Amino lipid, method for preparing amino lipid, lipid nanoparticle, pharmaceutical composition, method for treating or preventing infectious diseases, method for gene therapy, uses of amino lipid, and method for administering a medicament to a subject.

Description

1 / 72 “AMINO LIPID, METHOD FOR PREPARING AMINO LIPID, LIPID NANOPARTICLE, PHARMACEUTICAL COMPOSITION, METHOD FOR TREATING OR PREVENTING INFECTIOUS DISEASES, METHOD FOR Gene therapy, uses of amino lipids, and methods for administering a drug to a subject. Field of Invention

[001] The present invention relates to the technical field of biochemistry, and in particular to an amino lipid, a lipid nanoparticle and the use thereof. Background of the Invention

[002] Lipid nanoparticles (LNPs) are a novel biomolecular delivery technology for nucleic acids, and LNPs generally consist of four components: (1) ionizable lipids, which can self-assemble with mRNA to form virus-sized particles and release the mRNA from the endosome into the cytoplasm; (2) lipids conjugated to polymers, which can increase the half-life of LNPs in the bloodstream; (3) steroids, which can increase the stability of the nanoparticles; and (4) neutral phospholipids, which contribute to the formation of lipid bilayer structures.

[003] LNPs protect both mRNA from degradation by RNA enzymes and mRNA molecules from recognition by TLRs, preventing over-activation of the innate immune system. Among the components of LNPs, the selection of ionizable lipids has the greatest impact on LNPs. Ionizable lipids play a role in promoting cellular uptake, as well as assisting drug molecules in escaping endosomes, simultaneously affecting the efficiency of nucleic acid drug encapsulation, the efficiency of in vivo nucleic acid drug delivery, as well as cytotoxicity and the like.

[004] Currently, there are vaccines against the neocoronavirus. Petition 870250088241, dated 09 / 29 / 2025, p. 73 / 159 2 / 72 The COVID-19 vaccines from Moderna and BioNTech & Pfizer, both using LNP technology for mRNA drug delivery, thus achieving prevention of the COVID-19 neocoronavirus, demonstrate the great potential of LNP application in the area of ​​mRNA drugs and vaccines. However, currently, LNP still presents low encapsulation efficiency, low endosome escape rate, low expression level, low safety, and other deficiencies, and the direction of LNP delivery system development focuses mainly on lipid and ionizable formulations.

[005] Consequently, the development of new ionizable lipid compounds is of great importance for the development of nucleic acid drugs. Brief Description of the Invention

[006] In the first aspect, the present invention provides an amino lipid having the structure of formula (I), or an isomer, a pharmaceutically acceptable salt, a prodrug or a solvent thereof: m5 / L2, _.n.„ / L3„ h RM! M2M3R2(D where 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 cycloalkyl comprising at least one heteroatom, wherein substituteable carbon atoms or heteroatoms in the cycloalkyl are unsubstituted or substituted by one or more hydroxyl groups, C1-C4 alkyl, C2-C4 alkenyl, C3-C8 cycloalkyl, or Cs-Cs cycloalkenyl; M, M2, Ms and M4. They are identical or different from each other, and each is independently selected from C1-C24 alkylene, C3-C24 cycloalkylene, C2-C24 alkenylene, or C3-C24 cycloalkenylene; Petition 870250088241, dated 09 / 29 / 2025, p. 74 / 159 3 / 72 R1 and R2 are identical or different from each other, and each is selected independently from H, C1-C24 alkyl, C3-C24 cycloalkyl, C2-C24 alkenyl, or C3-C24 cycloalkenyl; L1, L2, L3 and L4 are identical or different from SS-; R, R' and R'' are identical or different from each other, and each is selected independently from H, C1-C10 alkyl, C3-C10 cycloalkyl, C3-C10 alkenyl, C3-C10 cycloalkenyl, C1-C10 alkyl terminated with a tertiary amino group, C3-C10 cycloalkyl terminating with a tertiary amino group, C3-C10 alkenyl terminating with a tertiary amino group, or cycloalkyl comprising at least one heteroatom, the cycloalkyl being unsubstituted or substituted by one or more C1-C4 alkyl, C2C4 alkenyl, C3-C8 cycloalkyl or C3-C8 cycloalkenyl groups; The M5 groups are selected independently from a single linkage, C1-C16 alkylene, C2-C16 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene.

[007] In the second aspect, the present invention provides a method for preparing the aforementioned amino lipid. The synthesis mainly involves three reaction steps: the first step is a ring-opening reaction; the second step is a condensation reaction; and the third step is a substitution reaction.

[008] In some embodiments of the present invention, the method for preparing the amino lipid comprises the following steps: S1: subject an epoxide compound and a carboxylic acid to a ring-opening reaction to prepare RMi-Mi-OH as intermediate 1; S2: submit intermediate 1 and a carboxylic acid compound Petition 870250088241, dated 09 / 29 / 2025, page 75 / 159 4 / 72 as a starting material to a condensation reaction in the presence of a condensing agent to give R1-Li-Mi-L2-M2-leaving group as intermediate 2; and S3: subjecting intermediate 2 and an amino compound as a starting material to one or more substitution reactions to obtain the target product; or comprises the following steps: S1': perform an oxidation reaction of a diol subjected to TBS protection and subject the oxidation product and a Grignard reagent to an addition reaction, to provide HO-Mi-OTBS as intermediate 1'; S2': subject intermediate 1' and a carboxylic acid compound to a condensation reaction in the presence of a condensing agent, to give the TBSO-M1-L2-M2-leaving group as intermediate 2'; S3': remove the TBS protecting group from the intermediate 2' TBSO-MiL2-M2-leaving group, allowing the resulting intermediate to undergo a condensation reaction with a carboxylic acid in the presence of a condensing agent, to generate the leaving group R1-L1-M1-L2-M2 as intermediate 2; and S4': subject intermediate 2 and an amino compound as a starting material to one or more substitution reactions to obtain the target product.

[009] In the third aspect, the present invention provides a lipid nanoparticle comprising any of the amino lipids mentioned above.

[0010] The lipid nanoparticle according to the present invention further comprises a steroid, a neutral lipid and / or a lipid conjugated to a polymer;

[0011] The lipid conjugated to the polymer has a chemical formula Petition 870250088241, dated 09 / 29 / 2025, page 76 / 159 5 / 72 of PYL, where P is a hydrophilic polymeric fraction, Y is an optional ligand, and L is a lipid fraction.

[0012] In the fourth aspect, the present invention provides a pharmaceutical composition comprising the aforementioned lipid nanoparticle and a pharmaceutically acceptable carrier.

[0013] In the fifth aspect, the present invention further provides a method for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases using the aforementioned amino lipid or lipid nanoparticle or pharmaceutical composition.

[0014] The present invention further provides a method for gene therapy, gene vaccination, antisense therapy, nucleic acid delivery or treatment by means of RNA interference, using the aforementioned amino lipid, lipid nanoparticle or pharmaceutical composition.

[0015] The present invention further provides the use of the aforementioned amino lipid or lipid nanoparticle in the preparation of a medicament to treat or prevent infectious diseases, cancer, genetic diseases, allergies, toxicity and autoimmune diseases.

[0016] The present invention further provides the use of the aforementioned amino lipid or lipid nanoparticle in the preparation of a medicament for gene therapy, gene vaccination, antisense therapy, nucleic acid delivery or treatment by means of RNA interference.

[0017] In the sixth aspect, the present invention provides a method for administering a drug to a subject, comprising administering to the subject a drug formulated in the aforementioned lipid nanoparticle.

[0018] The amino lipid of the present invention is self-assembled with steroids, neutral lipids, and lipids conjugated to polymers to form LNPs, which can further increase the level of translational expression of the charged nucleic acid within cells, improving the effectiveness of formulations of Petition 870250088241, dated 09 / 29 / 2025, page 77 / 159 6 / 72 nucleic acid-LNP and provide a theoretical basis for personalized treatment with nucleic acid-LNP formulations. Brief Description of the Drawings

[0019] Figure 1 shows the 1H-NMR spectrum of E12LA6B6O3 in Example 2.

[0020] Figure 2 shows the tumor size monitoring curve of mice bearing the tumor after intramuscular injection of the OVA mRNA vaccine in Example 39.

[0021] Figure 3 shows the survival curve of tumor-bearing mice after intramuscular injection of the OVA mRNA vaccine in Example 39. Detailed Description of the Invention Definitions

[0022] For greater clarity and ease of reading, the following scientific information and definitions are provided. Any technical characteristics mentioned or disclosed herein may be part of each embodiment of the present invention or may be interpreted as part of each embodiment of the present invention. Other definitions and explanations may be provided in the context of the present invention.

[0023] Unless defined otherwise, or unless the specific context requires otherwise, all technical terms used herein have the same meaning as those commonly understood by those qualified in the relevant technical field.

[0024] Unless the context indicates or requires otherwise, the terms “include”, “contain” and “comprise”, as well as similar expressions, should be interpreted in this descriptive report and claims as “including, but not limited to” in an open and inclusive manner.

[0025] Expressions such as “a form of realization”, “forms of Petition 870250088241, dated 09 / 29 / 2025, p. 78 / 159 7 / 72 embodiment” or “a specific embodiment” means that a specific characteristic, property or feature, or a specific group or combination of such characteristics, features or properties, as described in conjunction with the corresponding expression, exists in at least one feature, characteristic or property of the present invention. These expressions appearing in different parts of the description do not necessarily refer to the same feature. Furthermore, specific characteristics, features or properties may be combined in any suitable manner in one or more features.

[0026] Unless explicitly stated otherwise in the context, the singular forms “a”, “an” and “the” should be understood as including plural meanings.

[0027] When the term “neutral” is applied to compounds such as lipids or steroids, or groups or fractions, it indicates that the compound is neither cationic nor anionic, such as compounds that do not have ionizable functional groups under physiological conditions, for example, hydrocarbons; or it indicates that the compound is cationic and anionic under typical physiological conditions, i.e., zwitterionic, such as typical natural phosphatidylcholine.

[0028] As used herein, “lipid” refers to a group of organic compounds derived from fatty acids (such as esters), typically characterized by being insoluble in water but soluble in various organic solvents. Lipids are typically classified into at least three types: (1) “simple lipids,” including fats, oils, and waxes; (2) “complex lipids,” including phospholipids and glycolipids; and (3) “derived lipids,” such as steroids. With respect to glycolipids, in certain embodiments, LNP comprises glycolipids (e.g., GM1 monosialoganglioside).

[0029] In this context, the prefix “poly” refers to multiple atoms or groups in a compound that possess their respective properties. In Petition 870250088241, dated 09 / 29 / 2025, page 79 / 159 8 / 72 However, the absence of the prefix should not be interpreted as excluding the plural. For example, polycationic compounds are also cationic compounds and can be called cationic compounds.

[0030] The term “nucleic acid” refers to any compound that contains DNA or RNA or is composed of them. This term can also be used for oligonucleotides.

[0031] Immune System: The immune system can protect organisms from infection. If pathogens break through an organism's physical barriers and enter it, the innate immune system provides an immediate but non-specific response. If the 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 enhanced response is then retained in the form of immunological memory after the pathogen is eliminated, allowing the adaptive immune system to mount a faster and stronger attack each time it encounters the pathogen. Thus, the immune system comprises the innate immune system and the adaptive immune system. Each of these components includes so-called humoral and cellular components.

[0032] Adaptive Immune System: The adaptive immune system consists of highly specialized systemic cells and processes that can eliminate or prevent pathogenic growth. The adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens (immunity) and mount a stronger attack each time they encounter the pathogen. This system is highly adaptive due to somatic hypermutation (an increase in the frequency of somatic mutations) and V(D)J recombination (irreversible genetic recombination of antigen receptor gene fragments). This mechanism allows that Petition 870250088241, dated 09 / 29 / 2025, page 80 / 159 9 / 72 A small number of genes produce 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 descendants (daughters) of that cell inherit genes that encode the same receptor specificity, including memory B cells and memory T cells, which are essential for long-lasting specific immunity. The immune network theory is a theory about how the adaptive immune system works, based on the interactions between the variable regions of T cells, B cells, and the receptors for molecules produced by T cells and B cells that have variable regions.

[0033] The term “vaccine” is generally understood as a preventive or therapeutic material that provides at least one antigen or antigenic function. Antigens or antigenic functions can stimulate the body’s adaptive immune system to provide an adaptive immune response.

[0034] The term “antigen” generally refers to a substance that can be recognized by the immune system, preferably the adaptive immune system, and that can trigger an antigen-specific immune response, such as the formation of antibodies and / or antigen-specific T cells as part of the adaptive immune response.

[0035] The term “artificial mRNA” (sequence) can generally be understood as an mRNA molecule that does not exist naturally. In other words, artificial mRNA molecules can be understood as unnatural mRNA molecules. Such mRNA molecules may be unnatural due to their individual sequences (not occurring naturally) and / or due to other modifications, such as structural modifications of nucleotides that do not occur naturally. Typically, artificial mRNA molecules can be designed and / or produced using genetic engineering methods to match the desired artificial nucleotide sequence (sequence). Petition 870250088241, dated 09 / 29 / 2025, page 81 / 159 10 / 72 heterologous). In this context, an artificial sequence is typically a sequence that does not occur naturally, that is, it differs from the wild-type sequence by at least one nucleotide. The term "wild-type" can be understood as a sequence that exists in nature.

[0036] The term “pharmaceutically acceptable salt” refers to a form of a compound that does not cause significant irritation to the organism receiving the administration and does not cause the compound to lose its biological activity and properties.

[0037] The compounds described in this invention can exist in multiple crystalline forms, that is, different crystalline lattice arrangements of the same elements of the compound. Polymorphs typically exhibit different X-ray diffraction spectra, infrared spectra, melting points, densities, hardnesses, crystalline forms, optical and electrical properties, stabilities, and solubilities. Different factors, such as recrystallization solvents, crystallization rates, and storage temperatures, can result in recrystallization products dominated by a single crystalline form. It should be understood that the amino lipids described in the present invention include all these crystalline forms.

[0038] The compounds described in the present invention may contain chiral centers and / or axial chirality and, therefore, may exist in the form of racemic compounds, racemic mixtures, simple enantiomers, diastereomeric compounds and simple diastereomers, as well as in the form of 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 pure compounds, are included within the scope of the present invention. The term “each isomer,” as defined in the present invention, includes all such isomeric forms of the compounds. Petition 870250088241, dated 09 / 29 / 2025, page 82 / 159 11 / 72

[0039] The compounds described in this invention may also exist in various hydrates or solvates, wherein the solvates contain stoichiometric or non-stoichiometric amounts of solvent and are selectively formed during crystallization with pharmaceutically acceptable solvents such as water or ethanol. When the solvent is water, hydrates are formed, and when the solvent is ethanol or other solvents, solvates are formed.

