrected 15.08.2025 according to Rule 26] The amino lipid E12LA6B6O3A7 can be prepared by the same method by replacing 3-(4-phenyl-piperazin-1-yl)-propionic acid in Example 21 with 1-methylpiperidine-4-carboxylic acid, and its structural formula is as follows:

[0272] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.87-0.92(m,12H),1.26-1.41(m,48H),1.57-1.83(m,20H),2.24-2.40(m,16H),2.45 -2.48(m,2H),2.80-2.84(m,2H),4.01-4.06(m,2H),4.10-4.13(m,2H),4.21-4.25(m,2H),5.06-5.11(m,2H). ESI-MS C 58 H 109 N2O 10 + [M+H] + Calculated value 993.8, measured value 993.7.

[0273] [Corrected 15.08.2025 according to Rule 26] Example 28 Synthesis of amino lipid E12LA6B6O3A8

[0274] [Corrected 15.08.2025 according to Rule 26] The amino lipid E12LA6B6O3A8 can be prepared by the same method by replacing 3-(4-phenyl-piperazin-1-yl)-propionic acid in Example 21 with 1-piperidinepropionic acid, and its structural formula is as follows:

[0275] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.85-0.92(m,12H),1.24-1.36(m,44H),1.40-1.45(m,6H),1.54-1.69(m,12H),2.27-2.30(m,8 H),2.34-2.52(m,12H),2.63-2.67(m,2H),4.00-4.04(m,2H),4.07-4.10(m,8H),4.19-4.23(m,2H),5.03-5.09(m,2H). ESI-MS C 59 H 111 N2O 10 + [M+H] +Calculated value 1007.8, measured value 1007.9.

[0276] [Corrected 15.08.2025 according to Rule 26] Example 29 Synthesis of amino lipid E12LA12B6O30A9

[0277] [Corrected 15.08.2025 according to Rule 26] Step 1: Synthesis of compound E12LA12B6O30A9-1

[0278] [Corrected 15.08.2025 according to Rule 26] According to the method of Example 16, E12LA12B6O30A9-1 was synthesized.

[0279] [Corrected 15.08.2025 according to Rule 26] Step 2: Synthesis of compound E12LA12B6O3A9

[0280] [Corrected 15.08.2025 according to Rule 26] A dichloromethane solution of E12LA12B6O30A9-1 (1.06 g, 1 mmol, 1.0 eq), N,N'-dicyclohexylcarbodiimide (0.41 g, 2 mmol, 2.0 eq), DMAP (0.12 g, 0.1 mmol, 0.1 eq), and 3-dimethylaminopropanol (0.16 g, 1.5 mmol, 1.5 eq) was stirred at room temperature for 4 hours. TLC showed that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated and purified using a flash column chromatography system (dichloromethane:methanol = 50:1 to 10:1) to obtain compound E12LA12B6O3A9-1 (0.92 g, 80%). 1 HNMR (400MHz, CDCl3): δ0.86-0.91(m,12H),1.24-1.42(m,68H),1.45-1.72(m,20H),1.84-1.86(m,2H),2.26(s,6H),

[0281] [Corrected 15.08.2025 according to Rule 26] 2.30-2.42 (m, 12H), 2.60-2.63 (m, 2H), 3.78-3.81 (m, 2H), 4.01-4.15 (m, 4H), 4.22-4.26 (m, 2H), 5.06-5.12 (m, 2H). ESI-MS C 69 H 133 N2O 10 + [M+H] + Calculated value 1050.0, measured value 1050.3.

[0282] [Corrected 15.08.2025 according to Rule 26] Example 30 Synthesis of amino lipid E12LA12B6O30A10

[0283] [Corrected 15.08.2025 according to Rule 26] The amino lipid E12LA12B6O30A10 can be prepared by the same method by replacing 3-dimethylaminopropanol in Example 29 with 3-dimethylaminopropylamine, and its structural formula is as follows:

[0284] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.86-0.91(m,12H),1.24-1.42(m,68H),1.45-1.84(m,22H),2.28(s,6H),2.32-2.44(m,12H) ,2.60-2.63(m,2H),3.50-3.54,(m,2H),3.78-3.82(m,2H),4.00-4.04(m,2H),4.22-4.26(m,2H),5.06-5.12(m,2H). ESI-MS C 69 H 134 N3O9 + [M+H] + Calculated value 1149.0, measured value 1149.0.

[0285] [Corrected 15.08.2025 according to Rule 26] Example 31 Synthesis of amino lipid E12LA12B6O3A11

[0286] [Corrected 15.08.2025 according to Rule 26] Step 1: Synthesis of compound E12LA12B6O3A11-1

[0287] [Corrected 15.08.2025 according to Rule 26] According to the method of Example 9, E12LA6B6O3A11-1 was synthesized.

[0288] [Corrected 15.08.2025 according to Rule 26] Step 2: Synthesis of compound E12LA6B6O3A11-1-2

[0289] [Corrected 15.08.2025 according to Rule 26] A 50 mL round-bottom flask was purged and filled with nitrogen. Compound E12LA6B6O3A11-1 (5.18 g, 5 mmol, 1.0 eq) and anhydrous dichloromethane (20 mL) were added. The mixture was stirred at 0°C for 10 minutes. Trifluoromethanesulfonic anhydride (2.12 g, 7.5 mmol, 1.5 eq) was added dropwise. The mixture was stirred at 0°C for 10 minutes. 2,6-dimethylpyridine (0.80 g, 7.5 mmol, 1.5 eq) was added and the reaction was stirred at room temperature overnight. The reaction solution was washed once with water and once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 50:1 to 20:1) to obtain compound E12LA6B6O3A11-2 (4.38 g, 75%). ESI-MS C 64 H 121 F3NO 11 S + [M+H] + Calculated value 1168.9, measured value 1169.0.

