A lipid compound for enhancing the level of immune response and a preparation method and application thereof

Abstract

The application discloses a lipid compound for enhancing the level of immune response, and a preparation method and application thereof. The lipid compound of the application has a symmetrical tail carbon chain, and includes a compound shown in formula IX. The application also provides a lipid nanoparticle, a drug delivery system and a pharmaceutical composition containing the lipid compound. The lipid compound of the application can promote cell mitochondrial activation, enhance the level of cell metabolism, thereby significantly improving the immune response, and has a wide application prospect in the fields of mRNA vaccines, protein vaccines and CAR-T cell therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a lipid compound for enhancing immune response levels, its preparation method, and its application. Background Technology

[0002] Messenger RNA (mRNA)-based therapeutic and preventative strategies, such as mRNA vaccines and chimeric antigen receptor T-cell (CAR-T) therapy, have shown great potential in the treatment of infectious diseases, cancer, and genetic disorders. The core of these technologies lies in the efficient and safe delivery of nucleic acid molecules encoding specific antigens or CARs to target cells, such as dendritic cells or T cells. Lipid nanoparticles (LNPs), as one of the most advanced and mature non-viral delivery systems currently available, play a crucial role in protecting nucleic acids from degradation, promoting cellular uptake, and endosome escape.

[0003] In the components of LNPs (Liquid Nucleic Acids), ionizable lipids are the core functional components. Their mechanism of action involves carrying a positive charge in an acidic pH environment, thereby condensing negatively charged nucleic acid molecules (such as mRNA) and promoting endosome escape. Currently, the most commonly used ionizable lipids, such as ALC-0315 and SM-102, employ an asymmetric tail-chain structure design. However, this design primarily focuses on achieving efficient encapsulation of nucleic acids through Coulomb forces and leveraging their ionization properties to protonate in the acidic environment of the endosome, thereby disrupting the endosome membrane and releasing the nucleic acids. In other words, existing ionizable lipid designs mainly focus on improving delivery efficiency itself, and their function is generally limited to serving as a structural component. These lipids lack the ability to actively regulate the functional state of immune cells, making it difficult to synergistically enhance the body's immune response against specific antigens while delivering antigen information.

[0004] Therefore, there is an urgent need for a lipid compound and its preparation method to enhance the level of immune response. Summary of the Invention

[0005] To address at least some of the technical problems in the prior art, the present invention provides a lipid compound for enhancing immune response levels, a method for its preparation, and its applications. Specifically, the present invention includes the following:

[0006] In a first aspect, the present invention provides a lipid compound for enhancing immune response levels, the lipid compound comprising a main chain and tail branches located at both ends of the main chain, wherein the main chain and the tail branches are covalently linked by carbonyl groups, wherein the main chain has a bilaterally symmetrical structure along its center, and the tail branches include a first branch and a second branch having the same structure, the first branch and the second branch being carbon chains having 6-16 carbon atoms, the structure of the main chain being as shown in Formula Ia, and the structure of the carbonyl groups being as shown in Formula Ib. Formula Ia, Formula Ib; Among them, R1 and R2 are the same and are carbon chains with 5-8 carbon atoms, R3 is a carbon chain with 4-6 carbon atoms and has hydroxyl groups at the end; R4 and R5 are independently selected from carbon atoms, nitrogen atoms, oxygen atoms, sulfur atoms, methylene, methine or imino.

[0007] In some embodiments, the lipid compound for enhancing immune response levels according to the present invention is selected from compounds represented by Formula IX: Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, Equation VII, Formula VIII, Formula IX, Formula X.