[0040] The term “prodrug” is also known as precursor drug, drug precursor, or prokinetic drug, etc., and refers to a compound obtained through chemical structural modification of a drug that is inactive or has low activity in vitro, but releases an active drug through enzymatic or non-enzymatic conversion in vivo to exert its pharmacological effect. Prodrugs are divided into two main categories: vehicle prodrugs and biological prodrugs. Vehicle prodrugs are compounds with active components covalently linked to a carrier that transports them. Within the body, the carrier is removed by simple hydrolysis, allowing the active compound to exert its pharmacological effects. Vehicle prodrugs are often less active or inactive compared to their parent compounds. The structure of the carrier is typically lipophilic, necessary to be non-toxic to the body and capable of readily releasing the active compound.Biological prodrugs differ from vehicle prodrugs because the active substance does not need to be temporarily bound to a vehicle, but exerts its effects through changes in its own molecular structure. Biological prodrugs themselves are inactive, and the active compounds are their metabolites in the body.

[0041] Through research, it was unexpectedly discovered that the use of novel amino lipids and / or lipid nanoparticles provided by the present invention can effectively overcome the shortcomings of the technologies Petition 870250088241, dated 09 / 29 / 2025, page 83 / 159 12 / 72 existing drawbacks, such as low encapsulation efficiency, low endosome escape rate, low expression levels, and low safety, thus promoting the development of LNPs in the areas of mRNA drugs and vaccines. Amino lipid

[0042] Amino lipids are preferentially cationizable, that is, when the pH is reduced below the pKa of the ionizable group of the lipid, the amino lipid becomes protonated and, when it carries a positive charge, the lipid can bind to negatively charged nucleic acids.

[0043] In one aspect, the present invention provides an amino lipid having the structure of formula (I), or an isomer, a pharmaceutically acceptable salt, a prodrug or a solvent thereof: XG m5 / k / L2. ^n... ^l3i4.„ RM! M2M3R2(D where 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 cycloalkyl (preferably a 3- to 10-membered cycloalkyl, more preferably a 4- to 6-membered cycloalkyl) comprising at least one heteroatom (preferably the heteroatom is N or O, preferably 1 or 2 heteroatoms selected from N or O); substituteable carbon atoms or heteroatoms in the cycloalkyl are unsubstituted or substituted by one or more C1-C4 alkyl, C2-C4 alkenyl, C3-C8 cycloalkyl, or Cs-Cs cycloalkenyl groups; M, M2, Ms and M4. They are identical or different from each other, and each is independently selected from C1-C24 alkylene, C3-C24 cycloalkylene, C2-C24 alkenylene, or C3-C24 cycloalkenylene; R1 and R2 are identical or different from each other, and each is Petition 870250088241, dated 09 / 29 / 2025, p. 84 / 159 13 / 72 selected independently from H, C1-C24 alkyl, C3-C24 cycloalkyl, C2-C24 alkenyl, or C3-C24 cycloalkenyl; L1, L2, L3 and L4 are identical or different from SS-; R, R' and R'' are identical or different from each other, and each is independently selected from H, C1-C10 alkyl (preferably C1-C6 alkyl, more preferably C1-C4 alkyl), C3-C10 cycloalkyl, C3-C10 alkenyl, C3-C10 cycloalkenyl, C1-C10 alkyl terminated with a tertiary amino group, C3-C10 cycloalkyl terminated with a tertiary amino group, C3-C10 alkenyl terminated with a tertiary amino group, or cycloalkyl comprising at least one heteroatom, the cycloalkyl being unsubstituted or substituted by one or more C1-C4 alkyl, C2-C4 alkenyl, C3-C8 cycloalkyl, or C3-C8 cycloalkenyl groups;Preferably, a C1-C10 alkyl terminated with -N(C1-6 alkyl)2, or a C1-C10 alkyl terminated with a 3- to 8-membered cycloalkyl (preferably a 4- to 6-membered cycloalkyl) containing 1 to 2 nitrogen atoms in the ring, wherein the C3-C10 cycloalkyl and the 3- to 8-membered cycloalkyl (preferably a 4- to 6-membered cycloalkyl) may optionally be replaced by C1-C6 alkyl; M5 is selected from a single bond, C1-C16 alkylene, C2-C16 alkenylene, C3-C8 cycloalkylene or C3-C8 cycloalkenylene.

[0044] It should be noted that, unless explicitly prohibited, the alkyl, alkenyl, alkylene, and alkenylene groups mentioned in this invention, which contain multiple carbon atoms, may be linear or branched chain. Furthermore, the alkyl, alkenyl, alkylene, alkenylene, cycloalkyl, cycloalkenyl, cycloalkylene, and cycloalkenylene groups mentioned in this invention may be unsubstituted. Petition 870250088241, dated 09 / 29 / 2025, p. 85 / 159 14 / 72 or substituted, the substituents being any suitable substituents, i.e., any linear or branched chain alkyl, aryl, heteroalkyl or heteroaromatic structures, which may optionally contain other functional groups, such as ester or amide groups.

[0045] In some preferred embodiments, G is selected from H, OR or NR'R”, or cycloalkyl comprising at least one heteroatom, wherein the heteroatom is O or N; wherein R, R' and R” are identical or different from each other, and each is selected independently from H, C1-C2 alkyl, C3-C8 cycloalkyl, C3-C8 alkenyl, or C3-C8 cycloalkenyl.

[0046] In other preferred embodiments, G is selected from H, OR or NR'R”, where R' and R” are identical or different from each other, and each is selected independently from H or C1-C4 alkyl; -that is, NR'R” can be NH2, NHCH3, NHC2H5, NHC3H7, NHC4H9, N(CH3)2, CH3-N-C2H5, CH3-N-C3H7, CH3-N-C4H9, C2H5-NC2H5, C2H5-N-C3H7, C2H5-N-C4H9, C3H7-N-C3H7, C3H7-N-C4H9 or C4H9-NC4H9.

[0047] Alternatively, G is selected from substituted or unsubstituted 5-membered oxacycloalkyl, substituted or unsubstituted 5-membered azacycloalkyl, substituted or unsubstituted 6-membered azacycloalkyl, substituted or unsubstituted 6-membered diazocycloalkyl, or substituted or unsubstituted 6-membered azaoxacycloalkyl. Preferably, the The 5-membered oxacycloalkyl is '—', the 5-membered azacycloalkyl is a 6-membered azacycloalkyl is The 6-membered diazocycloalkyl is '—', the 6-membered azaoxacycloalkyl is , wherein the * site is attached to Ms. Petition 870250088241, dated 09 / 29 / 2025, p. 86 / 159 15 / 72

[0048] In some embodiments, when G is a 6-membered substituted diazocycloalkyl, the substituent is positioned on the nitrogen atom that is not bonded to M5.

[0049] 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 cycloalkyl (preferably a 3- to 10-membered cycloalkyl, more preferably a 4- to 6-membered cycloalkyl) containing 1 or 2 nitrogen atoms in the ring; substituteable carbon atoms or heteroatoms in the cycloalkyl are unsubstituted or substituted by one or more C1-C4 alkyl, C2-C4 alkenyl, C3-C8 cycloalkyl, or C3-C8 cycloalkenyl groups. C1-C10 terminated with N(C1-6 alkyl)2, or C1-C10 alkyl terminated with a 4- to 6-membered cycloalkyl containing 1 to 2 nitrogen atoms in the ring, a C3Cw cycloalkyl groups with 4 to 6 members can optionally be replaced by C1-C6 alkyl groups.

[0050] In some embodiments, M5 is selected from a single linkage, C2-C16 alkylene, C2-C16 alkenylene, C4-C8 cycloalkylene or C3-C8 cycloalkenylene.

[0051] More preferably, M5 is selected from a single linkage, C2-C16 alkylene or C4-C6 cycloalkylene. More preferably, M5 is selected from a single linkage, C2-C6 alkylene or C4-C6 cycloalkylene; or selected from C3-C5 alkylene.

[0052] In a preferred embodiment, in formula (I), the / M5. GN formed by linking M5 and G is a selection from A1 to A38: Petition 870250088241, dated 09 / 29 / 2025, p. 87 / 159 16 / 72 A33A34A35 A36 A37 A38 / M5 X GN

[0053] Preferably, it is a selection of A1-A18, A22-A24 and A28-A38. / M5 GN

[0054] More preferably, it is a selection of A15, A16, A17, A23, A29, A30, A33, A37, A38 and A42.

[0055] In some embodiments of the present invention, in GN formula (I), formed by the bonding of Mse G is selected from A39 to A52: Petition 870250088241, dated 09 / 29 / 2025, p. 88 / 159 17 / 72 A48 A49 A50 A51 A52

[0056] In some embodiments of the present invention, Li, l_2, Ls and l_4 are identical or different and independently selected from -C(=O)O-, -OC(=O)-, -C(=O)NR- or -NRC(=O)-, where, when Li, l_2, L3 and l_4 are independently selected from -C(=O)NR- or -NRC(=O)-, R is independently selected from H or C1-C10 alkyl. Preferably, R is H.

[0057] In a preferred embodiment of the present invention, L1 and l_4 are the same and are -C(=O)O- or -OC(=O)-.

[0058] In some embodiments of the present invention, M1, M2, M3 and M4 are identical or different, M1 and M4 are each independently selected from branched-chain C4-C22 alkylene, branched-chain C4-C22 cycloalkylene, branched-chain C4-C22 alkenylene or branched-chain C4-C22 cycloalkenylene, and M2 and M3 are each independently selected from C4-C22 alkylene, C4-C22 cycloalkylene, C4-C22 alkenylene or C4-C22 cycloalkenylene.

[0059] Even more preferably, M1, M2, Ms and M4 are identical or different, and M1 and M4 are each independently selected from Petition 870250088241, dated 09 / 29 / 2025, p. 89 / 159 18 / 72 of branched-chain C4-C22 alkylene, or branched-chain C4-C22 alkenylene, preferably branched-chain C6-C16 alkylene; and M2 and M3 are each independently selected from C4-C22 alkylene or C4-C22 alkenylene, preferably C3-C8 alkylene.

[0060] More preferably, in some embodiments of the present invention, M2 is the same as M3 and is a C4-C22 alkylene.

[0061] In some embodiments of the present invention, Mi is the same as M4e is a branched-chain C4-C22 alkylene.

[0062] In some embodiments of the present invention, R1 and R2 are identical or different, and each is independently selected from C4-C22 alkyl or C4-C22 alkenyl, preferably C5-C12 alkyl.

[0063] In preferred embodiments, the fragment R1-Li-MiL2-M2- is R1-C(=O)OMi-OC(=O)-M2-, the fragment R2-L4-M4-L3-M3- is R2C(=O)OM4-OC(=O)-M3-. More preferably, Mi, M2, M3 and M4 are identical or different, and Mi and M4 are each independently selected from branched-chain C4-C22 alkylene or branched-chain C4-C22 alkenylene; and M2 and M3 are each independently selected from C4-C22 alkylene or C4-C22 alkenylene; R1 and R2 are identical or different, and each is independently selected from C4-C22 alkyl or C4-C22 alkenyl.

[0064] Those skilled in the art may combine the above preferred embodiments for different groups, according to common knowledge, to obtain more preferred embodiments of the compounds of the present invention.

[0065] In a preferred embodiment of the present invention, the structure of formula (I) is selected from the following structures: Petition 870250088241, of 29 / 09 / 2025, p. 90 / 159 19 / 72 E8LA6B6O3 E8LA8B6O3 E8LA10B6O3 E8LA12B6O3 The CBHi7 Ο E10LA6B6O3 WITH CBH17 C7H15 E10LA8B6O3 C0H1T <^0^07Η15Ο WITH. E10LA10B603 E10LA12B6O3 E12LA6B6O3 E12LA8B6O3 CgHi9 CgHis E12LA10B6O3 E8CA5B6O3 E10CA5B6O3 E12LA6B4O2 E12LA8B5O2 E12LA6B6O2 WITH O _ I f^O^CeHn C10H21 ~ C10H21 E12LA6B7O2 E12LA6B8O2 E12LA6B4O3 E12LA6B5O3 E12LA8B7O3 E12LA6B8O3 E12LA6B4O4 O Ç^O^CsH, C10H21 CsHu E12LA6B5O4 CinH?i THE E12LA8B6O4 E12LA8B7O4 Ç10H21 E12LA8B8O4

[0066] In a preferred embodiment of the present invention, the structure of formula (I) is selected from the following Petition 870250088241, dated 09 / 29 / 2025, page 91 / 159 20 / 72 structures: HO^χ^N o o E12LA12B6O3 C6Hi3 CjH^I / < / θγΑθ5Ηιι E12LA6B6O3A4 o E8CA5B6O3 o o E12LA6B6O3A5 E12LA6B6O3 the i / N The Ç10H21 C5Hii ÇlOH21 THE E12LA6B6O9 the E12LA6B6O3A6 THE E12LA6B6O10 E8LA6B6O3A6 the E12LA6B6O12 the E12LA6B6O13 E12LA6B6O3A7 the E12LA6B6O3A8 Petition 870250088241, de 29 / 09 / 2025, pág. 92 / 159 21 / 72 The Ç10H21oA / °YC5Hl1θ10H21 o the E12LA12B6O30A9 E12LA12B6O30A10 E12LA12B6O30 the E12LA12B6O31 K3LA6B6O3 E12LA12B6O3A11 the the E12N1LA6B6O3 K4LA6B6O3 KLA6B6O3 E12N1LA8B6O3 E12N2LA6B6O3 ÇioH2i o E12LA6B6O3A2 % θ10H21s's .ο. .0. E12S1LA6B6O3 C5Hii

[0067] Experts in the field can combine the forms of Petition 870250088241, dated 09 / 29 / 2025, page 93 / 159 22 / 72 preferred embodiments above for different groups based on common knowledge to obtain more preferred embodiments of the compounds of the present invention. Method of preparing amino lipids

[0068] The present invention provides a method for preparing the aforementioned amino lipid, comprising the following steps: S1: subject an epoxide compound and a carboxylic acid to a ring-opening reaction to prepare R1-Li-Mi-OH as intermediate 1; S2: subject intermediate 1 and a carboxylic acid compound as a starting material to a condensation reaction in the presence of a condensing agent to give the R1-Li-Mi-L2-M2-leaving group as intermediate 2; and S3: subjecting intermediate 2 and an amino compound as a starting material to one or more substitution reactions to obtain the target product; or comprising the following steps: Si': perform an oxidation reaction of a diol subjected to TBS protection and subject the oxidation product and a Grignard reagent to an addition reaction, to provide HO-Mi-OTBS as intermediate 1'; S2': subject intermediate 1' and a carboxylic acid compound to a condensation reaction in the presence of a condensing agent, to give the TBSO-Mi-L2-M2-leaving group as intermediate 2'; S3': remove the TBS protecting group from the intermediate 2' TBSO-MiL2-M2-leaving group, allowing the resulting intermediate to undergo a condensation reaction with a carboxylic acid in the presence of a condensing agent, to generate the R1-Li-Mi-L2-M2-leaving group as intermediate 2; and S4': submit intermediate 2 and an amino compound as a Petition 870250088241, dated 09 / 29 / 2025, page 94 / 159 23 / 72 starting material to one or more substitution reactions, to obtain the target product.