[0290] [Corrected 15.08.2025 according to Rule 26] Step 3: Synthesis of compound E12LA6B6O3A11-3

[0291] [Corrected 15.08.2025 according to Rule 26] A 100 mL pressure bottle was added with E12LA6B6O3A11-2 (4.38 g, 3.75 mmol, 1.0 eq), 20 mL of tetrahydrofuran, and 5 mL of ammonia water (25% by mass in water). The mixture was stirred at 100°C overnight. The reaction solution was washed once with water and once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and purified by flash column chromatography (dichloromethane:methanol = 20:1 to 5:1) to obtain compound E12LA6B6O3A11-3 (1.71 g, 44%). ESI-MS C 63 H 123 N2O8 + [M+H] + Calculated value 1035.9, measured value 1035.9.

[0292] [Corrected 15.08.2025 according to Rule 26] Step 4: Synthesis of compound E12LA6B6O3A11

[0293] [Corrected 15.08.2025 according to Rule 26] A dichloromethane solution of E12LA6B6O3A11-3 (1.71 g, 1.65 mmol, 1.0 eq), N,N'-dicyclohexylcarbodiimide (0.68 g, 3.3 mmol, 2.0 eq), DMAP (0.20 g, 0.16 mmol, 0.1 eq), and 4-dimethylaminobutyric acid (0.32 g, 2.48 mmol, 1.5 eq) was stirred at room temperature for 4 hours. TLC showed that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated and purified using a flash column chromatography system (dichloromethane:methanol = 50:1 to 10:1) to obtain compound E12LA6B6O3A11 (1.48 g, 78%). 1 H NMR (400MHz, CDCl3): δ0.85-0.91(m,12H),1.24-1.41(m,68H),1.45-1.82(m,22H),2.27(s,6H),2.32-2.42(m,12H) ,2.61-2.63(m,2H),3.36-3.42,(m,2H),3.77-3.79(m,2H),4.01-4.04(m,2H),4.23-4.26(m,2H),5.04-5.10(m,2H). ESI-MS C 69 H 134 N3O9 + [M+H] + Calculated value 1049.0, measured value 1049.1.

[0294] [Corrected 15.08.2025 according to Rule 26] Example 32 Synthesis of amino lipid E12N1LA6B6O3

[0295] [Corrected 15.08.2025 according to Rule 26] Step 1: Synthesis of compound E12N1LA6B6O3-2

[0296] [Corrected 15.08.2025 according to Rule 26] 1,2-epoxydodecane (3.69 g, 20 mmol, 1.0 eq), 20 mL of ammonia (25% by mass in water), 10 mL of ethanol, and 10 mL of water were stirred at 60°C for 16 hours. The reaction solution was dried by spin drying, 3 mL of ethanol and 30 mL of n-heptane were added, and the mixture was stirred at 60°C for 4 hours. The mixture was filtered and the filter cake was dried at 50°C for 10 hours to obtain compound E12N1LA6B6O3-2 (3.46 g, 86%). ESI-MS C 12 H 28 NO + [M+H] + Calculated value 202.2, measured value 202.2.

[0297] [Corrected 15.08.2025 according to Rule 26] Step 2: Synthesis of compound E12N1LA6B6O3-3

[0298] [Corrected 15.08.2025 according to Rule 26] A solution of compound E12N1LA6B6O3-2 (3.46 g, 17.2 mmol, 1.5 eq), hexanoic acid (1.34 g, 11.5 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.30 g, 17.2 mmol, 1.5 eq), N,N-diisopropylethylamine (2.22 g, 17.2 mmol, 1.5 eq), 4-dimethylaminopyridine (2.10 g, 17.2 mmol, 1.5 eq) in dichloromethane (40 mL) was stirred at room temperature overnight. The reaction solution was extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (n-heptane:ethyl acetate = 10:1 to 3:1) to obtain compound E12N1LA6B6O3-3 (1.10 g, 32%). ESI-MS C 18 H 38 NO2 + [M+H] + Calculated value 300.3, measured value 300.2.

[0299] [Corrected 15.08.2025 according to Rule 26] Step 3: Synthesis of compound E12N1LA6B6O3-4

[0300] [Corrected 15.08.2025 according to Rule 26] A solution of compound E12N1LA6B6O3-3 (1.10 g, 3.68 mmol, 1.0 eq), hexanoic acid (0.51 g, 4.42 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.41 g, 7.36 mmol, 2.0 eq), N,N-diisopropylethylamine (1.90 g, 14.2 mmol, 4.0 eq), 4-dimethylaminopyridine (0.04 g, 0.37 mmol, 0.1 eq) in dichloromethane (20 mL) was stirred at room temperature overnight. TLC showed that the reaction was complete. The reaction solution was extracted three times with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (n-heptane:ethyl acetate = 100:1 to 20:1) to obtain compound E12N1LA6B6O3-4 (1.42 g, 81%). ESI-MS C 24 H 47 BrNO3 + [M+H] +Calculated value 476.3, measured value 476.3.

[0301] [Corrected 15.08.2025 according to Rule 26] Step 4: Synthesis of compound E12N1LA6B6O3

[0302] [Corrected 15.08.2025 according to Rule 26] A solution of compound E12N1LA6B6O3-4 (1.42 g, 2.99 mmol, 3.0 eq), 3-aminopropanol (0.075 g, 1.00 mmol, 1.0 eq), potassium carbonate (0.28 g, 2.00 mmol, 2.0 eq), and sodium iodide (0.15 g, 1.00 mmol, 1.0 eq) in acetonitrile (10 mL) was stirred at 75°C overnight. After concentration, the reaction mixture was purified using a flash column chromatography system (dichloromethane:methanol = 100:1 to 20:1) to obtain compound E12N1LA6B6O3 (0.68 g, 78%). 1 H NMR (400MHz, CDCl3): δ0.86-0.93(m,12H),1.25-1.37(m,48H),1.41-1.69(m,16 H),2.14-2.18(m,8H),2.30-2.43(m,6H),3.34-3.48(m,4H),4.90-4.96(m,2H). ESI-MS C 51 H 100 N3O7 + [M+H] + Calculated value 866.8, measured value 866.7.