[0008] A second aspect of this invention provides a method for preparing the lipid compound represented by Formula IX, wherein... Compound I is prepared by the following method: (I-1) React a carboxylic acid compound and a bromoalkane in an organic solvent at 10-30°C for 1-5 h to obtain intermediate I-1; (I-2) In the presence of a catalyst, the intermediate I-1 and the amino alcohol are reacted in an organic solvent at 50-70°C for 10-20 h to obtain compound I; Compound II is prepared by the following method: (II-1) A brominated amine compound is reacted with an activated ester compound in an organic solvent at 10-30°C for 2-10 h to obtain urethane intermediate II-1; (II-2) The carbamate intermediate II-1 is reacted with a dialkylamine compound in an organic solvent at 70-100°C for 10-20 h to obtain intermediate II-2; (II-3) In the presence of a catalyst, the intermediate II-2 and the amino alcohol are reacted in an organic solvent at 50-90°C for 10-20 h to obtain compound II; Compound III is prepared by the following method: (III-1) The secondary alcohol compound and the imidazole derivative are reacted in an organic solvent at 40-80°C for 2-8 h to obtain intermediate III-1; (III-2) React intermediate III-1 with a haloalcohol compound in an organic solvent at 10-40°C for 10-20 h to obtain intermediate III-2; (III-3) In the presence of a catalyst, the intermediate III-2 is reacted with an amino alcohol in an organic solvent at 60-100°C for 10-20 h to obtain compound III; Compound IV is prepared by the following method: (IV-1) React the triamine compound and carbonate in an organic solvent at 10-30°C for 5-15 h to obtain an amino carbonate intermediate; (IV-2) After reacting the amino alcohol compound with the carbonate, it is then reacted with the sulfonyl chloride compound in an organic solvent at 10-30°C for 1-10 h to obtain the sulfonyl ester intermediate. (IV-3) The sulfonate intermediate is reacted with the aminocarbonate intermediate in an organic solvent at 70-100°C for 36-48 h to obtain the carbamate intermediate; (IV-4) The carbamate intermediate and the carboxylic acid compound are reacted in an organic solvent at 10-30°C for 1-5 h to obtain a triamine intermediate; (IV-5) The triamine intermediate is reacted with an activated carboxylic acid compound in an organic solvent at 10-30°C for 5-15 h to obtain compound IV; Compound V is prepared by the following method: (V-1) Triamine compounds and amino alcohol compounds are reacted in an organic solvent at 10-30°C for 5-15 h to obtain triamine intermediate V-1; (V-2) The alcohol and carbonate compounds are reacted in an organic solvent at -25℃ to (-15℃) for 0.5-2 h, and then reacted at 10-30℃ for 1-5 h to obtain the active carbonate intermediate V-2; (V-3) React intermediate V-1 and intermediate V-2 in an organic solvent at 10-30°C for 5-15 h to obtain compound V; Compound VI is prepared by the following method: (VI-1) A bromoacid compound and an acyl chloride compound are reacted in an organic solvent at 10-30°C for 1-6 h to obtain an acyl chloride intermediate; (VI-2) React the thiol compound and the acyl chloride intermediate in an organic solvent at 10-30°C for 5-15 h to obtain the brominated sulfate intermediate; (VI-3) The brominated thioester intermediate is reacted with an amino alcohol in an organic solvent at 70-100°C for 24-36 h to obtain the thioester intermediate. (VI-4) The thioester intermediate is reacted with the bromosulfate intermediate in an organic solvent at 70-100°C for 36-48 h to obtain compound of formula VI. Formula VII is prepared by the following method: (VII-1) Under alkaline conditions, an alkyl alcohol compound and an acyl chloride reagent are reacted in an organic solvent at 10-30°C for 1-5 h to obtain a chloroformate; (VII-2) React the chloroformate and the bromoal compound in an organic solvent at 10-30°C for 5-15 h to give a 5-bromopentyl carbonate intermediate; (VII-3) React the 5-bromopentyl carbonate intermediate with an amino alcohol compound in an organic solvent at 70-100°C for 36-47 h to obtain the compound of formula VII; Formula VIII is prepared by the following method: (VIII-1) Under alkaline conditions, an alkyl alcohol compound and an acyl chloride reagent are reacted in an organic solvent at 10-30°C for 1-5 h to obtain a chloroformate; (VIII-2) React the chloroformate and the bromoalcohol compound in an organic solvent at 10-30°C for 5-15 h to give a 5-bromopentyl carbonate intermediate; (VIII-3) The 5-bromopentyl carbonate intermediate is reacted with an amino alcohol compound in an organic solvent at 70-100°C for 48-59 h to obtain compound of formula VIII; Formula IX is prepared by the following method: (IX-1) Under alkaline conditions, an alkyl alcohol compound and an acyl chloride reagent are reacted in an organic solvent at 10-30°C for 1-5 h to obtain a chloroformate; (IX-2) The chloroformate and the bromoal compound are reacted in an organic solvent at 10-30°C for 5-15 h to give a 5-bromopentyl carbonate intermediate; (IX-3) The 5-bromopentyl carbonate intermediate is reacted with an amino alcohol compound in an organic solvent at 70-100°C for 60 h to obtain compound of formula IX; Formula X is prepared by the following method: (X-1) Under alkaline conditions, an alkyl alcohol compound and an acyl chloride reagent are reacted in an organic solvent at 10-30°C for 1-5 h to obtain a chloroformate; (X-2) The chloroformate and the bromoal compound are reacted in an organic solvent at 10-30°C for 5-15 h to give a 5-bromopentyl carbonate intermediate; (X-3) The 5-bromopentyl carbonate intermediate is reacted with an amino alcohol compound in an organic solvent at 70-100°C for 61-72 h to obtain compound X.