[0069] Preferably, the carboxylic acid in the S2' step is the H-L2M2-leaving group.

[0070] Preferably, the carboxylic acid in the S3' step is R1-Li-H.

[0071] Preferably, the leaving group is a halogen.

[0072] Preferably, all intermediates and target products are purified by column chromatography.

[0073] In some embodiments, the preparation method comprises the following steps: - Ring-opening reaction: Carboxylic acid compound (1.0 eq), epoxy compound as starting material (0.6-3.0 eq), ferric trichloride (0.5-10 mol%), pyridine (0.2-20 mol%) are mixed and stirred at room temperature overnight. After the reaction is complete, purification by column chromatography is performed to obtain intermediate 1, with a yield of 56% to 99.0%; - Condensation reaction: Intermediate 1 (1.0 eq), halocarboxylic 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 are mixed and stirred at room temperature overnight, and purification by column chromatography is performed to provide intermediate 2, with a yield of 46.0%-96.4%; - Substitution reaction: Intermediate 2 (1.0-5.0 eq), potassium carbonate (1.0-5.0 eq), amine compounds (1.0 eq), sodium iodide (1.0-3.0 eq) and acetonitrile are mixed, followed by overnight stirring at 20-100 °C and purification by column chromatography, to obtain amino lipid, with a yield of 32.0%-92.1%.

[0074] In some embodiments, the preparation method includes the following steps: Petition 870250088241, dated 09 / 29 / 2025, page 95 / 159 24 / 72 - Addition reaction: The diol subjected to TBS protection is subjected to an oxidation reaction with PCC. The oxidation product (0.9-1.2 eq) is stirred in anhydrous tetrahydrofuran at -20 °C for 2 to 20 minutes, followed by the addition of Grignard reagent (1.2-1.5 eq). The mixture is stirred from -20 °C to 0 °C for 1 to 6 hours, and purification by column chromatography is performed to obtain the intermediate 1'; - Condensation reaction 1: Intermediate 1' (1.0 eq), halocarboxylic 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 are mixed sequentially and stirred at room temperature overnight. The result is purified by column chromatography to obtain intermediate 2'; - Condensation reaction 2: Intermediate 2' (1.0 eq) is treated with ammonium fluoride (5.0-20.0 eq) to remove the TBS protection, then 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 overnight at room temperature. The resulting product is purified by column chromatography to obtain intermediate 2; - Substitution reaction: Intermediate 2 (1.0-5.0 eq), potassium carbonate (1.0-5.0 eq), amine compounds (1.0 eq), sodium iodide (1.0-3.0 eq), and acetonitrile are mixed sequentially and stirred overnight at 20-100 °C. The resulting product is purified by column chromatography to obtain the amino lipid. Lipid nanoparticles

[0075] The present invention provides a lipid nanoparticle comprising any of the above amino lipids.

[0076] It will be understood that all the above options and preferences for amino lipids also apply to the lipid nanoparticles of the present invention comprising amino lipids. Petition 870250088241, dated 09 / 29 / 2025, page 96 / 159 25 / 72

[0077] In some embodiments of the present invention, the lipid nanoparticle further comprises a steroid, a neutral lipid and / or a lipid conjugated to a polymer. Steroid

[0078] A “steroid” is an organic compound with four rings arranged in a specific molecular configuration. It contains the following carbon skeleton:

[0079] Steroids and neutral steroids include natural steroids and their analogues (such as amphiphilic lipid cholesterol hemisuccinate (CHEMS), a succinate compound esterified to the β-hydroxyl group of cholesterol, as a cholesterol derivative). Neutral steroids may be steroids that do not contain ionizable atoms or groups under physiological conditions, or they may be zwitterionic steroids. In a preferred embodiment, neutral steroids do not contain atoms or groups that are ionizable under physiological conditions. In some preferred embodiments, the steroid or steroid analogue is cholesterol. The terms “steroid” and “neutral steroid” are used interchangeably in this document. Neutral lipids

[0080] The “neutral lipids” of the present invention, also referred to as “auxiliary lipids,” are preferably phospholipids or neutral phospholipids. As used in this document, a “neutral phospholipid” is an amphiphilic compound composed of molecules that typically have 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. The term “phospholipid” or “neutral phospholipid” in this invention is used in this context. Petition 870250088241, dated 09 / 29 / 2025, page 97 / 159 26 / 72 invention includes natural and synthetic phospholipids. Lipid conjugated with polymer

[0081] The term “polymer-conjugated lipid” refers to molecules that simultaneously contain a lipid moiety and a polymer moiety. Preferably, the polymer-conjugated lipid is a PEGylated lipid or PEG-lipid. The terms “PEGylated lipid” or “PEG-lipid” refer to molecules that simultaneously contain a lipid moiety and a polyethylene glycol moiety. PEGylated lipids are known in the art and include PEG-DMG, etc.

[0082] In specific embodiments, the lipid conjugated to the polymer has the chemical formula PYL, where P is a hydrophilic polymeric moiety, Y is an optional ligand, and L is a lipid moiety.

[0083] Specifically, the hydrophilic polymeric fraction P may be polyethylene glycol (PEG). In specific embodiments, the average molecular weight of the PEG fraction 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. Therefore, PEG may be the PEG commonly referred to as “PEG 2000”.

[0084] In another embodiment, the hydrophilic polymeric fraction P in the lipid conjugated to the polymer may also be a substantially hydrophilic polymer different from the hydrophilic polymeric fraction mentioned above. That is, the hydrophilic polymeric fraction P in the lipid conjugated to the polymer may be based on poly(epoxypropane), poly(vinylpyrrolidone), poly(vinyl alcohol), poly-N-(2-hydroxypropyl)methacrylamide, hydroxyethyl starch modification (HEsylation, according to PMID 24681396), PAsylation (i.e., proline-alanine-serine), the XTEN method known in the art (i.e., PEG-based peptides), poly(sarcosine), or poly(vinyl acetate).

[0085] Specifically, the optional linker Y can be any useful spacer structure, such as those typically found in lipids. Petition 870250088241, dated 09 / 29 / 2025, pp. 98 / 159 27 / 72 Pegylated compounds, including but not limited to those based on succinimide, amine, ether, ester, anhydride, aldehyde, ketone, amide, carbamate, or combinations thereof.

[0086] Specifically, the L lipid fraction can be derived from phospholipids, sphingolipids, or ceramides. As used in this document, the term “derived from phospholipids or ceramides” includes free radicals of phospholipids and ceramides. An example is a lipid conjugated to a polymer containing a phosphatidylethanolamine fraction or a phosphatidylglycerol fraction.

[0087] In a preferred embodiment, the lipid conjugated to the polymer is a PEGylated lipid. PEGylated lipids include, but are not limited to, the following PEGylated lipids: PEGylated diacylglycerol lipid (PEGDAG); PEGylated ceramide lipid (PEG-Cer); PEGylated phosphatidylethanolamine lipid (PEG-PE); PEGylated succinylglycerol lipid (PEG-S-DAG); PEGylated dialkylpropyl carbamate lipid; 1,2-dimyristyl-rac-glycero-3-methoxy polyethylene glycol (“PEG-DMG” or “DMG-PEG”).

[0088] In a more preferred embodiment, the lipid conjugated to the polymer is DMG-PEG2000.

[0089] 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.

[0090] In some embodiments of the present invention, the molar ratio of amino lipid, steroid, neutral lipid and polymer-conjugated lipid in the lipid nanoparticle is 30-70:30-65:0-30:0.2-5. More preferably, it is 30-60:35-60:0-20:0.3-3.

[0091] In a preferred embodiment of the present invention, in the lipid nanoparticle, the amino lipid is the aforementioned preferred amino lipid, the steroid is cholesterol, the neutral lipid is phospholipid, and the lipid conjugated to the polymer is PEGylated lipid; the molar ratio of amino Petition 870250088241, dated 09 / 29 / 2025, page 99 / 159 28 / 72 lipid, cholesterol, phospholipid and PEGylated lipid is 40-50:40-45:10-15:0.5-2.

[0092] In a preferred embodiment, the PEGylated lipid in the lipid nanoparticle is DMG-PEG2000.

[0093] The lipid nanoparticle of the present invention is not limited to any specific form and should be interpreted as including any form produced when the amino lipid is combined with one or more other lipids, such as in an aqueous environment and / or in the presence of nucleic acid compounds. For example, liposomes, lipid complexes, lipoplexes and the like fall within the scope of lipid nanoparticles.

[0094] The lipid nanoparticles of the present invention can be combined with at least one pharmaceutically acceptable carrier or excipient to form a pharmaceutical composition. Thus, the composition can be a dry composition, such as a powder or granule, 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 dissolved or dispersed form (e.g., suspension or emulsion). 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. This formulation is also preferred for compositions containing bioactive components, as described in more detail below.

[0095] As used herein, “nanoparticles” are submicrometer particles of any structure or shape. Submicrometer particles may also be called colloids or colloidal particles. Based on the materials, structure, or morphology of the nanoparticles, nanoparticles can be classified into several types, such as nanocapsules, vesicles, liposomes, lipid nanoparticles, micelles, cross-linked micelles, lipoplexes, polymers, or hybrid or composite materials. These are just a few examples of possible names. Petition 870250088241, dated 09 / 29 / 2025, pp. 100 / 159 29 / 72 for specific types of nanoparticles are mentioned. “Lipid nanoparticles” (LNPs) are nanoparticles formed from lipids, typically containing at least one amphiphilic film-forming lipid and optionally other lipids, and optionally also containing filler materials such as nucleic acid compounds. As used herein, the terms “lipid nanoparticles” or “LNPs” include any subtype and morphology of nanoparticles formed or co-formed with lipids, such as liposomes and lipoplexes.

[0096] As defined above, lipid nanoparticles include any type of nanoparticle formed or co-formed by lipids. Specifically, lipid nanoparticles can be co-formed by lipid combinations containing at least one vesicle-forming amphiphilic lipid. Liposomes and lipoplexes are examples of lipid nanoparticles.

[0097] Preferably, in some embodiments of the present invention, the lipid nanoparticles also contain bioactive components.

[0098] A bioactive component refers to any compound or material with biological activity, such that the compound or material can be used for the prevention, management, improvement, treatment, or therapy of a disease or condition in a subject (e.g., an animal, particularly a human subject).

[0099] In some embodiments, the bioactive component is a nucleic acid compound selected from the group consisting of artificial mRNA, chemically modified or unmodified messenger RNA containing at least one coding sequence, self-replicating RNA, circular RNA, viral RNA, and replicon RNA; or any combination thereof. Preferably, the bioactive component is mRNA or an mRNA compound.

[00100] In some embodiments, the bioactive component is selected from small interfering RNA (siRNA), RNA Petition 870250088241, dated 09 / 29 / 2025, pp. 101 / 159 30 / 72 asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[00101] In embodiments of the present invention, the mRNA includes one or more stem-loop chain termination nucleotides, a polyA sequence, a polyadenylation signal and / or a 5' cap structure.

[00102] In embodiments 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%.

[00103] In one embodiment of the present invention, the weight / weight ratio of the lipid component to the bioactive component in the lipid nanoparticle is from 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.

[00104] In one embodiment of the present invention, the N:P ratio in the lipid nanoparticle is from about 2:1 to about 30:1. More preferably, the N:P ratio is about 5.67:1.

[00105] In one embodiment of the present invention, the average size of the lipid nanoparticles is from about 70 nm to about 100 nm.

[00106] In one embodiment of the present invention, the polydispersity index of the lipid nanoparticles is from about 0.10 to about 0.20.

[00107] In embodiments of the present invention, the lipid nanoparticles have a zeta potential of about -10 mV to about +20 mV.

[00108] In preferred embodiments, the Petition 870250088241, dated 09 / 29 / 2025, pp. 102 / 159 31 / 72 The bioactive component is complexed or combined 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 terms “complexed” or “combined” refer to the formation of a larger, non-covalently linked complex, or to the assembly of the bioactive component with one or more lipids in a substantially stable manner. Use

[00109] The present invention provides a method for treating or preventing infectious diseases, cancer, tumorous diseases, genetic diseases, allergies, toxicity, and autoimmune diseases using the aforementioned amino lipid or lipid nanoparticle or pharmaceutical composition.

[00110] Consequently, the present invention provides the use of the aforementioned amino lipid or lipid nanoparticle or pharmaceutical composition in the preparation of a medicament to prevent or treat infectious diseases, cancer, tumorous diseases, genetic diseases, allergies, toxicity, and autoimmune diseases.

[00111] Infectious diseases include infectious diseases caused by viruses, bacteria, or protozoa. Viruses include, but are not limited to, SARS-CoV-2 coronavirus, 2019-nCoV 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, enteropathogenic Escherichia coli, Lassa virus (LASV), Middle East Respiratory Syndrome coronavirus (MERS-CoV), Mycobacterium tuberculosis, Nipah virus, Norovirus, rabies virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, Vaccinia virus, yellow fever virus and Zika virus. Petition 870250088241, dated 09 / 29 / 2025, pp. 103 / 159 32 / 72

[00112] Types of cancer include lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, leukemia, and prostate cancer.

[00113] The present invention also provides a method for gene therapy, gene vaccination, antisense therapy, nucleic acid delivery or treatment by means of RNA interference, using the aforementioned amino lipid or lipid nanoparticle or pharmaceutical composition.

[00114] Consequently, the present invention also provides the use of the aforementioned amino lipid or lipid nanoparticle in the preparation of a medicament for gene therapy, gene vaccination, antisense therapy, nucleic acid delivery or treatment by means of RNA interference.