[0303] [Corrected 15.08.2025 according to Rule 26] Example 33 Synthesis of amino lipid E12N1LA8B6O3

[0304] [Corrected 15.08.2025 according to Rule 26] The amino lipid E12N1LA8B6O3 can be prepared by the same method by replacing the hexanoic acid in Example 32 with octanoic acid, and its structural formula is as follows:

[0305] [Corrected 15.08.2025 in accordance with Article 26] 1 H NMR (400MHz, CDCl3): δ0.86-0.93(m,12H),1.25-1.37(m,48H),1.42-1.70(m,18 H),2.14-2.18(m,8H),2.31-2.43(m,6H),3.35-3.48(m,4H),4.90-4.96(m,2H). ESI-MS C 55 H 108 N3O7+ [M+H] + Calculated value 922.8, measured value 923.0.

[0306] [Corrected 15.08.2025 according to Rule 26] Example 34 Synthesis of amino lipid E12N2LA6B6O3

[0307] [Corrected 15.08.2025 according to Rule 26] Step 1: Synthesis of compound E12N2LA6B6O3-1

[0308] [Corrected 15.08.2025 according to Rule 26] According to Step 1 and Step 2 of Example 32, E12N2LA6B6O3-1 was synthesized.

[0309] [Corrected 15.08.2025 according to Rule 26] Step 2: Synthesis of compound E12N2LA6B6O3-2

[0310] [Corrected 15.08.2025 according to Rule 26] A 50 mL round-bottom flask was purged and filled with nitrogen. Compound E12N2LA6B6O3-1-1 (1.50 g, 5 mmol, 1.0 eq) and anhydrous dichloromethane (20 mL) were added. The mixture was stirred at 0°C for 10 minutes. Trifluoromethanesulfonic anhydride (2.12 g, 7.5 mmol, 1.5 eq) was added dropwise. The mixture was stirred at 0°C for 10 minutes. 2,6-dimethylpyridine (0.80 g, 7.5 mmol, 1.5 eq) was added and the reaction was stirred at room temperature overnight. The reaction solution was washed once with water and once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and purified using flash column chromatography (dichloromethane:methanol = 50:1 to 20:1) to obtain compound E12N2LA6B6O3-2 (1.51 g, 70%). ESI-MS C 19 H 37 F3NO4S + [M+H] + Calculated value 432.2, measured value 432.2.

[0311] [Corrected 15.08.2025 according to Rule 26] Step 3: Synthesis of compound E12N2LA6B6O3-3

[0312] [Corrected 15.08.2025 according to Rule 26] A 100 mL pressure bottle was added with E12N2LA6B6O3-2 (1.51 g, 3.50 mmol, 1.0 eq), 20 mL of tetrahydrofuran, and 5 mL of ammonia water (25% by mass in water). The mixture was stirred at 100°C overnight. The reaction solution was washed once with water and once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain compound E12N2LA6B6O3-3 (0.47 g, 45%). ESI-MS C 18 H 39 N2O + [M+H] + Calculated value 299.3, measured value 299.3.

[0313] [Corrected 15.08.2025 according to Rule 26] Step 4: Synthesis of compound E12N2LA6B6O3-4

[0314] [Corrected 15.08.2025 according to Rule 26] A solution of compound E12N2LA6B6O3-3 (0.74 g, 1.58 mmol, 3.0 eq), N,N'-dicyclohexylcarbodiimide (0.65 g, 3.15 mmol, 2.0 eq), DMAP (0.20 g, 0.16 mmol, 0.1 eq), N,N-diisopropylethylamine (0.82 g, 6.32 mmol, 4.0 eq), and 6-bromohexanoic acid (0.46 g, 2.37 mmol, 1.5 eq) in dichloromethane was stirred at room temperature for 4 hours. TLC showed that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated and purified by flash column chromatography (n-heptane:ethyl acetate = 50:1 to 1:1) to give compound E12N2LA6B6O3-4 (1.48 g, 88%). ESI-MS C 24 H 48 BrN2O2 + [M+H] + Calculated value 475.3, measured value 475.3.

[0315] [Corrected 15.08.2025 according to Rule 26] Step 5: Synthesis of compound E12N2LA6B6O3

[0316] [Corrected 15.08.2025 according to Rule 26] A solution of compound E12N2LA6B6O3-4 (1.48 g, 1.39 mmol, 3.0 eq), 3-aminopropanol (0.035 g, 0.46 mmol, 1.0 eq), potassium carbonate (0.13 g, 0.92 mmol, 2.0 eq), and sodium iodide (0.069 g, 0.46 mmol, 1.0 eq) in acetonitrile (10 mL) was stirred at 75°C overnight. After concentration, the reaction mixture was purified using a flash column chromatography system (dichloromethane:methanol = 100:1 to 20:1) to obtain compound E12N2LA6B6O3 (0.97 g, 81%). 1 H NMR (400MHz, CDCl3): δ0.86-0.92(m,12H),1.25-1.37(m,42H),1.41-1.69(m,18H),2.06- 2.18(m,12H),2.30-2.42(m,4H),3.32-3.48(m,4H),3.70-3.74(m,2H),4.90-4.96(m,2H). ESI-MS C 51 H 102 N5O5 + [M+H] + Calculated value 864.8, measured value 864.7.