[0009] In some embodiments, according to the preparation method of the present invention, the organic solvent comprises at least one selected from dichloromethane, N,N-dimethylformamide, toluene, acetonitrile, n-hexane, and tetrahydrofuran; and the catalyst comprises at least one selected from alkali metal halides and organic bases.

[0010] A third aspect of the present invention provides lipid nanoparticles comprising the lipid compounds described in the first aspect of the present invention.

[0011] A fourth aspect of the present invention provides a drug delivery system comprising a lipid compound as described in the first aspect of the present invention or lipid nanoparticles as described in the third aspect of the present invention.

[0012] A fifth aspect of the present invention provides a pharmaceutical composition comprising the lipid compound described in the first aspect of the present invention, the lipid nanoparticles described in the third aspect of the present invention, or the drug delivery system described in the fourth aspect of the present invention.

[0013] A sixth aspect of the present invention provides the use of a lipid compound or lipid nanoparticles in the preparation of a drug delivery system, wherein the lipid compound is the lipid compound described in the first aspect of the present invention, and the lipid nanoparticles are the lipid nanoparticles described in the third aspect of the present invention.

[0014] A seventh aspect of the present invention relates to the use of a lipid compound, lipid nanoparticles, or a drug delivery system in the preparation of a pharmaceutical composition, wherein the lipid compound is the lipid compound described in the first aspect of the present invention, the lipid nanoparticles are the lipid nanoparticles described in the third aspect of the present invention, and the drug delivery system is the drug delivery system described in the fourth aspect of the present invention.

[0015] In some embodiments, according to the seventh aspect of the invention, the pharmaceutical composition comprises at least one of a small molecule drug, a nucleic acid drug, and a protein / peptide drug.

[0016] An eighth aspect of the present invention provides a method for enhancing cellular immune responses in vitro, comprising the step of contacting cells in vitro with a lipid compound described in the first aspect of the present invention, lipid nanoparticles described in the third aspect of the present invention, or a drug delivery system described in the fourth aspect of the present invention.

[0017] The present invention has found that lipid compounds with symmetrical tail carbon chains can promote mitochondrial activation and enhance cellular metabolism, thereby significantly improving the immune response and showing broad application prospects in the fields of mRNA vaccines, protein vaccines and CAR-T cell therapy. Attached Figure Description

[0018] Figure 1 The results of detecting antigen-specific IFN-γ secreting cells in spleen cells are shown.

[0019] Figure 2 The results of detecting antigen-specific IL-2 secreting cells in spleen cells are shown.

[0020] Figure 3 The results of LNP transfection in vitro are shown, where A represents the proportion of eGFP-positive cells and B represents the average fluorescence intensity.

[0021] Figure 4 The results of in vivo mRNA delivery for each group of LNPs are shown.

[0022] Figure 5 The effects of LNPs in each group on the delivery of herpes zoster gE vaccine mRNA are shown. In this study, A represents the results of antigen-specific IFN-γ secreting cells in spleen cells, and B represents the results of antigen-specific IL-2 secreting cells in spleen cells.

[0023] Figure 6 The results of flow cytometry analysis of the proportion of CAR-positive cells at different time points are shown.

[0024] Figure 7 The results of flow cytometry analysis of the proportion of CAR-positive cells at 24 h are shown.

[0025] Figure 8 The flow cytometry pseudocolor images of CAR-positive cell expression in each group at 24 h are shown. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention.