[00115] The present invention also provides a method for administering a drug to a subject, comprising: administering the drug formulated in the aforementioned lipid nanoparticle to the subject.

[00116] When administering the drug, the method of administration is determined based on the drug formulation. The drug formulation is related to the excipients and / or vehicles. General routes for systemic administration include: transdermal, oral, and parenteral routes, including subcutaneous, intravenous, intramuscular, intra-arterial, intradermal, and intraperitoneal injections and / or intranasal routes of administration. Routes of local administration generally include intradermal, transdermal, subcutaneous, or intramuscular injection; or intralesional, intracranial, pulmonary, intracardiac, intratumoral, or sublingual injection. When the drug is in vaccine form, the preferred routes of administration are intramuscular and intradermal injection.

[00117] The following examples are provided to illustrate the Petition 870250088241, dated 09 / 29 / 2025, pp. 104 / 159 33 / 72 present invention, but do not intend to limit the scope of the invention.

[00118] Unless otherwise indicated, the techniques or conditions used in the examples are those described in the literature of the field or as described in the product manuals. Unless otherwise indicated, the reagents or instruments used are conventional products available through regular channels.

[00119] For illustrative purposes, the following examples show general methods for preparing the compounds provided by the present invention. For more detailed descriptions of each reaction step, see the following examples. Those skilled in the art will understand 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 easily be used to provide various derivatives and / or reaction conditions. Furthermore, in combination with the disclosure of the following examples, many compounds prepared by the methods described below can be further modified using conventional chemical methods known to those skilled in the art to obtain other compounds within the scope of the present invention.

[00120] The abbreviations used correspond to the following substances: Py: Pyridine; EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; DMAP: 4-Dimethylaminopyridine; DIPEA: Diisopropylethylamine; DCM: Dichloromethane; MeCN: Acetonitrile; DSPC: Distearoylphosphatidylcholine; Petition 870250088241, dated 09 / 29 / 2025, pp. 105 / 159 34 / 72 DMG-PEG2000: 1,2-dimyristyl-rac-glycero-3-methoxy polyethylene glycol 2000. Preparation of aminolipid compounds Example 1 Synthesis of the amino lipid E8LA12B6O3 Step 1: E8LA12 Synthesis: C6H13 E8 ο CiiH23^OH LA12 FeCl3, Py rt, 12 h E8LA12

[00121] 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 reaction solution was stirred at room temperature overnight. After the reaction was complete, 1.2 g of the intermediate E8LA12 (colorless oily liquid) was obtained by purification by column chromatography separation, with a yield of 91%. Step 2: Synthesis of E8LA12B6: CeHi3 EDCI, DMAP, DIPEA DCM, rt, 12 h E8LA12B6 E8LA12 B6

[00122] 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 to a 25 mL reaction tube. The reaction solution was stirred at room temperature overnight. After the reaction was complete, 1.65 g of the intermediate E8LA12B6 (colorless oily liquid) was obtained by purification by column chromatography separation, with a yield of 89%. Step 3: Synthesis of E8LA12B6O3: E8LA12B6 03 E8LA12B6O3 O Petition 870250088241, dated 09 / 29 / 2025, pp. 106 / 159 35 / 72

[00123] 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) were added sequentially to a 25 mL reaction tube. The reaction solution was stirred at 75 °C overnight. After the reaction was complete, 0.94 g of E8LA12B6O3 (colorless oily liquid) was obtained by purification via column chromatography separation, with a yield of 68%.

[00124] 1H NMR (400 MHz, CDCla): δ 5.08-5.03 (m, 2H), 4.22 (dd, J1 = 11.6 Hz, J 2 = 3.6 Hz, 2H), 4.02 (dd, J1 = 12.0 Hz, J 2 = 6.4 Hz, 2H), 3.82 (t, J = 5.6 Hz, 2H), 2.7 Hz, 7 (t (t, J = 6.0 Hz, 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), 0.02C NMR (t, J = 1). MHz, CDCla): δ 173.1, 70.7, 65.5, 59.0, 58.5, 57.2, 34.2, 33.9, 31.9, 31.8, 30.7, 30.3, 29.6, 29.0.3, 29.0.3, 25.0, 25.3, 24.7, 22.7, 14.1. ESI-MS C55H106NO9+ [M+H]+calculated 924.7862, found 924.7850. Example 2 Synthesis of the amino lipid E12LA6B6O3 Stage 1: Synthesis of E12LA6: o O.nu+ΗFeCl3'Py- A10 21CsmAoH rt, 12 hC5H110| C10H21 E12 LA6 E12LA6

[00125] 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 reaction solution was stirred at room temperature overnight. After the reaction was complete, 1.02 g of the intermediate E12LA6 (colorless oily liquid) was obtained by purification via column chromatography separation with a yield of 85%. Petition 870250088241, dated 09 / 29 / 2025, pp. 107 / 159 36 / 72 Step 2: Synthesis of E12LA6B6: o Ci0H2i E12LA6 EDCI, DMAP, DIPEA DCM, rt, 12 h * E12LA6B6

[00126] 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 to a 25 mL reaction tube. The reaction solution was stirred at room temperature overnight. After the reaction was complete, 1.08 g of the intermediate E12LA6B6 (colorless oily liquid) was obtained by purification via column chromatography separation, with a yield of 76%. Step 3: Synthesis of E12LA6B6O3: O Ç10H21 o --_ O Ã K2CO3, Nal HnΛN oC5Hi1 + HO NHíMeCN, 75 °C, 12 h XOC,oh21 O C10H21 ---O^CsH,,0Y E12LA6B6 03 E12LA6B6O3 O

[00127] 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 to a 25 mL reaction tube. The reaction solution was stirred at 75 °C overnight. After the reaction was complete, 458 mg of E12LA6B6O3 (colorless oily liquid) was obtained by purification via column chromatography separation, with a yield of 66%.

[00128] 1H NMR (400 MHz, CDCla): 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 = 5.2Hz, 2H), 2.73-2.70 (m, 2H), 2.52-2.48 (m, 4H), 2.33-2.28 (m, 8H), 1.741.50 (m, 14H), 1.42-1.25 (m, 48H), 0.91-0.86 (m, 12H). (As shown in Figure 1) ESI-MS CõiH98NO9+[M+H]+calculated 868.7236, found 868.7234. Example 3 Synthesis of the amino lipid E8LA8B6O3

[00129] The amino lipid E8LA8B6O3 can be prepared from Petition 870250088241, dated 09 / 29 / 2025, pp. 108 / 159 37 / 72 according to the same method as Example 1, except for the substitution of lauric acid in Example 1 with caprylic acid, and its structural formula is as follows: O OβH13 CtH15

[00130] 1H NMR (400 MHz, CDCI3): 0 5.08-5.02 (m, 2H), 4.22 (dd, Ji = 12.0 Hz, J2 — 3.6 Hz, 2H), 4.01 (dd, Ji = 11.6 Hz, J2 = 6.0 Hz, 2H), 3.81 (t, J = 5.2 Hz, 2H), 2.98 (t, J = 6.0 Hz, 2H), 2.79 (t, J = 7.6 Hz, 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 C47H9oN09+[M+H]+calculated 812.6610, found 812.6598. Example 4 Synthesis of the amino lipid E8LA10B6Q3

[00131] The amino lipid E8LA10B6O3 can be prepared according to the same method as in Example 1, except that lauric acid in Example 1 is replaced by capric acid, and its structural formula is as follows:

[00132] 1H NMR (400 MHz, CDCI3) : δ 0.85-0.89 (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, 8H), 2.77-2.79 (m, 4H), 2.96-2.98 (m, 2H), 3.79-3.80 (m, 2H), 4.01-4.05 (m, 2H), 4.214.24 (m, 2H), 5.03-5.08 (m, 2H). ESl-MSCsiHgsNOg* [M+H]+calculated 868.7, found 868.7. Example 5 Synthesis of the amino lipid E10LA6B6Q3

[00133] The amino lipid E10LA6B6O3 can be prepared according to the same method as in Example 2, except that 1,2-epoxidodecane in Example 2 is replaced by 1,2-epoxidodecane, and its structural formula is Petition 870250088241, dated 09 / 29 / 2025, pp. 109 / 159 38 / 72 as follows: O

[00134] 1Η NMR (400 MHz, CDCI3): 5 5.08-5.02 (m, 2Η), 4.22 (dd, Ji = 12.0 Hz, J2 = 3.2 Hz, 2H), 4.01 (dd, Ji = 11.6 Hz, J2 = 6.4 Hz, 2H), 3.79 (t, J = 5.2 Hz, 2H), 2.82 (t, J = 5.6 Hz, 2H), 2.62 (t, J = 8.0 Hz, 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 C47H9oN09+[M+H]+calculated 812.6610, found 812.6634. Example 6 Synthesis of the amino lipid E10LA8B6Q3

[00135] The amino lipid E10LA8B6O3 can be prepared according to the same method as in Example 1, except by replacing 1,2-epoxyoctane in Example 1 with 1,2-epoxycane and by replacing lauric acid in Example 1 with n-octanoic acid, and its structural formula is as follows: the -C7H15

[00136] 1H NMR (400 MHz, CDCI3) : δ 0.84-0.89 (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, 8H), 2.78-2.80 (m, 4H), 2.96-2.98 (m, 2H), 3.80-3.81 (m, 2H), 4.01-4.04 (m, 2H), 4.204.24 (m, 2H), 5.03-5.07 (m, 2H). ESI-MSC5iHg8NOg+[M+H]+calculated 868.7, found 868.7. Example 7 Synthesis of the amino lipid E10LA10B6Q3

[00137] The amino lipid E10LA10B603 can be prepared according to the same method as in Example 1, except by replacing 1,2-epoxyoctane in Example 1 with 1,2-epoxycane and by replacing lauric acid in Example 1 with capric acid, and its structural formula is as follows: Petition 870250088241, dated 09 / 29 / 2025, pp. 110 / 159 39 / 72

[00138] 1H NMR (400 MHz, CDCI3): δ 0.86-0.92 (m, 12H), 1.251.40 (m, 56H), 1.56-1.71 (m, 12H), 1.87-1.89 (m, 2H), 2.27-2.32 (m, 8H), 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-MSCssHioeNO^ [M+H]+calculated 924.8, found 924.9. Example 8 Synthesis of the amino lipid E12LA8B6O3

[00139] The amino lipid E12LA8B6O3 can be prepared according to the same method as in Example 2, except by replacing the nhexanoic acid in Example 2 with n-octanoic acid, and its structural formula is as follows:

[00140] 1H NMR (400 MHz, CDCI3): δ 5.07-5.04 (m, 2H), 4.23 (dd, Ji = 12.0 Hz, J2 = 3.6 Hz, 2H), 4.01 (dd, Ji = 12.0 Hz, J2 = 6.4 Hz, 2H), 3.80 (t, J = 5.2 Hz, 2H), 2.86 (t, J = 5.6 Hz, 2H), 2.66 (t, J = 7.6 Hz, 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 C55Hio6N09+[M+H]+calculated 924.7862, found 924.7891. Example 9 Synthesis of compound E12LA12B6O3 E12LA12B6O3 Petition 870250088241, dated 09 / 29 / 2025, pp. 111 / 159 40 / 72 Step 1: Synthesis of compound E12LA12B6O3-2

[00141] 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. The reaction solution was 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 C24H49O3+ [M+H]+calculated 385.4, found 385.4. Step 2: Synthesis of compound E12LA12B6O3-3

[00142] 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 sequentially to a 25 mL reaction tube. The reaction solution was 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 = 100:1 to 20:1) to obtain the compound E12LA12B6O3-3 (1.65 g, 92.0%). ESI-MS C30H58BrO4+[M+H]+calculated 561.4, found 561.4. Step 3: Synthesis of E12LA12B6O3

[00143] 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 sequentially to a 25 mL reaction tube. The reaction solution was stirred at 70–80 °C overnight. After the reaction was complete, 0.94 Petition 870250088241, dated 09 / 29 / 2025, pp. 112 / 159 41 / 72 g of E12LA12B6O3 (0.94 g, 68%) was obtained by column chromatography separation.

[00144] 1H NMR (400 MHz, CDCI3) : δ 0.87-0.91 (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, 8H), 2.38-2.42 (m, 4H), 2.61 -2.64 (m, 2H), 3.78-3.81, (m, 2H), 4.01 -4.06 (m, 2H), 4.204.24 (m, 2H), 5.04-5.10 (m, 2H). ESI-MS C63Hi22NO9+[M+H]+calculated 1036.9, found 1037.0. Example 10 Synthesis of the amino lipid E8CA5B6O3

[00145] The amino lipid E8CA5B6O3 can be prepared according to the same method as in Example 9, except by replacing 1,2-epoxyodecane in Example 9 with 1,2-epoxyoctane and by replacing lauric acid in Example 9 with 2-pentylheptanoic acid, and its structural formula is as follows: C6H13CjH^ THE

[00146] 1H NMR (400 MHz, CDCI3) : δ 0.85-0.90 (m, 15H), 1.24-1.36 (m, 54H), 1.53-1.74 (m, 16H), 2.27-2.33 (m, 8H), 2.83 (s, 6H), 3,984.03 (m, 2H), 4.21-4.25 (m, 2H), 5.02-5.07 (m, 2H). ESI-MS C53Hio2N08+[M+H]+calculated 924.8, found 924.8. Example 11 Synthesis of the amino lipid E12LA6B6O9

[00147] The amino lipid E12LA6B6O9 can be prepared according to the same method as in Example 2, except by replacing the 3-aminopropanol in Example 2 with n-pentylamine, and its structural formula is as follows: Petition 870250088241, dated 09 / 29 / 2025, pp. 113 / 159 42 / 72

[00148] 1H NMR (400 MHz, CDCI3) : δ 0.84-0.90 (m, 12H), 1.24-1.31 (m, 54H), 1.53-1.65 (m, 14H), 1.99-2.01 (m, 2H), 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). ESIMS C54H102NO9+ [M+H]+calculated 880.8, found 880.8 Example 12 Synthesis of the amino lipid E12LA6B6O10

[00149] The amino lipid E12LA6B6O10 can be prepared according to the same method as in Example 2, except that the 3-aminopropanol in Example 2 is replaced by trans-p-aminocyclohexanol, and its structural formula is as follows:

[00150] 1H NMR (400 MHz, CDCla) : δ 0.84-0.90 (m, 12H), 1.24-1.31 (m, 54H), 1.53-1.65 (m, 14H), 1.99-2.01 (m, 2H), 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). ESIMS C54Hi02NO9+[M+H]+calculated 908.8, found 908.7. Example 13 Synthesis of the amino lipid E12LA6B6O12