[0317] [Corrected 15.08.2025 according to Rule 26] Example 35 Synthesis of amino lipid E12S1LA6B6O3

[0318] [Corrected 15.08.2025 according to Rule 26] Step 1: Synthesis of compound E12S1LA6B6O3-2

[0319] [Corrected 15.08.2025 according to Rule 26] Ferric chloride (16.2 mg, 0.1 mmol, 0.025 eq), pyridine (4 mg, 0.05 mmol, 0.0125 eq), hexanoic acid (464 mg, 4.0 mmol, 1.0 eq), and 1,2-epoxydodecane (884 mg, 4.8 mmol, 1.2 eq) were added sequentially to a 25 mL reaction tube and stirred at room temperature overnight. After completion of the reaction, the reaction solution was concentrated and purified using a flash column chromatography system (n-heptane:ethyl acetate = 50:1 to 10:1) to obtain compound E12S1LA6B6O3-2 (1.2 g, 90.0%). ESI-MS C 18 H 37 O3 + [M+H] + Calculated value 301.3, measured value 301.3.

[0320] [Corrected 15.08.2025 according to Rule 26] Step 2: Synthesis of compound E12S1LA6B6O3-3

[0321] [Corrected 15.08.2025 according to Rule 26] E12S1LA6B6O3-2 (1.2 g, 3.6 mmol, 1.0 eq), triethylamine (0.36 g, 3.6 mmol, 1.0 eq), methanesulfonyl chloride (0.49 g, 4.32 mmol, 1.2 eq), and 10 mL of dichloromethane were added to a 25 mL reaction tube. The mixture was stirred at room temperature for 2 hours. The reaction solution was washed once with water and once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. 10 mL of N,N-dimethylformamide and sodium hydrosulfide (0.24 g, 4.32 mmol, 1.2 eq) were stirred at 45°C overnight. The reaction solution was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (n-heptane:ethyl acetate = 100:1 to 10:1) to obtain compound E12S1LA6B6O3-3 (0.69 g, 61%). ESI-MS C 18 H 37 O2S + [M+H] + Calculated value 317.3, measured value 317.2.

[0322] [Corrected 15.08.2025 according to Rule 26] Step 3: Synthesis of compound E12S1LA6B6O3-4

[0323] [Corrected 15.08.2025 according to Rule 26] In a 25 mL reaction tube, E12S1LA6B6O3-3 (0.69 g, 2.2 mmol, 1.0 eq), dibromohydantoin (0.13 g, 0.44 mmol, 0.2 eq), 3-bromopropane-1-thiol (0.41 g, 2.64 mmol, 1.2 eq), and 10 mL of dichloromethane were added. The mixture was stirred at room temperature for 1 hour. The reaction solution was extracted three times with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by flash column chromatography (n-heptane:ethyl acetate = 100:1 to 10:1) to obtain compound E12S1LA6B6O3-4 (0.70 g, 68%). ESI-MS C 21 H 42 BrO2S2 + [M+H] + Calculated value 469.2, measured value 469.2.

[0324] [Corrected 15.08.2025 according to Rule 26] Step 4: Synthesis of compound E12S1LA6B6O3

[0325] [Corrected 15.08.2025 according to Rule 26] A solution of compound E12S1LA6B6O3-4 (0.70 g, 1.50 mmol, 3.0 eq), 3-aminopropanol (0.038 g, 0.50 mmol, 1.0 eq), potassium carbonate (0.14 g, 1.00 mmol, 2.0 eq), and sodium iodide (0.075 g, 0.50 mmol, 1.0 eq) in acetonitrile (10 mL) was stirred at 75°C overnight. After concentration, the reaction mixture was purified using a flash column chromatography system (dichloromethane:methanol = 100:1 to 20:1) to obtain compound E12S1LA6B6O3 (1.09 g, 80%). 1 H NMR (400MHz, CDCl3): δ0.86-0.91(m,12H),1.25-1.35(m,40H),1.41-1.54(m,12H),1.59-1.67(m,10 H),2.09-2.18(m,10H),2.34-2.40(m,4H),2.50-2.54(m,2H),3.64-3.72(m,2H),4.00-4.06(m,4H). ESI-MS C 49 H 98 NO5S4 + [M+H] + Calculated value 908.6, measured value 908.7.

[0326] [Corrected 15.08.2025 according to Regulation 26] Preparation of lipid nanoparticles

[0327] [Corrected 15.08.2025 according to Rule 26] Example 36 Preparation of lipid nanoparticles

[0328] [Corrected 15.08.2025 according to Rule 26] The amino lipid compound of the present invention (or DLin-MC3 or SM-102 (both purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.)), phospholipid (DSPC), cholesterol, and pegylated lipid (DMG-PEG2000) were mixed and dissolved in anhydrous ethanol at a molar ratio of 47.5:10:41:1.5. The resulting ethanol solution and luciferase mRNA (TriLink) dissolved in citrate buffer (50 mM, pH = 4.0) were mixed in a microfluidic chip at a volume ratio of 1:3 using a microfluidic preparation system to prepare a crude lipid nanoparticle solution. The solution was then dialyzed in 1X PBS at 4°C for 6 h using a dialysis cassette (Fisher, MWCO 20,000). The solution was then filtered through a 0.22 μm microporous membrane before use. The mass ratio of the amino lipid compound to luciferase mRNA (Luc mRNA) was approximately 40:1.

[0329] [Corrected 15.08.2025 according to Rule 26] The obtained lipid nanoparticles were characterized and the results are shown in Table 1.

[0330] [Corrected 15.08.2025 according to Rule 26] The particle size and PDI (polydispersity index) of the prepared lipid nanoparticles were measured using a Nano-ZSZEN3600 (Malvern). 20 μL of the lipid nanoparticle (LNP) solution was taken for particle size measurement, and the measurement was repeated three times, with each cycle lasting 30 seconds.