[0029] lipid compounds In one aspect, the present invention provides a lipid compound (sometimes referred to as "ionizable lipid") for enhancing immune response levels, the lipid compound comprising a main chain and tail branches located at both ends of the main chain, wherein the main chain and the tail branches are covalently linked by carbonyl groups, wherein the main chain has a bilaterally symmetrical structure along its center, and the tail branches comprise a first branch and a second branch having the same structure, wherein the first and second branches are carbon chains having 6-16 (preferably 6-10, for example 6, 7, 8, 9, 10) carbon atoms, the structure of the main chain is shown in Formula Ia, and the structure of the carbonyl groups is shown in Formula Ib. Formula Ia, Formula Ib; R1 and R2 are identical and are carbon chains with 5-8 (preferably 5-6, for example 5 or 6) carbon atoms, R3 is a carbon chain with 4-6 (preferably 4) carbon atoms and has hydroxyl groups at the end; R4 and R5 are independently selected from carbon atoms, nitrogen atoms, oxygen atoms, sulfur atoms, methylene, methine or imino.

[0030] In a preferred embodiment, the lipid compound is selected from the compounds shown in Formula IX: Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, Equation VII, Formula VIII, Formula IX, Formula X.

[0031] This invention has found that lipid compounds with symmetrical tail carbon chain structures can improve their binding ability to endosomes, promote communication between mitochondria and other intracellular structures, regulate cellular metabolic efficiency, and thus enhance the level of immune response.

[0032] Preparation method In one aspect, the present invention provides a method for preparing a lipid compound.

[0033] In a preferred embodiment, the compound of formula I is prepared by the following method: (I-1) A carboxylic acid compound (e.g., but not limited to 2-hexyloctanoic acid) and a bromoalkane (e.g., but not limited to 1,6-dibromohexane) are reacted in an organic solvent at 10-30°C (preferably 11-29°C, even more preferably 12-28°C, further preferably 13-27°C, more preferably 14-26°C, even more preferably 15-25°C, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 1-5 h (e.g. 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 h) to obtain intermediate I-1; (I-2) In the presence of a catalyst, the intermediate I-1 and an amino alcohol (e.g., but not limited to 4-aminobut-1-ol) are reacted in an organic solvent at 50-70°C (preferably 51-69°C, even more preferably 52-68°C, further preferably 53-67°C, more preferably 54-66°C, even more preferably 55-65°C, for example 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65°C) for 10-20 h (preferably 11-20 h, even more preferably 12-20 h, for example 12, 13, 14, 15, 16, 17, 18, 19, 20 h) to obtain compound I.