[00151] The amino lipid E12LA6B6O12 can be prepared according to the same method as in Example 2, except by replacing 3-aminopropanol in Example 2 with 3-dimethylaminopropylamine, and its structural formula is as follows:

[00152] 1H NMR (400 MHz, CDCla) : δ 0.86-0.94 (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), Petition 870250088241, dated 09 / 29 / 2025, pp. 114 / 159 43 / 72 2.70-2.76 (m, 2H), 3.37-3.41 (m, 4H), 3.99-4.04 (m, 2H), 4.24-4.34 (m, 6H), 5.005.06 (m, 2H). ESI-MS C53Hi03N2O8+[M+H]+calculated 895.8, found 895.8. Example 14 Synthesis of the amino lipid E12LA6B6O13

[00153] The amino lipid E12LA6B6O13 can be prepared according to the same method as in Example 2, except by replacing 3-aminopropanol in Example 2 with 1-(2-aminoethyl)piperidine, and its structural formula is as follows:

[00154] 1H NMR (400 MHz, CDCla) : δ 0.82-0.92 (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), 5,035.08 (m, 2H). ESI-MS C55H105N2O8+ [M+H]+calculated 921.8, found 921.8. Example 15 Synthesis of the amino lipid E12LA6B6O15

[00155] The amino lipid E12LA6B6O15 can be prepared according to the same method as in Example 2, except by replacing the 3-aminopropanol in Example 2 with N-(2-aminoethyl)-4-hydroxypiperidine, and its

[00156] 1H NMR (400 MHz, CDCla) : δ 0.86-0.90 (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 C55H105N2O9+ [M+H]+calculated 937.8, found 937.9. Petition 870250088241, dated 09 / 29 / 2025, pp. 115 / 159 44 / 72 Example 16 Synthesis of the amino lipid E12LA12B6O30

[00157] The amino lipid E12LA12B6O30 can be prepared according to the same method as in Example 2, except that the 3-aminopropanol in Example 2 is replaced by β-alanine, and its structural formula is as follows:

[00158] 1H NMR (400 MHz, CDCla) : δ 0.86-0.92 (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), 5.025.08 (m, 2H). ESI-MS C64Hi22NOi0+[M+H]+calculated 1064.9, found 1065.0. Example 17 Synthesis of the amino lipid E12LA6B6O31

[00159] The amino lipid E12LA12B6O31 can be prepared according to the same method as in Example 2, except by replacing the 3-aminopropanol in Example 2 with 4-aminobutyronitrile, and its structural formula is as follows:

[00160] 1H NMR (400 MHz, CDCla) : δ 0.86-0.91 (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), 2.60-2.6-2.42 (m, 3.8H), (m, 2H), 4.00-4.04 (m, 2H), 4.18-4.22 (m, 2H), 5.02-5.09 (m, 2H). ESI-MS C64Hi2iN20s+[M+H]+calculated 1045.9, found 1045.9. Petition 870250088241, of 29 / 09 / 2025, p. 116 / 159 45 / 72 Example 18 Synthesis of the amino lipid K3LA6B6O3 WITH' ΌΗ TBSCI WITH' PCC OTBS ----0' CioH2iMgBr OTBS --------* WITH' Ch10X21 OTBS K3LA6B603-1 K3LA6B6O3-2 K3LA6B6O3-3 K3LA6B6O3-4 Br COOH Br O CiqH2i O CsHn 0' CgHn O’ OH Ch10X21 Br Ό' OH K3LA6B6O3-7 K3LA6B6O3-6 H2N OH nh4f Br O Ch10X2i YOU ARE' OTBS K3LA6B6O3-5 YOU ARE K3LA6B6O3 Ch10X21 9 Ch10X21 0 Ό CsHn CgHn Step 1: Synthesis of compound K3LA6B6O3-2

[00161] Imidazole (6.54 g, 96 mmol, 1.2 eq) was added to a 250 mL round-bottom flask, and the flask was filled with nitrogen by evacuation. Dichloromethane (60 mL), N,N-dimethylformamide (30 mL), and 1,3-propylene glycol (6.09 g, 80 mmol, 1.0 eq) were added sequentially, and the mixture was stirred at 0 °C for 10 minutes. tert-Butyldimethylsilyl chloride (12.06 g, 80 mmol, 1.0 eq) was dissolved in dichloromethane (60 mL) and added dropwise to the mixture. 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 a compound K3LA6B6O3-2 (10.96 g, 72%), ESI-MS CgH23O2Si+ [M+H]+ calculated 191.1, found 191.1. Step 2: Synthesis of the compound K3LA6B6O3-3

[00162] 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 Petition 870250088241, dated 09 / 29 / 2025, pp. 117 / 159 46 / 72 dichloromethane (100 mL) was stirred at room temperature for 5 hours. The reaction mixture was filtered, and then the filtrate was concentrated and purified using a flash column chromatography system (n-heptane: ethyl acetate = 100:1 to 20:1) to obtain a compound K3LA6B6O3-3 (8.69 g, 80%), ESIMS C9H21O2Si+[M+H]+ calculated 189.1, found 189.1. Step 3: Synthesis of the compound K3LA6B6O3-4

[00163] A 500 mL round-bottom flask was taken and filled with nitrogen by evacuation. The 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. 70 mL of decylmagnesium bromide solution (1 mol / L anhydrous tetrahydrofuran solution, 1.5 eq) were added dropwise to the mixture. The mixture was stirred at -20 °C to 0 °C for 3 hours, and 50 mL of saturated ammonium chloride solution were added for quenching. The resulting 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 20:1) to obtain a compound. K3LA6B6O3-4 (9.93 g, 65%), ESI-MS CwH43O2Si+[M+H]+calculated 331.3, found 331.4. Step 4: Synthesis of the compound K3LA6B6O3-5

[00164] 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) and 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 Petition 870250088241, dated 09 / 29 / 2025, pp. 118 / 159 47 / 72 a flash column chromatography system (n-heptane:ethyl acetate = 100:1 to 20:1) to obtain a compound K3LA6B6O3-5 (12.79 g, 84%), ESI-MS C25H52BrO3Si+[M+H]+calculated 507.3, found 507.2. Step 5: Synthesis of the compound K3LA6B6O3-6

[00165] A solution of K3LA6B6O3-5 (12.79 g, 25.2 mmol, A solution of 1.0 eq), ammonium fluoride (9.33 g, 252 mmol, 10.0 eq) and methanol (75 mL) were refluxed at 65 °C and stirred for 3 hours. TLC showed that 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 a compound K3LA6B6O36 (8.72 g, 88%), ESI-MS CwH38BrO3+[M+H]+calculated393.2, found 393.2. Step 6: Synthesis of the compound K3LA6B6O3-7

[00166] 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) and 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 a compound K3LA6B6O3-7 (7.86 g, 72%), ESI-MS C25H48BrO4+[M+H]+calculated 491.3, found 491.2. Step 7: Synthesis of the compound K3LA6B6O3

[00167] A solution of the 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), sodium iodide (0.80 g, 5.33 mmol, 1.0 Petition 870250088241, dated 09 / 29 / 2025, pp. 119 / 159 48 / 72 eq) in acetonitrile (10 mL) was stirred at 75 °C overnight. After the reaction solution was concentrated, it was purified using a flash column chromatography system (dichloromethane:methanol = 100:1 to 20:1) to obtain a compound K3LA6B6O3-7 (3.58 g, 75%), 1H NMR (400 MHz, CDCla): δ 0.85-0.90 (m, 12H), 1.24-1.36 (m, 48H), 1.56-1.73 (m, 18H), 2.25-2.29 (m, 8H), 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 C53Hio2N09+[M+H]+calculated 896.8, found 896.9. Example 19 Synthesis of the amino lipid K4LA6B6O3

[00168] The amino lipid K4LA6B6O3 can be prepared according to the same method as in Example 18, except by replacing the 1,3-propylene glycol in Example 18 with 1,4-butylene glycol, and its structural formula is as follows:

[00169] 1H NMR (400 MHz, CDCla) : δ 0.85-0.91 (m, 12H), 1.26- 1.41 (m, 48H), 1.56-1.72 (m, 18H), 2.27-2.33 (m, 8H), 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). ESIMS C55Hio6N09+[M+H]+calculated 924.8, found 924.8. Example 20 Synthesis of the amino lipid K5LA6B6O3

[00170] The amino lipid K5LA6B6O3 can be prepared according to the same method as in Example 18, except by replacing 1,3-propylene glycol in Example 18 with 1,5-pentanediol, and its structural formula is as follows: Petition 870250088241, dated 09 / 29 / 2025, pp. 120 / 159 49 / 72

[00171] 1H NMR (400 MHz, CDCI3) : δ 0.86-0.91 (m, 12H), 1.26- 1.41 (m, 54H), 1.50-1.67 (m, 18H), 2.27-2.34 (m, 8H), 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). ESIMS C57H110NO9+ [M+H]+calculated 952.8, found 952.8. Example 21 Synthesis of the amino lipid E12LA6B6O3A2 CinHíi O Ç10H21 Step 1: Synthesis of compound E12LA6B6O3

[00172] According to the method in Example 2, E12LA6B6O3 was synthesized. Step 2: Synthesis of compound E12LA6B6O3A2

[00173] A 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), 3-(4-phenyl-piperazin-1-yl)-propionic acid (0.26 g, 1.5 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 then 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%).1H NMR (400 MHz, CDC1a): δ 0.85-0.90 (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 C59Hii2N30io+[M+H]+calculated 1022.8, found 1022.9. Example 22 Synthesis of the amino lipid E12LA6B6O3A3

[00174] The amino lipid E12LA6B6O3A3 can be prepared according to the same method as in Example 21, except by replacing the 3-(4-phenyl-piperazin-1-yl)-propionic acid in Example 21 with 4-(4-methyl-1 Petition 870250088241, dated 09 / 29 / 2025, pp. 121 / 159 50 / 72 piperazine)butyric acid, and its structural formula is as follows:

[00175] 1H NMR (400 MHz, CDCI3) : δ 0.85-0.90 (m, 12H), 1.25-1.45 (m, 50H), 1.55-1.66 (m, 12H), 1.69-1.83 (m, 4H), 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). ESIMS C6oHii4N30io+[M+H]+calculated 1036.8, found 1036.8. Example 23 Synthesis of the amino lipid E12LA6B6O3A4

[00176] The amino lipid E12LA6B6O3A4 can be prepared according to the same method as in Example 21, except by replacing the 3-(4-phenyl-piperazin-1-yl)-propionic acid in Example 21 with 3-(dimethylamino)propionic acid, and its structural formula is as follows:

[00177] 1H NMR (400 MHz, CDCla) : δ 0.84-0.89 (m, 12H), 1.24-1.35 (m, 50H), 1.41-1.48 (m, 6H), 1.52-1.65 (m, 12H), 2.23 (s, 6H), 2.262.30 (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 C56Hio7N20io+[M+H]+calculated 967.8, found 967.8. Example 24 Synthesis of the amino lipid E12LA6B6O3A5

[00178] The amino lipid E12LA6B6O3A5 can be prepared according to the same method as in Example 21, except by replacing the 3-(4-phenyl-piperazin-1-yl)-propionic acid in Example 21 with 5-(dimethylamino)pentanoic acid, and its structural formula is as follows: Petition 870250088241, dated 09 / 29 / 2025, pp. 122 / 159 51 / 72

[00179] 1H NMR (400 MHz, CDCI3) : δ 0.84-0.89 (m, 12H), 1.24-1.35 (m, 48H), 1.41-1.75 (m, 18H), 2.21 (s, 6H), 2.22-2.30 (m, 12H), 2,352.37 (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 C58HiiiN20io+[M+H]+calculated 995.8, found 995.9. Example 25 Synthesis of the amino lipid E12LA6B6O3A6

[00180] The amino lipid E12LA6B6O3A3 can be prepared according to the same method as in Example 21, except by replacing the 3-(4-phenyl-piperazin-1-yl)-propionic acid in Example 21 with 7-(dimethylamino)heptanoic acid, and its structural formula is as follows:

[00181] 1H NMR (400 MHz, CDCla) : δ 0.84-0.91 (m, 12H), 1.23-1.37 (m, 50H), 1.39-1.49 (m, 6H), 1.52-1.64 (m, 14H), 2.21 (s, 6H), 2.252.30 (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 C6oHii5N2Oi0+[M+H]+calculated 1023.8, found 1023.8. Example 26 Synthesis of the amino lipid E8LA6B6O3A6

[00182] The amino lipid E8LA6B6O3A6 can be prepared according to the same method as in Example 21, except that E12LA6B6O3 in Example 21 is replaced by E8LA6B6O3, and its structural formula is as follows: Petition 870250088241, dated 09 / 29 / 2025, pp. 123 / 159 52 / 72

[00183] 1H NMR (400 MHz, CDCI3) : δ 0.86-0.91 (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 Cõ2H99N2Ow+[M+H]+calculated 911.7, found 911.9. Example 27 Synthesis of the amino lipid E12LA6B6O3A7

[00184] The amino lipid E12LA6B6O3A7 can be prepared according to the same method as in Example 21, except by replacing the 3-(4-phenyl-piperazin-1-yl)-propionic acid in Example 21 with 1-methylpiperidine-4-carboxylic acid, and its structural formula is as follows: THE

[00185] 1H NMR (400 MHz, CDCla) : δ 0.87-0.92 (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), 5.065.11 (m, 2H). ESI-MS C58Hi09N2Oio+[M+H]+calculated 993.8, found 993.7. Example 28 Synthesis of the amino lipid E12LA6B6O3A8

[00186] The amino lipid E12LA6B6O3A8 can be prepared according to the same method as in Example 21, except by replacing the 3-(4-phenyl-piperazin-1-yl)-propionic acid in Example 21 with 1-piperidine propionic acid, and its structural formula is as follows:

[00187] 1H NMR (400 MHz, CDCla) : δ 0.85-0.92 (m, 12H), Petition 870250088241, dated 09 / 29 / 2025, pp. 124 / 159 53 / 72 1.24-1.36 (m, 44H), 1.40-1.45 (m, 6H), 1.54-1.69 (m, 12H), 2.27-2.30 (m, 8H), 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 C59HiiiN20io+[M+H]+calculated 1007.8, found 1007.9. Example 29 Synthesis of the amino lipid E12LA12B6O30A9 Step 1: Synthesis of compound E12LA12B6O30A9-1