[0331] [Corrected 15.08.2025 according to rule 26] The encapsulation efficiency was determined according to the standard procedure of the Quant-iT RiboGreen RNA kit.

[0332] [Corrected 15.08.2025 according to Regulation 26] Table 1 Characterization data of LNPs prepared using representative amino lipid compounds

[0333] [Corrected 15.08.2025 according to Rule 26] It can be seen from the above results that the encapsulation efficiency of the lipid nanoparticles provided by the present invention is higher than that of DLin-MC3, and the encapsulation efficiency of most of the lipid nanoparticles is higher than that of SM-102.

[0334] [Corrected 15.08.2025 according to Rule 26] Example 37 Transfection of lipid nanoparticles prepared from amino lipid compounds on primary BMDC cells

[0335] [Corrected 15.08.2025 according to rule 26] Animal preparation: 6-week-old female C57BL / 6 mice weighing approximately 20 g were selected and kept in an SPF-grade cage. Animal experiments were conducted in strict accordance with the guidelines of national health institutions and animal ethics requirements.

[0336] [Corrected 15.08.2025 according to rule 26] Cell acquisition: C57BL / 6 mice were killed by cervical dislocation and immersed in 75% alcohol for 5 minutes for disinfection. The thigh and tibia of the mouse were dissected and the attached muscles were removed to expose the bone. The bone marrow in the tibia was blown out with a 1 mL syringe filled with PBS. The obtained bone marrow was blown out and filtered through a 50 μm filter to remove impurities. Red blood cell lysis buffer (3-4 mL) was added to the obtained filtrate and the mixture was allowed to stand for 5 minutes. The supernatant was removed by centrifugation at 800 g for 5 minutes. The obtained cells were placed in 1640 culture medium (containing 10% fetal bovine serum, 20 ng / mL GMCSF, 10 ng / mL The cells were resuspended in 1% IL4 and seeded into 6-well plates at a seeding density of 100,000 cells / mL of culture medium. The plates were placed in a 37°C cell culture incubator containing 5% CO2. Half of the medium was changed every 2 days. On the seventh day, the suspended cells and loosely adhered cells were collected and seeded into 96-well all-white ELISA plates at a seeding density of 20,000 cells per well in a volume of 100 μL of culture medium.

[0337] [Corrected 15 August 2025 in accordance with Rule 26] Cell transfection: Add luciferase mRNA-encapsulated lipid nanoparticles to a 96-well all-white ELISA plate containing primary cells, controlling the volume of mRNA-lipid nanoparticles added to 10 μL per well. Incubate at 37°C in a 5% CO2 incubator for 12 hours.

[0338] [Corrected 15.08.2025 according to Rule 26] Transfection efficiency test: Add 20 μL of substrate ONE-Glo to each well of a 96-well all-white ELISA plate. TM Luciferase was detected after 1 min using a multifunctional microplate reader (Biorek SynergyH1). The expression intensity of Luc mRNA transfected into BMDCs by lipid nanoparticles (LNPs) composed of representative amino lipid compounds is shown in Table 2, with DLin-MC3 and SM0102 used as controls.

[0339] [Corrected 15.08.2025 according to Rule 26] Table 2 Expression intensity of representative amino lipid compounds transfected on BMDC

[0340] [Corrected 15.08.2025 according to Rule 26] It can be seen that the expression intensity of the amino lipids provided by the present invention is significantly better than that of DLin-MC3, and the expression intensity of some of the amino lipids is better than that of SM-102.

[0341] [Corrected 15.08.2025 according to Rule 26] Example 38 Evaluation of luciferase mRNA delivery performance of lipid nanoparticles in vivo

[0342] [Corrected 15.08.2025 according to Rule 26] Preparation of lipid nanoparticles: same as Example 36.

[0343] [Corrected 15.08.2025 according to Regulation 26] Animal experiments

[0344] [Corrected 15.08.2025 according to Regulation 26] Animal Preparation: Six-week-old female C57BL / 6 mice weighing approximately 20 g were housed in an SPF-grade enclosure. Animal experiments were conducted in strict accordance with the guidelines of national health agencies and animal ethics requirements.

[0345] [Corrected 15 August 2025 according to Rule 26] In vivo delivery: Five C57BL / 6 mice were randomly selected from each group and injected intramuscularly with a lipid nanoparticle solution at a dose of 0.5 mg / kg mRNA. 12 hours later, each mouse was injected intraperitoneally with 200 μL of 10 mg / mL D-luciferin potassium salt. Five minutes later, the mice were placed under an in vivo imaging system (IVIS-200, Xenogen), and the total fluorescence intensity of each mouse was observed and photographed. The expression intensity of Luc mRNA delivered by intramuscular injection using LNPs composed of representative amino lipid compounds is shown in Table 3. DLin-MC3 and SM-102 were used as controls.

[0346] [Corrected 15.08.2025 according to Rule 26] Table 3 Expression intensity of Luc mRNA delivered by intramuscular injection of LNPs composed of representative amino lipids

[0347] [Corrected 15.08.2025 according to Rule 26] Example 39 Evaluation of the in vivo immune and tumor therapeutic effects of lipid nanoparticles prepared from amino lipid compounds

[0348] [Corrected 15 August 2025 according to Rule 26] Preparation method: The amino lipid compound of the present invention was mixed with DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 47.5:10:41:1.5 and dissolved in anhydrous ethanol. Using a microfluidic preparation system, the resulting ethanol solution and OVA mRNA dissolved in citric acid buffer (50 mM, pH 4.0) were mixed in a microfluidic chip at a volume ratio of 1:3 to prepare lipid nanoparticles. The particles were then dialyzed in 1X PBS at 4°C for 6 hours using a dialysis cassette (Fisher, MWCO 20,000) and filtered through a 0.22 μm microporous membrane before use. The mass ratio of the amino lipid compound to ovalbumin mRNA (OVA mRNA) was approximately 40:1.