[0034] In a preferred embodiment, the compound of formula II is prepared by the following method: (II-1) A brominated amine compound (e.g., but not limited to 6-bromohexane-1-amine) is reacted with an activated ester compound (e.g., but not limited to 4-nitrophenyl chloroformate) in an organic solvent at 10-30°C (preferably 11-29°C, more preferably 12-28°C, further preferably 13-27°C, more preferably 14-26°C, even more preferably 15-25°C, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 2-10 h (preferably 3-9 h, e.g. 3, 4, 5, 6, 7, 8, 9 h) to obtain urethane intermediate II-1; (II-2) The carbamate intermediate II-1 is reacted with a dialkylamine compound (e.g., but not limited to dihexylamine) in an organic solvent at 70-100°C (preferably 72-98°C, more preferably 74-96°C, even more preferably 76-94°C, more preferably 78-92°C, even more preferably 80-90°C, for example 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90°C) for 10-20 h (preferably 11-20 h, more preferably 12-20 h, for example 12, 13, 14, 15, 16, 17, 18, 19, 20 h) to obtain intermediate II-2; (II-3) In the presence of a catalyst, the intermediate II-2 and an amino alcohol (e.g., but not limited to 4-aminobut-1-ol) are reacted in an organic solvent at 50-90°C (preferably 52-88°C, more preferably 54-86°C, further preferably 56-84°C, more preferably 58-82°C, and even more preferably 60-80°C, for example 62, 64, 66, 68, 70, 72, 74, 76, 78, 80°C) for 10-20 h (preferably 11-20 h, more preferably 12-20 h, for example 12, 13, 14, 15, 16, 17, 18, 19, 20 h) to obtain compound II; In a preferred embodiment, the compound of formula III is prepared by the following method: (III-1) A secondary alcohol compound (e.g., but not limited to 7-tridecyl alcohol) and an imidazole derivative (e.g., but not limited to N,N'-carbonyldiimidazole) are reacted in an organic solvent at 40-80°C (preferably 42-78°C, more preferably 44-76°C, further preferably 46-74°C, more preferably 48-72°C, and even more preferably 50-70°C, e.g. 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70°C) for 2-8 h (e.g., 2, 3, 4, 5, 6, 7, 8 h) to obtain intermediate III-1; (III-2) The intermediate III-1 is reacted with a haloalcohol compound (e.g., but not limited to 5-bromopent-1-ol) in an organic solvent at 10-40°C (preferably 12-38°C, more preferably 14-36°C, further preferably 16-34°C, more preferably 18-32°C, even more preferably 20-30°C, for example 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30°C) for 10-20 h (preferably 11-20 h, more preferably 12-20 h, for example 12, 13, 14, 15, 16, 17, 18, 19, 20 h) to obtain intermediate III-2; (III-3) In the presence of a catalyst, the intermediate III-2 is reacted with an amino alcohol (e.g., but not limited to 4-aminobut-1-ol) in an organic solvent at 60-100°C (preferably 62-98°C, more preferably 64-96°C, even more preferably 66-94°C, more preferably 68-92°C, even more preferably 70-90°C, for example 72, 74, 76, 78, 80, 82, 84, 86, 88, 90°C) for 10-20 h (preferably 11-20 h, even more preferably 12-20 h, for example 12, 13, 14, 15, 16, 17, 18, 19, 20 h) to obtain compound III.