[00188] According to the method of Example 16, E12LA12B6O30A9-1 was synthesized. Step 2: Synthesis of compound E12LA12B6O3A9

[00189] A 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) in dichloromethane was stirred at room temperature for 4 hours. TLC showed that the reaction was complete. The reaction solution was filtered, and then the filtrate was concentrated and purified using a flash column chromatography system. (dichloromethane:methanol = 50:1 to 10:1) to obtain a compound E12LA12B6O3A9-1 (0.92 g, 80%), 1H NMR (400 MHz, CDCla): δ 0.86-0.91 (m, 12H), 1.24-1.42 (m, 68H), 1.45-1.72 (m, 20H), 1.84-1.86 (m, 2H), 2.26 (s, 6H), 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 C69Hi33N20io+[M+H]+calculated 1050.0, found 1050.3. Example 30 Synthesis of the amino lipid E12LA12B6O30A10

[00190] The amino lipid E12LA12B6O30A10 can be prepared according to the same method as in Example 29, except for the substitution of 3-dimethylaminopropanol in Example 29 by 3Petition 870250088241, dated 29 / 09 / 2025, page 125 / 159 54 / 72 dimethylaminopropylamine, and its structural formula is as follows:

[00191] 1H NMR (400 MHz, CDCla) : δ 0.86-0.91 (m, 12H), 1.24-1.42 (m, 68H), 1.45-1.84 (m, 22H), 2.28 (s, 6H), 2.32-2.44 (m, 12H), 2,602.63 (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 C69H134N3O9+ [M+H]+calculated 1149.0, found 1149.0. Example 31 Synthesis of the amino lipid E12LA12B6O3A11 E12LA12B6O3A11-1 Tf2O TfOs^-χ / N E12LA12B6O3A11-2 E12LA12B6O3A11 NH3·H2O E12LA12B6O3A11-3 Step 1: Synthesis of compound E12LA12B6O3A11-1

[00192] According to the method in Example 9, E12LA6B6O3A11-1 was synthesized. Step 2: Synthesis of compound E12LA6B6O3A11-1-2

[00193] A 50 mL round-bottom flask was taken and filled with nitrogen by evacuation. The compound E12LA6B6O3A11-1 (5.18 g, 5 mmol, 1.0 eq) and anhydrous dichloromethane (20 mL) were added to the flask and the mixture was shaken at 0 °C for 10 minutes. Anhydride Petition 870250088241, dated 09 / 29 / 2025, pp. 126 / 159 55 / 72 trifluoromethanesulfonic acid (2.12 g, 7.5 mmol, 1.5 eq) was added dropwise to the mixture and the resulting product 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 solution was stirred at room temperature overnight. The reaction solution was washed once with water and a saturated sodium chloride solution, respectively, dried over anhydrous sodium sulfate, concentrated and purified using a flash column chromatography system (dichloromethane:methanol = 50:1 to 20:1) to obtain a compound E12LA6B6O3A11-2 (4.38 g, 75%), ESI-MS C64Hi2iF3NOiiS+[M+H]+ calculated 1168.9, found 1169.0. Step 3: Synthesis of compound E12LA6B6O3A11-3

[00194] E12LA6B6O3A11-2 (4.38 g, 3.75 mmol, 1.0 eq), 20 mL of tetrahydrofuran and 5 mL of ammoniacal water (25% by mass aqueous solution) were added to a 100 mL pressure flask, and the mixture was stirred at 100 °C overnight. The reaction solution was washed once with water and a saturated sodium chloride solution, respectively, dried over anhydrous sodium sulfate, concentrated and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain a compound E12LA6B6O3A11-3 (1.71 g, 44%), ESI-MS C63Hi23N2Os+[M+H]+ calculated 1035.9, found 1035.9. Step 4: Synthesis of compound E12LA6B6O3A11

[00195] A 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), 4-dimethylaminobutyric acid (0.32 g, 2.48 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 then the filtrate was concentrated and purified using a flash column chromatography system (dichloromethane:methanol = 50:1 to 10:1) to obtain a compound E12LA6B6O3A11 (1.48 g, 78%), 1H NMR (400 MHz, CDCl3): δ Petition 870250088241, dated 09 / 29 / 2025, pp. 127 / 159 56 / 72 0.85-0.91 (m, 12H), 1.24-1.41 (m, 68H), 1.45-1.82 (m, 22H), 2.27 (s, 6H), 2.322.42 (m, 12H), 2.61-2.63 (m, 2H), 3.36-3.42, (m, 2H), 3.77-3.79 (m, 2H), 4.014.04 (m, 2H), 4.23-4.26 (m, 2H), 5.04-5.10 (m, 2H). ESI-MS C69Hi34N3O9+[M+H]+calculated 1049.0, found 1049.1. Example 32 Synthesis of the amino lipid E12N1LA6B6O3 E12N1LA6B6O3-1 E12N1LA6B6O3-2 E12N1LA6B6O3-3 E12N1LA6B6O3 E12N1LA6B6O3-4 Step 1: Synthesis of compound E12N1LA6B6O3-2

[00196] A mixture of 1,2-epoxydecane (3.69 g, 20 mmol, 1.0 eq), 20 mL of aqueous ammonia (25% by mass aqueous solution), 10 mL of ethanol, and 10 mL of water were stirred at 60 °C for 16 hours. The reaction solution was centrifuged, 3 mL of ethanol and 30 mL of n-heptane were added, and the suspension was stirred at 60 °C for 4 hours. The resulting product was filtered, and the filter cake was dried at 50 °C for 10 hours to obtain a compound E12N1LA6B6O3-2 (3.46 g, 86%), ESI-MS Ci2H28NO+[M+H]+ calculated 202.2, found 202.2. Step 2: Synthesis of compound E12N1LA6B6O3-3

[00197] A solution of the 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) and 4-dimethylaminopyridine (2.10 g, Petition 870250088241, dated 09 / 29 / 2025, pp. 128 / 159 57 / 72 A solution of 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 a compound. E12N1LA6B6O3-3 (1.10 g, 32%), ESI-MS Ci8H38NO2+[M+H]+calculated 300.3, found 300.2. Step 3: Synthesis of compound E12N1LA6B6O3-4

[00198] A solution of the 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) and 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 a compound E12N1LA6B6O3-4 (1.42 g, 81%), ESI-MS C24H47BrNO3+[M+H]+calculated 476.3, found 476.3. Step 4: Synthesis of compound E12N1LA6B6O3

[00199] 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 of the reaction solution, it was purified using a flash column chromatography system (dichloromethane:methanol = 100:1 to 20:1) to obtain a compound E12N1LA6B6O3 (0.68 g, 78%).1H NMR (400 MHz, CDCl3) : δ 0.86-0.93 (m, 12H), 1.25-1.37 (m, 48H), 1.41-1.69 (m, 16H), 2.14-2.18 (m, 8H), Petition 870250088241, dated 09 / 29 / 2025, pp. 129 / 159 58 / 72 2.30-2.43 (m, 6H), 3.34-3.48 (m, 4H), 4.90-4.96 (m, 2H). ESI-MS C5iHiooN307+ [M+H]+calculated 866.8, found 866.7. Example 33 Synthesis of the amino lipid E12N1LA8B6O3

[00200] The amino lipid E12N1LA8B6O3 can be prepared according to the same method as in Example 32, except that hexanoic acid in Example 32 is replaced by caprylic acid, and its structural formula is as follows: the

[00201] 1H NMR (400 MHz, CDCla) : δ 0.86-0.93 (m, 12H), 1.25-1.37 (m, 48H), 1.42-1.70 (m, 18H), 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 C55Hio8N307+[M+H]+calculated 922.8, found 923.0. Example 34 Synthesis of the amino lipid E12N2LA6B6O3 THE CwH., / ACsHii OHH E12N2LA6B6O3-1 Tf2O THE ACsHii OTfH E12N2LA6B6O3-2 NH3-H2O 9 -------- c,oH21^ ΛX6h„ nh2h E12N2LA6B6O3-3 Br O ^OH OC10H21 H HNY 0 HNY o -C5H. Br' OC,“H21' ACsHii^γNHH THE E12N2LA6B6O3-4 E12N2LA6B6O3 Step 1: Synthesis of compound E12N2LA6B6O3-1

[00202] According to steps 1 and 2 of Example 32, E12N2LA6B6O3-1 was synthesized. Step 2: Synthesis of compound E12N2LA6B6O3-2

[00203] A 50 mL round-bottomed bottle was taken and Petition 870250088241, dated 09 / 29 / 2025, pp. 130 / 159 A 59 / 72 flask was filled with nitrogen by evacuation. The compound E12N2LA6B6O3-1-1 (1.50 g, 5 mmol, 1.0 eq) and anhydrous dichloromethane (20 mL) were added to the flask. The mixture was stirred at 0 °C for 10 minutes. Trifluoromethanesulfonic anhydride (2.12 g, 7.5 mmol, 1.5 eq) was added dropwise to the mixture and the resulting 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 solution was stirred at room temperature overnight. The reaction solution was washed once with water and saturated sodium chloride solution, respectively, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system. (dichloromethane:methanol = 50:1 to 20:1) to obtain a compound E12N2LA6B6O3-2 (1.51 g, 70%), ESI-MS CwH3?F3NO4S+[M+H]+calculated 432.2, found 432.2. Step 3: Synthesis of compound E12N2LA6B6O3-3

[00204] E12N2LA6B6O3-2 (1.51 g, 3.50 mmol, 1.0 eq), 20 mL of tetrahydrofuran and 5 mL of ammoniacal water (25% by mass aqueous solution) were added to a 100 mL pressure flask, and the mixture was stirred at 100 °C overnight. The reaction solution was washed once with water and a saturated sodium chloride solution, respectively, dried over anhydrous sodium sulfate, concentrated and purified using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain the compound E12N2LA6B6O3-3 (0.47 g, 45%), ESI-MS CisH39N2O+[M+H]+ calculated 299.3, found 299.3. Step 4: Synthesis of compound E12N2LA6B6O3-4

[00205] A solution of the 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 Petition 870250088241, dated 09 / 29 / 2025, pp. 131 / 159 60 / 72 was complete. The reaction solution was filtered, and then the filtrate was concentrated and purified using a flash column chromatography system (n-heptane:ethyl acetate = 50:1 to 1:1) to obtain a compound. E12N2LA6B6O3-4 (1.48 g, 88%), ESI-MS C24H48BrN2O2+[M+H]+calculated 475.3, found 475.3. Step 5: Synthesis of compound E12N2LA6B6O3

[00206] A solution of the 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), sodium iodide (0.069 g, 0.46 mmol, 1.0 eq) in acetonitrile (10 mL) was stirred at 75 °C overnight. After concentration of the reaction solution, it was purified using a flash column chromatography system (dichloromethane:methanol = 100:1 to 20:1) to obtain a compound E12N2LA6B6O3 (0.97 g, 81%). 1H NMR (400 MHz, CDC1a): δ 0.86-0.92 (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 C5iHi02NõO5+[M+H]+calculated 864.8, found 864.7. Example 35 Synthesis of the amino lipid E12S1LA6B6O3 Step 1: Synthesis of compound E12S1LA6B6O3-2

[00207] 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 1,2Petition 870250088241, dated 09 / 29 / 2025, page 132 / 159 61 / 72 epoxidedecane (884 mg, 4.8 mmol, 1.2 eq) were sequentially added to a 25 mL reaction tube. The reaction solution was stirred at room temperature overnight. After the reaction was complete, the reaction solution was concentrated and purified using a flash column chromatography system. (n-heptane:ethyl acetate = 50:1 to 10:1) to obtain a compound E12S1LA6B6O3-2 (1.2 g, 90.0%). ESI-MS CisH37O3+[M+H]+calculated 301.3, found 301.3. Step 2: Synthesis of compound E12S1LA6B6O3-3

[00208] 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 a saturated sodium chloride solution, respectively, 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 added and the mixture was 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 a compound E12S1LA6B6O3-3 (0.69g, 61%), ESI-MS CisH37O2S+[M+H]+calculated 317.3, found 317.2. Step 3: Synthesis of compound E12S1LA6B6O3-4

[00209] 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 to a 25 mL reaction tube. The mixture was stirred at room temperature for 1 hour. The reaction solution was extracted three times with ethyl acetate, washed once. Petition 870250088241, dated 09 / 29 / 2025, pp. 133 / 159 62 / 72 with saturated saline solution, dried over anhydrous sodium sulfate, concentrated and purified using a flash column chromatography system (nheptane:ethyl acetate = 100:1 to 10:1) to obtain a compound E12S1LA6B6O3-4 (0.70g, 68%), ESI-MS C21H42BrO2S2+[M+H]+calculated 469.2, found 469.2. Step 4: Synthesis of compound E12S1LA6B6O3

[00210] 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 of the reaction solution, it was purified using a flash column chromatography system (dichloromethane:methanol = 100:1 to 20:1) to obtain compound E12S1LA6B6O3 (1.09 g, 80%). 1H NMR (400 MHz, CDC1a): δ 0.86-0.91 (m, 12H), 1.25-1.35 (m, 40H), 1.41-1.54 (m, 12H), 1.59-1.67 (m, 10H), 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 C49H98NOõS4+[M+H]+calculated 908.6, found 908.7. Preparation of lipid nanoparticles Example 36 Preparation of lipid nanoparticles

[00211] The aminolipid compound of the present invention (or DLin-MC3 or SM-102 (both acquired from AVT (Shanghai) Pharmaceutical Technology Co., Ltd.), phospholipid bicarbonate (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 citric acid buffer (50 mM, pH = 4.0) dissolved with Luc-mRNA (TriLink) were mixed on a microfluidic chip at a volume ratio of 1:3 using a microfluidic preparation system to prepare a crude solution of lipid nanoparticles, which was then dialyzed in 1X PBS at 4 °C for Petition 870250088241, dated 09 / 29 / 2025, pp. 134 / 159 63 / 72 h using a dialysis cassette (Fisher, MWCO 20,000). The product was filtered through a 0.22 μm microporous membrane before use. The mass ratio of the aminolipid compound to the luciferase mRNA (Luc mRNA) was approximately 40:1.

[00212] The lipid nanoparticles obtained were characterized and the results are presented in Table 1.

[00213] The particle size and PDI (polydispersity index) of the prepared lipid nanoparticles were measured using the Nano-ZSZEN3600 (Malvern). 20 μL of lipid nanoparticle (LNP) solution were taken for particle size measurement and cycled three times with each cycle for 30 s.