[0349] [Corrected 15.08.2025 according to rule 26] Animal preparation: Female C57BL / 6 mice aged 5-6 weeks, weighing approximately 18-20 g, were selected and maintained in an SPF-grade enclosure. Animal experiments were conducted in strict accordance with the guidelines of the National Health Agency and animal ethics requirements.

[0350] [Corrected 15.08.2025 according to rule 26] In vivo delivery: B16-OVA melanoma cells (1.5×10 5 ) was injected subcutaneously into the outer thigh of the mouse. When the tumor grew to 50 mm 3 Vaccination was initiated at 6 or 7 days after tumor inoculation. Animals were immunized twice intramuscularly with LNP formulations containing 1 μg of OVA-mRNA, with the second injection 7 days apart. Tumor growth was measured three times a week using a digital caliper and calculated using the formula 0.5 × length × width × width. When the tumor volume reached 1500 mm 3 The mice were euthanized at 4 hr. Tumor growth rates in the E12LA6B6O3 and E10LA6B6O3 groups were significantly slower than in the MC3 group (as shown in Figure 2). Complete remission was achieved in 100% of the mice in the E12LA6B6O3 group and 80% of the mice in the E10LA6B6O3 group, respectively, significantly better than in the MC3 group (as shown in Figure 3).

[0351] [Corrected 15.08.2025 according to Rule 26] Example 40 Evaluation of the in vivo delivery performance of luciferase mRNA in LNPs composed of different phospholipid lipid ratios

[0352] [Corrected 15 / 08 / 2025 according to Rule 26] Preparation of lipid nanoparticles: Maintaining an ionizable lipid (E12LA6B6O3) content of 42.5% and a PEGylated lipid (DMG-PEG2000) content of 1.5%, the phospholipid ratios were adjusted to 15, 12.5, 10, 7.5, 5.0, 2.5, and 0. The effects of these adjusted phospholipid ratios on lipid nanoparticle quality control and in vivo luciferase mRNA delivery were evaluated by increasing or decreasing the phospholipid ratios relative to the cholesterol ratio. Groups are numbered B1-B7, and the specific ratios are shown in Table 4. ALC-0315 and SM-102 were used as positive controls.

[0353] [Corrected 15.08.2025 according to Rule 26] Table 4: Proportions of components under different phospholipid lipids

[0354] [Corrected 15.08.2025 according to Regulation 26] E12LA6B6O3, phospholipid (DSPC), cholesterol, and the PEGylated lipid DMG-PEG2000 were mixed and dissolved in anhydrous ethanol according to the molar ratios listed in Table 4. The resulting ethanol solution was mixed with luciferase mRNA (TriLink) dissolved in citric acid buffer (50 mM, pH 4.0) at a volume ratio of 1:3 in a microfluidic chip using a microfluidic preparation system to prepare a crude lipid nanoparticle solution. The solution was then dialyzed against 1X PBS at 4°C for 6 h using a dialysis cassette (Fisher, MWCO 20,000). The solution was then filtered through a 0.22 μm microporous filter membrane before use. The molar ratio of the amino lipid compound to the luciferase mRNA (Luc mRNA) was approximately 6.5:1. The quality control of lipid nanoparticles was detected on a Malvern particle size analyzer and an enzyme reader, and SM-102 and ALC-0315 (both purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.) were added as a control group.

[0355] [Corrected 15.08.2025 according to Regulation 26] Animal Preparation: Six-week-old female Balb / C mice weighing approximately 20 g were housed in an SPF-protected enclosure. Animal experiments were conducted in strict accordance with national health agency guidelines and animal ethics requirements.

[0356] [Corrected 15 August 2025 according to Rule 26] In vivo delivery: Three Balb / C mice were randomly selected from each group and injected with the lipid nanoparticle solution at a dose of 0.1 mg / kg mRNA via tail vein injection. Six hours later, 150 μL of 15 mg / mL D-luciferin potassium salt was injected intraperitoneally into each mouse. Ten minutes later, the mice were placed under an in vivo imaging system (IVIS Spectrum). The total fluorescence intensity of each mouse was observed and photographed. The expression intensity of Luc mRNA delivered by tail vein injection using LNPs with different DSPC content is shown in Table 5.

[0357] [Corrected 15.08.2025 according to Rule 26] Table 5: Quality control data and expression intensity of LNPs composed of different phospholipid content ratios

[0358] [Corrected on 15.08.2025 according to Rule 26] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. [Corrected 15.08.2025 in accordance with Rule 26] An amino lipid having the structure represented by general formula (I), or an isomer, pharmaceutically acceptable salt, prodrug or solvate thereof: in, G is selected from H, OR, CN, -C(=O)OR', -OC(=O)R', -C(=O)NR'R", -NR'C(=O)R", -NR'R", or a cyclic alkyl structure containing at least one heteroatom; the substitutable carbon atoms or heteroatoms in the cyclic alkyl structure are unsubstituted or substituted with one or more hydroxyl groups, C1-C4 alkyl groups, C2-C4 alkenyl groups, C3-C8 cycloalkyl groups, or C3-C8 cycloalkenyl groups; M1, M2, M3, and M4 are the same or different from each other and are each independently selected from C1-C 24 Alkylene, C3-C 24 Cycloalkylene, C2-C 24 Alkenylene or C3-C 24 cycloalkenylene; R 1 、R 2 are the same as or different from each other and are independently selected from H, C1-C 24 Alkyl, C3-C 24 Cycloalkyl, C2-C 24 Alkenyl or C3-C 24 cycloalkenyl; L1, L2, L3, L4 are the same as or different from each other and are independently selected from -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, -C(=O)NR-, -NRC(=O)-, -S(=O)-, -OS(=O)2-, -S(=O)2O-, -O-, -S- or -SS-; R, R', R" are the same or different from each other and are independently selected from H, Cl-C 10 Alkyl, C3-C 10 Cycloalkyl, C3-C 10 Alkenyl, C3-C 10 Cycloalkenyl, C1-C 10 Alkyl, C3-C 10 Cycloalkyl, C3-C 10 an alkenyl group, or a cyclic alkyl group containing at least one heteroatom, wherein the cyclic alkyl group is unsubstituted or substituted by one or more C1-C4 alkyl groups, C2-C4 alkenyl groups, C3-C8 cycloalkyl groups, or C3-C8 cycloalkenyl groups; M5 are each independently selected from a single bond, C1-C 16 Alkylene, C2-C 16 C3-C8 cycloalkylene or C3-C8 cycloalkenylene.