[0035] In a preferred embodiment, the compound of formula IV is prepared by the following method: (IV-1) A triamine compound (e.g., but not limited to di(6-aminohexyl)amine) and a carbonate (e.g., but not limited to di-tert-butyl carbonate) are reacted in an organic solvent at 10-30°C (preferably 11-29°C, even more preferably 12-28°C, further preferably 13-27°C, more preferably 14-26°C, even more preferably 15-25°C, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 5-15 h (e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 h) to obtain an amino carbonate intermediate; (IV-2) An amino alcohol compound (e.g., but not limited to 4-(N-tert-butoxycarbonylamino)-1-butanol) is reacted with a carbonate (e.g., but not limited to di-tert-butyl carbonate), and then reacted with a sulfonyl chloride compound (e.g., but not limited to p-toluenesulfonyl chloride) in an organic solvent at 10-30°C (preferably 11-29°C, more preferably 12-28°C, further preferably 13-27°C, more preferably 14-26°C, even more preferably 15-25°C, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 1-10 h (preferably 1-9 h, more preferably 1-8 h, more preferably 1-7 h, e.g. 1, 2, 3, 4, 5, 6, 7 h) to obtain a sulfonyl ester intermediate; (IV-3) The sulfonate intermediate is reacted with the aminocarbonate intermediate in an organic solvent at 70-100°C (preferably 72-98°C, more preferably 74-96°C, even more preferably 76-94°C, more preferably 78-92°C, even more preferably 80-90°C, for example 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90°C) for 36-48 h (for example 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 h) to obtain the carbamate intermediate; (IV-4) The carbamate intermediate and a carboxylic acid compound (e.g., but not limited to trifluoroacetic acid) are reacted in an organic solvent at 10-30°C (preferably 11-29°C, more preferably 12-28°C, even more preferably 13-27°C, more preferably 14-26°C, even more preferably 15-25°C, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 1-5 h (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 h) to obtain a triamine intermediate; (IV-5) The triamine intermediate is reacted with an activated carboxylic acid compound (e.g., but not limited to 2-hexyloctanoic acid) in an organic solvent at 10-30°C (preferably 11-29°C, more preferably 12-28°C, even more preferably 13-27°C, more preferably 14-26°C, even more preferably 15-25°C, for example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 5-15 h (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 h) to obtain the compound of formula IV; Compound V is prepared by the following method: (V-1) A triamine compound (e.g., but not limited to, di(6-aminohexyl)amine) is reacted with an amino alcohol compound (e.g., but not limited to, 4-amino-1-butanol) in an organic solvent at 10-30°C (preferably 11-29°C, even more preferably 12-28°C, further preferably 13-27°C, more preferably 14-26°C, even more preferably 15-25°C, for example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 5-15 h (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 h) to obtain triamine intermediate V-1; (V-2) An alcohol compound (e.g., but not limited to 7-tridecyl alcohol) and a carbonate compound (e.g., but not limited to pentafluorophenyl chloroformate) are reacted in an organic solvent at -25°C to (-15°C) (e.g., -25, -24, -23, -22, -21, -20, -19, -18, -17, -16, -15°C) for 0.5-2 h (e.g., 0.5, 0.8, 1, 1.5, 1.8, 2 h), and then further reacted at 10-30°C (preferably 11-29°C, more preferably 12-28°C, even more preferably 13-27°C, more preferably 14-26°C, and even more preferably 15-25°C, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 1-5 h. h (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 h) yields activated carbonate intermediate V-2; (V-3) The intermediates V-1 and V-2 are reacted in an organic solvent at 10-30°C (preferably 11-29°C, more preferably 12-28°C, even more preferably 13-27°C, more preferably 14-26°C, and even more preferably 15-25°C, for example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 5-15 h (for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 h) to obtain compound V; Compound VI is prepared by the following method: (VI-1) A bromoacid compound (e.g., but not limited to 8-bromooctanoic acid) and an acyl chloride compound (e.g., but not limited to oxaloyl chloride) are reacted in an organic solvent at 10-30°C (preferably 11-29°C, even more preferably 12-28°C, further preferably 13-27°C, more preferably 14-26°C, even more preferably 15-25°C, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 1-6 h (e.g. 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6 h) to obtain an acyl chloride intermediate; (VI-2) A thiol compound (e.g., but not limited to 7-tridecylthiol) and the acyl chloride intermediate are reacted in an organic solvent at 10-30°C (preferably 11-29°C, even more preferably 12-28°C, further preferably 13-27°C, more preferably 14-26°C, even more preferably 15-25°C, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 5-15 h (e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 h) to obtain a brominated sulfate intermediate; (VI-3) The brominated thioester intermediate is reacted with an amino alcohol (e.g., but not limited to 4-amino-1-butanol) in an organic solvent at 70-100°C (preferably 72-98°C, more preferably 74-96°C, even more preferably 76-94°C, more preferably 78-92°C, and even more preferably 80-90°C, e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90°C) for 24-36 h (e.g., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 h) to obtain the thioester intermediate; (VI-4) The thioester intermediate is reacted with the brominated sulfate intermediate in an organic solvent at 70-100°C (preferably 72-98°C, more preferably 74-96°C, even more preferably 76-94°C, more preferably 78-92°C, and even more preferably 80-90°C, for example 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90°C) for 36-48 h (for example 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 h) to obtain compound of formula VI; Formula VII is prepared by the following method: (VII-1) Under alkaline conditions, an alkyl alcohol compound (e.g., but not limited to 9-heptadecanool) and an acyl chloride reagent (e.g., but not limited to triphosgene) are reacted in an organic solvent at 10-30°C (preferably 11-29°C, even more preferably 12-28°C, further preferably 13-27°C, more preferably 14-26°C, even more preferably 15-25°C, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 1-5 h (e.g. 