[00214] Encapsulation efficiency was determined according to the standard procedure of the Quant-iT RiboGreen RNA kit. Table 1: Characterization data of LNPs prepared using Representative aminolipid compounds Item Amino lipid No. Average particle size (nm) PD I Encapsulation efficiency (%) LNP-1 E8LA8B6O3 84 0.0 7 90.03 LNP-2 E8LA10B6O3 73 0.0 9 93.95 LNP-3 E8LA12B6O3 72 0.0 9 95.67 LNP-4 E10LA6B6O3 92 0.0 7 95.41 LNP-5 E10LA8B6O3 71 0.1 1 92.23 LNP-6 E10LA10B6O3 70 0.0 6 95.90 LNP-7 E12LA6B6O3 75 0.0 8 98.26 LNP-8 E12LA8B6O3 73 0.1 0 94.90 LNP-9 E12LA12B6O3 73 0.0 9 97.5% LNP-10 E8CA5B6O3 69 0.0 6 95.8% LNP-12 E12LA6B6O9 95 0.1 93.2% Petition 870250088241, dated 09 / 29 / 2025, pp. 135 / 159 64 / 72 Item Amino lipid No. Average particle size (nm) PD I Encapsulation efficiency (%) 1 LNP-13 E12LA6B6O10 102 0.1 0 93.0% LNP-14 E12LA6B6O12 97 0.0 5 97.6% LNP-15 E12LA6B6O13 83 0.0 1 94.4% LNP-16 E12LA6B6O15 90 0.1 2 95.2% LNP-17 E12LA12B6O30 127 0.0 3 98.2% LNP-18 E12LA12B6O31 130 0.0 4 98.4% LNP-19 K3LA6B6O3 129 0.0 8 98.8% LNP-20 K4LA6B6O3 120 0.0 6 98.9% LNP-21 K5LA6B6O3 106 0.0 3 96.2% LNP-23 E12LA6B6O3A3 53 0.0 7 94.6% LNP-24 E12LA6B6O3A4 80 0.0 3 90.7% LNP-25 E12LA6B6O3A5 117 0.0 8 97.8% LNP-26 E12LA6B6O3A6 103 0.0 7 98.5% LNP-27 E8LA6B6O3A6 108 0.0 7 97.6% LNP-28 E12LA6B6O3A7 110 0.0 5 96.5% LNP-29 E12LA6B6O3A8 124 0.0 6 95.3% LNP-30 E12LA12B6O30 A9 121 0.1 4 92.4% LNP-31 E12LA12B6O30 A10 117 0.0 8 91.8% LNP-32 E12LA12B6O3A 11 132 0.0 9 95.6% LNP-33 E12N2LA6B6O3 168 0.0 6 90.3% LNP-1 comparative DLin-MC3 72 0.1 2 89.74 LNP-2 comparative SM-102 79 0.1 0 94.32

[00215] Based on the results above, it can be observed that Petition 870250088241, dated 09 / 29 / 2025, pp. 136 / 159 65 / 72 all lipid nanoparticles provided by the present invention have a superior encapsulation efficiency to that of DLin-MC3, and most lipid nanoparticles have a higher encapsulation efficiency than SM-102. Example 37 Transfection of lipid nanoparticles prepared from amino-lipid compounds into primary BMDC cells

[00216] Preparation of animals: female mice C57BL / 6 mice, 6 weeks old and weighing approximately 20 g, were selected and reared in a sun-protected feeding room (SPF). Animal experiments were conducted in strict accordance with National Institutes of Health guidelines and animal ethics requirements.

[00217] Cell acquisition: C57BL / 6 mice were sacrificed by cervical dislocation and immersed in 75% alcohol for 5 minutes for disinfection. The thigh and tibia of the mice were dissected and the attached muscles were removed to expose the bone. Bone marrow in the tibia was blown out using a 1 mL syringe filled with PBS. The obtained bone marrow was dispersed by blowing and filtered through a 50 μm filter mesh to remove impurities. Red blood cell lysis solution (3-4 mL) was added to the filtrate and left to stand for 5 minutes. Then, the supernatant was removed by centrifugation at 800 g for 5 minutes. The obtained cells were placed in culture medium 1640 (containing 10% fetal bovine serum, 20 ng / mL GMCSF, 10 ng / mL IL-4) for resuspension and inoculated into 6-well plates at a density of 100,000 cells / mL of culture medium.The plates were placed in a cell culture incubator containing 5% CO2 at 37 °C, and the medium was changed by half every 2 days. On the seventh day, suspended cells and loosely adherent cells were collected and inoculated onto completely blank 96-well ELISA plates at a density of 20,000 cells per well, with a culture medium volume of... Petition 870250088241, dated 09 / 29 / 2025, pp. 137 / 159 66 / 72 100 pL.

[00218] Cell transfection: Lipid nanoparticles encapsulating luciferase mRNA were added to a completely white 96-well ELISA plate with scattered primary cells, and the volume of mRNA lipid nanoparticles added to each well was controlled to be 10 pL. The plate was then placed in an incubator with a 5% CO2 concentration at 37 °C for 12 hours.

[00219] Transfection efficiency detection: 20 pL of ONE-Glo™ Luciferase substrate were added to each well of a completely blank 96-well ELISA plate, and the assay was performed using a multifunctional microplate reader (Biorek SynergyHI) after 1 minute. The intensity of expression of Luc mRNA transfected in BMDCs by lipid nanoparticles (LNPs) composed of representative aminolipid compounds is shown in Table 2, with DLin-MC3 and SM0102 as controls. Table 2: Expression intensity of transfection of representative aminolipid compounds in BMDcs Item Amino Lipid No. Fluorescence Intensity LNP-1 E8LA8B6O3 1.3E+05 LNP-2 E8LA10B6O3 5.2E+04 LNP-4 E10LA6B6O3 1.5E+05 LNP-6 E10LA10B6O3 6.9E+04 LNP-7 E12LA6B6O3 1.8E+05 LNP-8 E12LA8B6O3 7.6E+04 LNP-9 E12LA12B6O3 4.1E+05 LNP-10 E8CA5B6O3 2.3E+05 LNP-11 E12LA6B6O3 5.4E+05 Petition 870250088241, dated 09 / 29 / 2025, pp. 138 / 159 67 / 72 Item Amino Lipid No. Fluorescence Intensity LNP-12 E12LA6B6O9 2.2 E+05 LNP-14 E12LA6B6O12 4.9 E+04 LNP-18 E12LA12B6O31 9.6 E+04 LNP-19 K3LA6B6O3 5.4E+05 LNP-20 K4LA6B6O3 4.9 E+05 LNP-21 K5LA6B6O3 4.8E+05 LNP-22 E12LA6B6O3A2 4.0E+05 LNP-25 E12LA6B6O3A5 1.5 E+05 LNP-27 E8LA6B6O3A6 2.8E+05 LNP-28 E12LA6B6O3A7 7.3 E+04 LNP-29 E12LA6B6O3A8 1.9 E+05 LNP-30 E12LA12B6O30A9 2.7 E+05 LNP-31 E12LA12B6O30A10 4.1E+05 LNP-32 E12LA12B6O3A11 1.4E+05 LNP-1 comparative DLin-MC3 4.1E+04 LNP-2 compared SM-102 1.0E+05

[00220] It can be observed that the expression intensities of the amino lipids provided by the present invention are significantly better than those of DLin-MC3, and the expression intensities of some of the amino lipids are better than those of SM-102. Example 38 Evaluation of the in vivo delivery performance of LIPID nanoparticles for LUCIFERASE mRNA

[00221] Preparation of lipid nanoparticles: same as Petition 870250088241, dated 09 / 29 / 2025, pp. 139 / 159 68 / 72 Example 36. Experiments with animals

[00222] Preparation of animals: female mice C57BL / 6 mice, 6 weeks old and weighing approximately 20 g, were selected and reared in a sun-protected feeding room (SPF). Animal experiments were conducted strictly in accordance with National Institutes of Health guidelines and animal ethics requirements.

[00223] In vivo administration: Five C57BL / 6 mice were randomly selected from each group and injected with a lipid nanoparticle solution at a dose of 0.5 mg / kg mRNA by intramuscular injection. Twelve hours later, 200 μL of D-luciferin potassium salt at 10 mg / mL were injected into the peritoneal cavity of each mouse. Five minutes later, the mice were placed under an in vivo imaging system (IVIS-200, Xenogen) to observe the total fluorescence intensity of each mouse and take photographs for record-keeping. The mRNA expression intensity of Luc administered by intramuscular injection of LNPs composed of representative aminolipid compounds is shown in Table 3, with DLinMC3 and SM-102 as controls. Table 3: Expression intensity of Luc mRNA administered by Intramuscular injection of LNPs composed of representative amino lipids. No. Amino lipid No. Fluorescence intensity 1 E8LA8B6O3 7.7E+08 2 E10LA6B6O3 1.4E+09 3 E10LA10B6O3 1.2E+09 4 E12LA6B6O3 2.2E+09 5 E12LA8B6O3 8.2E+08 6 E8LA10B6O3 1.6E+09 Petition 870250088241, dated 09 / 29 / 2025, pages 140 / 159 69 / 72 No. Amino lipid No. Fluorescence intensity 7 E12LA6B6O10 1.6E+09 8 E10LA8B6O3 1.8E+09 9 E12LA6B6O3A6 1.5E+09 10 K3LA6B6O3 2.0E+09 11 DLin-MC3 7.6E+08 12 SM-102 1.4E+09 Example 39 Evaluation of immunological and tumor therapeutic effects in LIPID NANOPARTICLES PREPARED FROM AMINOLIPID COMPOUNDS

[00224] Preparation method: The aminolipid 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. The resulting ethanol solution and citric acid buffer (50 mM, pH = 4.0) dissolved with OVA mRNA were mixed on a microfluidic chip in a 1:3 ratio using a microfluidic preparation system for lipid nanoparticle preparation, and then dialyzed in 1X PBS at 4 °C for 6 h using a dialysis cassette (Fisher, MWCO 20,000) and filtered with a 0.22 μm microporous filter membrane before use. The mass ratio of the aminolipid compound to ovalbumin mRNA (OVA mRNA) was approximately 40:1.

[00225] Preparation of animals: Female mice C57BL / 6 mice aged 5 to 6 weeks, weighing approximately 18 to 20 g, were selected and kept in a feeding room with FPS. Animal experiments were conducted strictly in accordance with National Institutes of Health guidelines and animal ethics requirements.

[00226] In vivo administration: B16-OVA melanoma cells (1.5 x 105) were injected subcutaneously into the lateral thigh of mice. Vaccination was initiated when the tumor size reached 50 mm3. Petition 870250088241, dated 09 / 29 / 2025, pp. 141 / 159 70 / 72 (approximately 6 or 7 days after tumor inoculation). Animals were immunized twice by intramuscular injection of an LNP formulation containing 1 μg of OVA-mRNA, with a 7-day interval between the second and first injections. Tumor growth was measured three times a week using a digital caliper and calculated as 0.5 χ length χ width χ width. Mice were euthanized when the tumor volume reached 1500 mm3. The tumor growth rates of the E12LA6B6O3 group and the E10LA6B6O3 group were significantly slower than those of the MC3 group (as shown in Figure 2), and 100% (E12LA6B6O3 group) and 80% (E10LA6B6O3 group) of mice achieved complete remission, which was significantly better than the MC3 group (as shown in Figure 3). Example 40 Evaluation of in vivo delivery performance of LUCIFERASE mRNA in LNPs composed of different proportions of phospholipid lipids

[00227] Preparation of lipid nanoparticles: the ionizable lipid content (E12LA6B6O3) was maintained at 42.5%, the PEGylated lipid content DMG-PEG2000 was maintained at 1.5%, and the phospholipid-to-lipid ratio was adjusted to 15, 12.5, 10, 7.5, 5.0, 2.5, and 0. The increased or decreased molar ratio during the adjustment of the phospholipid-to-lipid molar ratio was applied to the cholesterol ratio to verify its effect on the quality control assessment of lipid nanoparticles and on the in vivo delivery performance of luciferase mRNA. The groups were numbered B1 to B7, and the specific proportions are shown in Table 4. ALC-0315 and SM-102 were used as positive controls. Table 4: Proportions of components under different phospholipids No. Ionizable lipid: phospholipid lipid: cholesterol: PEGylated lipid B1 40: 0: 58.5: 1.5 B2 40: 2.5: 56: 1.5 B3 40: 5: 53.5: 1.5 B4 40: 7.5: 51: 1.5 Petition 870250088241, dated 09 / 29 / 2025, pages 142 / 159 71 / 72 No. Ionizable lipid: phospholipid lipid: cholesterol: PEGylated lipid B5 40: 10: 48.5: 1.5 B6 40: 12.5: 46: 1.5 B7 40: 15: 43.5: 1.5

[00228] E12LA6B6O3, phospholipid bicarbonate (DSPC), cholesterol, and PEGylated lipid DMG-PEG2000 were mixed and dissolved in anhydrous ethanol according to the molar ratio in Table 4. The resulting ethanol solution and citric acid buffer (50 mM, pH = 4.0) dissolved with Luc-mRNA (TriLink) were mixed on a microfluidic chip at a volume ratio of 1:3 using a microfluidic preparation system to prepare a crude solution of lipid nanoparticles, which was then dialyzed in 1X PBS at 4 °C for 6 h using a dialysis cassette (Fisher, MWCO 20000). The product was filtered through a 0.22 μm microporous filter membrane before use. The base molar ratio of the amino lipid compound to the luciferase mRNA (Luc mRNA) is approximately 6.5:1. The quality control of the lipid nanoparticles was tested using a Malvern particle size analyzer and a microplate reader, and SM-102 and ALC-0315 (both acquired from AVT (Shanghai) Pharmaceutical Technology Co., Ltd.) were added as a control group.

[00229] Animal preparation: Female Balb / C mice, one week old, weighing approximately 20 g, were selected and reared in a feeding room with FPS. Animal experiments were conducted in strict accordance with National Institutes of Health guidelines and animal ethics requirements.