2. [Corrected 15.08.2025 according to Rule 26] The amino lipid according to claim 1, characterized in that G is selected from H, OR, CN, -C(=O)OR', -OC(=O)R', -C(=O)NR'R", -NR'C(=O)R", -NR'R" or a cyclic alkyl structure containing at least one heteroatom; the cyclic alkyl structure may be substituted with a carbon atom or a heteroatom, or may be substituted with one or more C1-C4 alkyl, C2-C4 alkenyl, C3-C8 cycloalkyl or C3-C8 cycloalkenyl; R, R', R" are the same or different and are each independently selected from H, C1-C 10 Alkyl, C3-C 10 Cycloalkyl, C3-C 10 Alkenyl or C3-C 10 Cycloalkenyl, or a cyclic alkyl group containing at least one heteroatom, wherein the cyclic alkyl group is unsubstituted or substituted by one or more C1-C4 alkyl groups, C2-C4 alkenyl groups, C3-C8 cycloalkyl groups, or C3-C8 cycloalkenyl groups.

3. [Corrected 15.08.2025 according to Rule 26] The amino lipid according to claim 1 or 2, characterized in that In the general formula (I), G is selected from H, OR, CN, -C(=O)OR', -OC(=O)R', -C(=O)NR'R", -NR'C(=O)R", -NR'R" or a cyclic alkyl structure containing at least one heteroatom; wherein the heteroatom is O or N; the cyclic alkyl is unsubstituted or substituted with one or more C1-C4 alkyl, C3-C8 cycloalkyl or hydroxyl groups; R, R', and R" are the same or different and are independently selected from H, C1-C8 alkyl, C3-C8 cycloalkyl, C3-C8 alkenyl or C3-C8 cycloalkenyl, and the C1-C8 terminal is connected to a tertiary amine group. 10 Alkyl, C3-C 10 Cycloalkyl or cyclic alkyl containing at least one heteroatom, wherein the cyclic alkyl is unsubstituted or substituted by one or more C1-C4 alkyl or C3-C8 cycloalkyl groups.

4. [Corrected 15.08.2025 according to Rule 26] The amino lipid according to claim 3, characterized in that In the general formula (I), G is selected from H, OH or NR'R", wherein R' and R" are the same or different from each other and are each independently selected from H or C1-C4 alkyl; Alternatively, G is selected from substituted or unsubstituted oxa five-membered cycloalkyl, aza five-membered cycloalkyl, aza six-membered cycloalkyl, diaza six-membered cycloalkyl or azaoxa six-membered cycloalkyl.

5. [Corrected 15.08.2025 according to Rule 26] The amino lipid according to claim 4, characterized in that In the general formula (I), when G is selected from a substituted diaza six-membered cycloalkyl group, the substituent position is the nitrogen atom that is not connected to M5.

6. [Corrected 15.08.2025 according to Rule 26] The amino lipid according to claim 3, characterized in that In the general formula (I), M5 is selected from a single bond, C2-C 16 Alkylene, C2-C 16 C4-C8 cycloalkylene or C3-C8 cycloalkenylene.

7. [Corrected 15.08.2025 according to Rule 26] The amino lipid according to claim 6, characterized in that In the general formula (I), M5 is selected from a single bond, C2-C 16 Alkylene or C4-C6 cycloalkylene.

8. [Corrected 15.08.2025 according to Rule 26] The amino lipid according to claim 7, characterized in that In the general formula (I), M5 and G are connected to form One selected from A1 to A38:

9. [Corrected 15.08.2025 according to Rule 26] The amino lipid according to claim 7, characterized in that In the general formula (I), M5 and G are connected to form Select one from A39 to A52:

10. [Corrected 15.08.2025 according to Rule 26] The amino lipid according to claim 8, characterized in that described Selected from one of A1-A18, A22-A24, A28-A38.

11. [Corrected 15.08.2025 according to Rule 26] The amino lipid according to claim 10, characterized in that described Selected from one of A15, A16, A17, A23, A29, A30, A33, A37, A38, A42.

12. [Corrected 15.08.2025 according to Rule 26] The amino lipid according to any one of claims 1 to 11, characterized in that In the general formula (I), L1, L2, L3, and L4 are the same as or different from each other and are independently selected from -C(=O)O-, -OC(=O)-, -C(=O)NR-, -NRC(=O)-, or -SS-; Preferably, L1 and L4 are the same and are -C(=O)O- or -OC(=O)-.

13. [Corrected 15.08.2025 according to Rule 26] The amino lipid according to claim 12, characterized in that When L1, L2, L3, L4 are independently selected from -C(=O)NR- or -NRC(=O)-, wherein R is independently selected from H or C1-C 10 alkyl.

14. [Corrected 15.08.2025 according to Rule 26] The amino lipid according to any one of claims 1 to 13, characterized in that In the general formula (I), M1, M2, M3, and M4 are the same or different from each other, and M1 and M4 are each independently selected from a C4-C 22 Alkylene, branched C4-C 22 Cycloalkylene, C4-C 22 Alkenylene or branched C4-C 22 Cycloalkenylene, M2 and M3 are each independently selected from C4-C 22 Alkylene, C4-C 22 Cycloalkylene, C4-C 22 Alkenylene or C4-C 22 Cycloalkenylene.