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 h) to obtain a chloroformate; (VII-2) The chloroformate and the bromoalcohol compound (e.g., but not limited to 5-bromo-1-pentanol) are reacted in an organic solvent at 10-30°C (preferably 11-29°C, even more preferably 12-28°C, further preferably 13-27°C, more preferably 14-26°C, even more preferably 15-25°C, for example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 5-15 h (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 h) to obtain a 5-bromopentyl carbonate intermediate; (VII-3) The 5-bromopentyl carbonate intermediate is reacted with an amino alcohol compound (e.g., but not limited to 4-amino-1-butanol) in an organic solvent at 70-100°C (preferably 72-98°C, more preferably 74-96°C, even more preferably 76-94°C, more preferably 78-92°C, even more preferably 80-90°C, e.g. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90°C) for 36-47 h (e.g. 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 h) to obtain the compound of formula VII; Formula VIII is prepared by the following method: (VIII-1) Under alkaline conditions, an alkyl alcohol compound (e.g., but not limited to 9-heptadecanool) and an acyl chloride reagent (e.g., but not limited to triphosgene) are reacted in an organic solvent at 10-30°C (preferably 11-29°C, even more preferably 12-28°C, further preferably 13-27°C, more preferably 14-26°C, even more preferably 15-25°C, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 1-5 h (e.g. 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 h) to obtain a chloroformate; (VIII-2) The chloroformate and the bromoalcohol compound (e.g., but not limited to 5-bromo-1-pentanol) are reacted in an organic solvent at 10-30°C (preferably 11-29°C, even more preferably 12-28°C, further preferably 13-27°C, more preferably 14-26°C, even more preferably 15-25°C, for example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 5-15 h (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 h) to obtain the 5-bromopentyl carbonate intermediate; (VIII-3) The 5-bromopentyl carbonate intermediate is reacted with an amino alcohol compound (e.g., but not limited to 4-amino-1-butanol) in an organic solvent at 70-100°C (preferably 72-98°C, more preferably 74-96°C, even more preferably 76-94°C, more preferably 78-92°C, even more preferably 80-90°C, e.g. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90°C) for 48-59 h (e.g. 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 h) to obtain compound VIII; Formula IX is prepared by the following method: (IX-1) Under alkaline conditions, an alkyl alcohol compound (e.g., but not limited to 9-heptadecanool) and an acyl chloride reagent (e.g., but not limited to triphosgene) are reacted in an organic solvent at 10-30°C (preferably 11-29°C, even more preferably 12-28°C, further preferably 13-27°C, more preferably 14-26°C, even more preferably 15-25°C, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 1-5 h (e.g. 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 h) to obtain a chloroformate; (IX-2) The chloroformate and a bromoalcohol compound (e.g., but not limited to 5-bromo-1-pentanol) are reacted in an organic solvent at 10-30°C (preferably 11-29°C, even more preferably 12-28°C, further preferably 13-27°C, more preferably 14-26°C, even more preferably 15-25°C, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 5-15 h (e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 h) to obtain a 5-bromopentyl carbonate intermediate; (IX-3) The 5-bromopentyl carbonate intermediate is reacted with an amino alcohol compound (e.g., but not limited to 4-amino-1-butanol) in an organic solvent at 70-100°C (preferably 72-98°C, more preferably 74-96°C, even more preferably 76-94°C, more preferably 78-92°C, and even more preferably 80-90°C, for example 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90°C) for 60 h to obtain the compound of formula IX; Formula X is prepared by the following method: (X-1) Under alkaline conditions, an alkyl alcohol compound (e.g., but not limited to 9-heptadecanool) and an acyl chloride reagent (e.g., but not limited to triphosgene) are reacted in an organic solvent at 10-30°C (preferably 11-29°C, even more preferably 12-28°C, further preferably 13-27°C, more preferably 14-26°C, even more preferably 15-25°C, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 1-5 h (e.g. 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 h) to obtain a chloroformate; (X-2) The chloroformate and the bromoalcohol compound (e.g., but not limited to 5-bromo-1-pentanol) are reacted in an organic solvent (e.g., but not limited to dichloromethane) at 10-30°C (preferably 11-29°C, even more preferably 12-28°C, further preferably 13-27°C, more preferably 14-26°C, even more preferably 15-25°C, e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25°C) for 5-15 h (e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 h) to obtain a 5-bromopentyl carbonate intermediate; (X-3) The 5-bromopentyl carbonate intermediate is reacted with an amino alcohol compound (e.g., but not limited to 4-amino-1-butanol) in an organic solvent at 70-100°C (preferably 72-98°C, more preferably 74-96°C, further preferably 76-94°C, more preferably 78-92°C, even more preferably 80-90°C, for example 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90°C) for 61-72 h (e.g., 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 h) to obtain compound X.