[00230] In vivo administration: Three Balb / C mice were randomly selected from each group and injected with a lipid nanoparticle solution at a dose of 0.1 mg / kg mRNA via tail vein injection. After 6 hours, 150 μL of D-luciferin potassium salt at 15 mg / mL was injected into the peritoneal cavity of each mouse. After 10 minutes, the Petition 870250088241, dated 09 / 29 / 2025, pp. 143 / 159 72 / 72 mice were placed under the in vivo imaging system (IVIS Spectrum) to observe the total fluorescence intensity of each mouse and take photos for record-keeping. The mRNA expression intensity of Luc administered by injection into the tail vein of LNPs with different DSPC contents is shown in Table 5. Table 5: Quality control data and intensity of expression of LNPs with different phospholipid lipid contents No. % of phospholipid content Particle size (nm) PDI Encapsulation efficiency (%) Fluorescence intensity B1 0 115.2 0.09 93.5 2.86E+11 B2 2.5 106.7 0.09 95.9 2.09E+11 B3 5 97.0 0.11 96.9 2.23E+11 B4 7.5 115.8 0.12 95.6 7.47E+10 B5 10 101.6 0.10 95.9 4.09E+10 B6 12.5 98.1 0.08 95.8 2.33E+10 B7 15 93.9 0.06 96.0 1.34E+10 ALC-0315 10 71.3 0.08 94.1 1.26E+10 SM-102 10 91.9 0.09 96.4 9.86E+9

[00231] Finally, it should be noted that the above examples serve only to illustrate the technical solutions of the present invention, and not to limit it. Although the present invention has been described in detail with reference to the examples mentioned above, those skilled in the art should understand that they may still modify the technical solutions described in the examples mentioned above or make equivalent substitutions for some of the technical features contained therein. However, such modifications or substitutions do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the examples of the present invention. Petition 870250088241, dated 09 / 29 / 2025, pages 144 / 159

Claims

1 / 12 Claims 1. AMINO LIPID, characterized by having the structure as shown in formula (I), or an isomer, a pharmaceutically acceptable salt, a prodrug or a solvent thereof: XG m5 / L2. ,-N,. / L3„ h R h / h M2 M3 R2 (D where 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 cycloalkyl comprising at least one heteroatom, wherein the substituteable carbon atoms or heteroatoms in the cycloalkyl are unsubstituted or substituted by one or more hydroxyl groups, C1-C4 alkyl, C2-C4 alkenyl, C3-C8 cycloalkyl, or Cs-Cs cycloalkenyl; M1, M2, M3 and M4 are identical or different from each other, and each is independently selected from C1-C24 alkylene, C3-C24 cycloalkylene, C2-C24 alkenylene or C3-C24 cycloalkenylene; R1 and R2 are identical or different from each other, and each is selected independently from H, C1-C24 alkyl, C3-C24 cycloalkyl, C2-C24 alkenyl, or C3-C24 cycloalkenyl;L1, L2, L3 and L4 are identical or different from SS-; R, R' and R" are identical or different from each other, and each is selected independently from H, C1-C10 alkyl, C3-C10 cycloalkyl, C3-C10 alkenyl, C3-C10 cycloalkenyl, C1-C10 alkyl terminated with a tertiary amino group, C3-C10 cycloalkyl terminated with a tertiary amino group, Petition 870250088241, dated 09 / 29 / 2025, page. C3-C8 cycloalkenyl;The M5 groups are selected independently from a single linkage, C1-C16 alkylene, C2-C16 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene.

2. 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 cycloalkyl comprising at least one heteroatom, wherein the substituteable carbon atoms or heteroatoms in the cycloalkyl are unsubstituted or substituted by one or more C1-C4 alkyl, C2-C4 alkenyl, C3-C8 cycloalkyl or C3-C8 cycloalkenyl groups; R, R' and R'' are identical or different from each other, and each is independently selected from H, C1-C10 alkyl, C3-C10 cycloalkyl, C3-C10 alkenyl or C3-C10 cycloalkenyl, or cycloalkyl comprising at least one heteroatom; the cycloalkyl is unsubstituted. or substituted by one or more C1-C4 alkyl, C2-C4 alkenyl, C3-C8 cycloalkyl, or C3-C8 cycloalkenyl groups.

3. AMINO LIPID, according to any one of claims 1 to 2, characterized in that, in formula (I), G being selected from H, OR, CN, -C(=O)OR', -OC(=O)R', -C(=O)NR'R”, -NR'C(=O)R”, NR'R”, or cycloalkyl comprising at least one heteroatom; wherein the heteroatom is O or N; and the cycloalkyl is unsubstituted or substituted by one or more C1-C4 alkyl, C3-C8 cycloalkyl or hydroxyl groups; R, R' and R'' are identical or different from each other, and each is independently selected from H, C1-C8 alkyl, C3-C8 cycloalkyl, C3-C8 alkenyl, or C3-C8 cycloalkenyl, C1-C10 alkyl terminated with an amino group. Petition 870250088241, of 29 / 09 / 2025, page.

4. AMINO LIPID, according to claim 3, characterized in that, in formula (I), G is selected from H, OR or NR'R”, where R' and R'' are identical or different from each other, and each is selected independently from H or C1-C4 alkyl; or G is selected from substituted or unsubstituted 5-membered oxacycloalkyl, substituted or unsubstituted 5-membered azacycloalkyl, substituted or unsubstituted 6-membered azacycloalkyl, substituted or unsubstituted 6-membered diazocycloalkyl, or substituted or unsubstituted 6-membered azaoxacycloalkyl.

5. AMINO LIPID, according to claim 4, characterized in that, in formula (I), when G is selected from 6-membered substituted diazocycloalkyl, the substituent being positioned on the nitrogen atom that is not bonded to M5.

6. AMINO LIPID, according to claim 3, characterized in that, in formula (I), M5 is selected from a single bond, C2-C16 alkylene, C2-C16 alkenylene, C4-C8 cycloalkylene or C3-C8 cycloalkenylene.

7. AMINO LIPID, according to claim 6, characterized in that, in formula (I), M5 is selected from a single bond, C2-C16 alkylene or C4-C6 cycloalkylene.

8. AMINO LIPID, according to claim 7, / M5. GN characterized in that, in formula (I), the bond formed by M5e G being selected from A1 to A38: Petition 870250088241, dated 29 / 09 / 2025, pp. 147 / 159 4 / 12 9. AMINO LIPID, according to claim 7, M5 g 'n^ characterized by, in formula (I), the ο formed by the linkage of Ms and G being a selected from A39 to A52: Petition 870250088241, dated 29 / 09 / 2025, p. 148 / 159 5 / 12 10. AMINO LIPID, according to claim 8, characterized by being selected from A1-A18, A22-A24 and A28-A38.

11. AMINO LIPID, according to claim 10, m5 G characterized to be selected from A15, A16, A17, A23, A29, A30, A33, A37, A38 and A42.

12. AMINO LIPID, according to any one of claims 1 to 11, characterized in that, in formula (I), Li, L2, L and L4 are identical or different and independently selected from 0(=0)O-, -00(=0)-, -C(=O)NR-, -NRC(=O)- or -SS-; Petition 870250088241, dated 09 / 29 / 2025, p. 149 / 159 6 / 12 preferably, by Li and L4 being equal and being -C(=O)O- or -OC(=O)-.

13. AMINO LIPID, according to claim 12, characterized in that, when Li, L2, L3 and L4 are selected independently of -C(=O)NR- or -NRC(=O)-, R is selected independently of H or C1-C10 alkyl.

14. AMINO LIPID, according to any one of claims 1 to 13, characterized in that, in formula (I), M1, M2, M3 and M4 are identical or different, M1 and M4 being each independently selected from branched-chain C4-C22 alkylene, branched-chain C4-C22 cycloalkylene, branched-chain C4-C22 alkenylene or branched-chain C4-C22 cycloalkenylene, and M2 and M3 being each independently selected from C4-C22 alkylene, C4-C22 cycloalkylene, C4-C22 alkenylene or C4-C22 cycloalkenylene.

15. AMINO LIPID, according to claim 14, characterized in that, in formula (I), M2 is the same as M3 and is a C4-C22 alkylene; and / or M1 is the same as M4 and is a branched-chain C4-C22 alkylene.

16. AMINO LIPID, according to any one of claims 2 to 15, characterized in that, in formula (I), R1 and R2 are identical or different, and each is independently selected from C4-C22 alkyl or C4-C22 alkenyl.

17. AMINO LIPID, according to claim 16, characterized in that, in formula (I), the fragment R1-L1-M1-L2-M2- is R1C(=O)O-M1-OC(=O)-M2-, the fragment R2-L4-M4-L3-M3- is R2-C(=O)O-M4OC(=O)-M3-.

18. AMINO LIPID, according to claim 17, Petition 870250088241, dated 09 / 29 / 2025, page. 150 / 159 7 / 12 characterized by the structure as shown in formula (I) being one selected from the following structures: the E8LA6B6O3 E8LA8B6O3 E8LA10B6O3 E8LA12B6O3 E1QLA6B6O3 O E10LA8B6O3 E10LA10B6O3 O E10LA12B6O3 O E12LA6B6O3 E12LA8B6O3 E12LA10B6O3 E8CA5B6O3 E10CA5B6O3 E12LA6B4O2 O E12LA6B5O2 E12LA6B6O2 E12LA6B7O2 O E12LA6B8O2 Ò C10H21 E12LA6B5O3 O C10H21 E12LA6B7O3 E12LA6B4O3 Ç10H21 O E12LA6B4O4 E12LA6B5O4 E12LA6B8O3 HO O C10H21 E12LA6B7O4 O E12LA6B8O4 E12LA6B6O4 Petition 870250088241, dated 09 / 29 / 2025, page. 151 / 159 8 / 12 E12LA12B6O3 E12LA6B6O3A3 C6Hi3 C5H11 O Ç10H21 °Α / ογ°5Ηΐ1 C6Hi3 0514^ E12LA6B6O3A4 E8CA5B6O3 C5H11 E12LA6B6O3 E12LA6B6O3A5 O Ç10H21 CsHii ÇlOH21 Ο E12LA6B6O9 E12LA6B6O10 E12LA6B6O3A6 E12LA6B6O3A7 E12LA6B6O12 E12LA6B6O13 Ç10H21 οΑ_ / ΟγΟ5Η11 Ο Ç10H21 E12LA6B6O3A8 Petition 870250088241, dated 09 / 29 / 2025, page.152 / 159 9 / 12 E12LA6B6O15 o E12LA12B6O30A9 E12LA12B6O30A10 NC E12LA12B6O30 o o E12LA12B6O31 E12LA12B6O3A11 o K3LA6B6O3 o E12N1LA6B6O3 o K4LA6B6O3 E12N1LA8B6O3 K5LA6B6O3 o E12LA6B6O3A2 E12N2LA6B6O3 E12S1LA6B6O3.

19. METHOD FOR PREPARING AMINO LIPID, as defined in any one of claims 1 to 18, characterized by Petition 870250088241, dated 09 / 29 / 2025, page 153 / 159 10 / 12 comprising the following steps: S1: subjecting an epoxide compound and a carboxylic acid to a ring-opening reaction to prepare RMi-Mi-OH as intermediate 1; S2: subjecting intermediate 1 and a carboxylic acid compound as a starting material to a condensation reaction in the presence of a condensing agent to give an R1-L1-M1-L2-M2-leaving group as intermediate 2; and S3: subjecting intermediate 2 and an amino compound as a starting material to one or more substitution reactions to obtain the target product; or S1': perform an oxidation reaction of a diol subjected to TBS protection and subject the oxidation product and a Grignard reagent to an addition reaction, to provide HO-Mi-OTBS as intermediate 1';S2': subject intermediate 1' and a carboxylic acid compound as a starting material to a condensation reaction in the presence of a condensing agent, to give the TBSO-M1-L2-M2-leaving group as intermediate 2'; S3': remove the TBS protecting group from the intermediate 2' TBSO-MiL2-M2-leaving group, allowing the resulting intermediate to react with a carboxylic acid to generate the R1-L1-M1-L2-M2-leaving group as intermediate 2; and S4': subject intermediate 2 and an amino compound as a starting material to one or more substitution reactions, to obtain the target product.

20. LIPID NANOPARTICLE, characterized by comprising the amino lipid, as defined in any one of claims 1 to 18.

21. NANOPARTICLE, according to claim 20, characterized in that the lipid nanoparticle further comprises a steroid, a neutral lipid and / or a lipid conjugated to the polymer; wherein the lipid conjugated to the polymer has a chemical formula of PYL, in which P is a hydrophilic polymeric fraction, Y is an optional ligand and L is a lipid fraction.

22. NANOPARTICLE, according to claim 21, characterized in that the steroid is cholesterol; and / or in that the neutral lipid is a phospholipid; and / or in that the lipid conjugated to the polymer is PEGylated lipids.

23. NANOPARTICLE, according to claim 21, characterized by the molar ratio of the amino lipid, steroid, neutral lipid and lipid conjugated to the polymer in the lipid nanoparticles being 3070:30-65:0-30:0.2-5.

24. PHARMACEUTICAL COMPOSITION, characterized by comprising the lipid nanoparticle, as defined in any one of claims 20 to 23, and a pharmaceutically acceptable carrier.

25. METHOD FOR TREATING OR PREVENTING INFECTIOUS DISEASES, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases, characterized by using the amino lipid, as defined in any one of claims 1 to 18, the lipid nanoparticle, as defined in any one of claims 20 to 23, or the pharmaceutical composition, as defined in claim 24.

26. METHOD, according to claim 25, characterized by the cancer including lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, leukemia and prostate cancer.

27. METHOD FOR GENE THERAPY, gene vaccination, antisense therapy, nucleic acid delivery or treatment by means of RNA interference, characterized by using the amino lipid, as defined in any of claims 1 to 18, the lipid nanoparticle, as defined in any of claims 20 to 23, or the pharmaceutical composition, as defined in claim 24.

28. USE OF AMINO LIPID, as defined in any one of claims 1 to 18, or of the lipid nanoparticle, as defined in any one of claims 20 to 23, or of the pharmaceutical composition, as defined in claim 24, characterized by being in the preparation of a medicament to treat or prevent infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases.

29. USE, according to claim 28, characterized by the cancer including lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, leukemia and prostate cancer.

30. USE OF AMINO LIPID, as defined in any one of claims 1 to 18, or of lipid nanoparticle, as defined in any one of claims 20 to 23, or of pharmaceutical composition, as defined in claim 24, characterized in being in the preparation of a medicament for gene therapy, gene vaccination, antisense therapy, nucleic acid delivery, or RNA interference treatment.

31. METHOD FOR ADMINISTERING A MEDICINE TO A SUBJECT, characterized by comprising administering to the subject a medicine formulated in lipid nanoparticles, as defined in any one of claims 20 to 23.

32. USE OF AMINO LIPID, as defined in any of claims 1 to 18, characterized by being for treating or preventing infectious diseases, cancer, genetic diseases, allergies, toxicity, and autoimmune diseases. Petition 870250088241, dated 09 / 29 / 2025, pp. 156 / 159

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