15. [Corrected 15.08.2025 according to Rule 26] The amino lipid according to claim 14, characterized in that In the general formula (I), M2 and M3 are the same and are C4-C 22 alkylene; And / or, M1 is the same as M4 and is a C4-C 22 Alkylene.

16. [Corrected 15.08.2025 according to Rule 26] The amino lipid according to any one of claims 2 to 15, characterized in that In the general formula (I), R 1 、R 2 are the same as or different from each other and are each independently selected from C4-C 22 Alkyl or C4-C 22 Alkenyl.

17. [Corrected 15.08.2025 according to Rule 26] The amino lipid according to claim 16, characterized in that In the general formula (I), R 1 -L1-M1-L2-M2- fragment is R 1 -C(=O)O-M1-OC(=O)-M2-,R 2 -L4-M4-L3-M3- fragment is R 2 -C(=O)O-M4-OC(=O)-M3-.

18. [Corrected 15.08.2025 according to Rule 26] The amino lipid according to claim 17, characterized in that The structure of general formula (I) is selected from one of the following structures:

19. [Corrected 15.08.2025 according to Rule 26] A method for preparing an amino lipid according to any one of claims 1 to 18, characterized in that The following steps are involved: S1: Epoxide and carboxylic acid undergo ring-opening reaction to prepare intermediate 1R 1 -L1-M1-OH; S2: Intermediate 1 and the carboxylic acid compound raw material undergo condensation reaction in the presence of a condensing agent to prepare intermediate 2R 1 -L1-M1-L2-M2-leaving group; S3: Intermediate 2 undergoes one or more substitution reactions with the amino compound raw material to prepare the target product; Or S1': The diol is protected by TBS and then oxidized, and the oxidation product undergoes an addition reaction with a Grignard reagent to prepare the intermediate 1'HO-M1-OTBS; S2': intermediate 1' and the carboxylic acid compound raw material undergo condensation reaction in the presence of a condensing agent to prepare intermediate 2'TBSO-M1-L2-M2-leaving group; S3': Intermediate 2'TBSO-M1-L2-M2-leaving group removes TBS protection and reacts with carboxylic acid to generate intermediate 2R 1 -L1-M1-L2-M2-leaving group; S4': Intermediate 2 undergoes one or more substitution reactions with the amino compound raw material to prepare the target product.

20. [Corrected 15.08.2025 according to Rule 26] A lipid nanoparticle, characterized in that The invention comprises the amino lipid according to any one of claims 1 to 18.

21. [Corrected 15.08.2025 according to Rule 26] The lipid nanoparticle according to claim 20, characterized in that The lipid nanoparticles further comprise steroids, neutral lipids and / or polymer-bound lipids; The polymer-bound lipid has the chemical formula PYL, where P is the hydrophilic polymer moiety, Y is an optional linker, and L is the lipid moiety.

22. [Corrected 15.08.2025 according to Rule 26] The lipid nanoparticle according to claim 21, characterized in that The steroid is cholesterol; and / or, the neutral lipid is a phospholipid; And / or, the polymer-bound lipid is a PEGylated lipid.

23. [Corrected 15.08.2025 according to Rule 26] The lipid nanoparticle according to claim 21, characterized in that In the lipid nanoparticles, the molar ratio of amino lipid, steroid, neutral lipid and polymer-bound lipid is 30-70:30-65:0-30:0.2-5.

24. [Corrected 15.08.2025 according to Rule 26] A pharmaceutical composition characterized in that The invention comprises the lipid nanoparticles according to any one of claims 20 to 23 and a pharmaceutically acceptable carrier.

25. [Corrected 15.08.2025 according to Rule 26] A method for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, or autoimmune diseases using the amino lipid according to any one of claims 1 to 18, the lipid nanoparticles according to any one of claims 20 to 23, or the pharmaceutical composition according to claim 24.

26. [Corrected 15.08.2025 according to Rule 26] The method according to claim 25, characterized in that Such cancers include lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer and prostate cancer.

27. [Corrected 15.08.2025 in accordance with Rule 26] A method for gene therapy, gene vaccination, antisense therapy, nucleic acid transfer or treatment by interfering RNA using the amino lipid of any one of claims 1 to 18 or the lipid nanoparticle of any one of claims 20 to 23 or the pharmaceutical composition of claim 24.

28. [Corrected 15.08.2025 according to Rule 26] Use of the amino lipid according to any one of claims 1 to 18, the lipid nanoparticle according to any one of claims 20 to 23, or the pharmaceutical composition according to claim 24 in the preparation of a medicament for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, or autoimmune diseases.

29. [Corrected 15.08.2025 under Rule 26] The use according to claim 28, characterized in that Such cancers include lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer and prostate cancer.

30. [Corrected 15.08.2025 according to Rule 26] Use of the amino lipid according to any one of claims 1 to 18, the lipid nanoparticle according to any one of claims 20 to 23, or the pharmaceutical composition according to claim 24, in the preparation of a medicament for gene therapy, gene vaccination, antisense therapy, nucleic acid transfer, or treatment by interfering RNA.

31. [Corrected 15.08.2025 under Rule 26] A method of delivering a pharmaceutical agent to a subject, characterized in that Comprising: administering to a subject an agent formulated in the lipid nanoparticles of any one of claims 20-23.

32. [Corrected 15.08.2025 according to Rule 26] An amino lipid according to any one of claims 1 to 18 for use in the treatment or prevention of infectious diseases, cancer, genetic diseases, allergies, toxicity, or autoimmune diseases.

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