[0036] In the preparation method of the present invention, the organic solvent includes, but is not limited to, dichloromethane, N,N-dimethylformamide, toluene, acetonitrile, n-hexane, tetrahydrofuran, etc.; the catalyst includes, but is not limited to, alkali metal halides (e.g., but not limited to potassium iodide) and organic bases (e.g., but not limited to 1,8-diazabicyclo[5.4.0]undec-7-ene), etc.; the reaction is preferably carried out in an alkaline environment, and the substance providing the alkaline environment is not particularly limited, such as potassium carbonate, triethylamine, pyridine, etc.

[0037] It is understood that, in order to accelerate the reaction, those skilled in the art can adjust the pH of the reaction system, activate the reaction raw materials with an activator, or perform stirring operations. In addition, in order to improve the purity of the product, those skilled in the art can perform purification processes such as washing, filtering, and drying on the reaction product, all of which are within the scope of protection of this application.

[0038] lipid nanoparticles In one aspect, the present invention provides lipid nanoparticles comprising the lipid compounds described herein.

[0039] In a preferred embodiment, the lipid nanoparticles of the present invention further comprise steroids, phospholipids, and PEG lipids, wherein the steroids include cholesterol and its derivatives, such as, but not limited to, cholesterol, dihydrocholesterol, epicholesterol, sterols, 7-dehydrocholesterol, 5-hydroxycholesterol, 24S-hydroxycholesterol, 27-hydroxycholesterol, 7α / 7β-hydroxycholesterol, 7-ketocholesterol, etc.; the phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin, such as, but not limited to, distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), bispentadecanoylphosphatidylcholine, dilaurylphosphatidylcholine (DLPC), palmitoyl oleyl oleate, etc. Phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-cholestenylhemisuccino-sn-glycerol-3-phosphate choline, 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearate-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoyl Phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytyl-phosphatidylethanolamine (DPyPE), etc.; the PEG lipids include, but are not limited to, DMG-PEG, DSG-PEG, DSPE-PEG, DOPE-PEG, DPPE-PEG, DMPE-PEG, Chol-PEG, etc.

[0040] In a preferred embodiment, the lipid nanoparticles of the present invention comprise 20-65 mol% (preferably 25-60 mol%, more preferably 30-55 mol%, even more preferably 35-50 mol%, and more preferably 40-50 mol%, for example 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 mol%) of a lipid compound, 10-50 mol% (preferably 15-45 mol%, more preferably 20-45 mol%, and more preferably 25-45 mol%, for example 25, 30, 35, 40, 45 mol%) of a steroid, 5-25 mol% (preferably 6-24 mol%, more preferably 7-23 mol%, even more preferably 8-22 mol%, and more preferably 9-20 mol%, for example 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mol%) of phospholipids and 0.1-10 PEG lipids in mol% (preferably 0.2-9 mol%, more preferably 0.3-8 mol%, even more preferably 0.4-6 mol%, more preferably 0.5-4 mol%, and even more preferably 0.5-3 mol%, for example 0.1, 1, 1.5, 2, 2.5, 3 mol%).

[0041] In a preferred embodiment, the molar ratio of lipid compound, steroid, phospholipid and PEG lipid in the lipid nanoparticles of the present invention is (30-55):(20-60):(5-30):(0.5-5), preferably (31-54):(21-59):(6-29):(0.6-4.5), even more preferably (32-53):(22-58):(7-28):(0.7-4), and more preferably (35-50):(24-55):(8-25):(1-3). Examples include 35:24:8:1, 40:24:8:1, 45:24:8:1, 50:24:8:1, 35:30:8:1, 40:35:8:1, 45:40:8:1, 50:50:8:1, 50:55:8:1, 35:30:10:1, 40:35:15:1, 45:40:20:1, 50:50:25:1, 35:24:8:1.5, 40:24:8:2, 45:24:8:2.5, and 50:24:8:3. In a preferred embodiment, the molar ratio of lipid compound, steroid, phospholipid, and PEG lipid in the lipid nanoparticles of the present invention is 45:44:9:2.

[0042] In some embodiments, the lipid nanoparticles of the present invention have a particle size of 20-200 nm, preferably 25-195 nm, even more preferably 30-190 nm, further preferably 35-185 nm, more preferably 40-180 nm, more preferably 45-175 nm, more preferably 50-170 nm, more preferably 55-165 nm, more preferably 60-160 nm, more preferably 65-155 nm, more preferably 70-150 nm, more preferably 75-145 nm, more preferably 80-140 nm, for example 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140.

[0043] The lipid nanoparticles of this invention can be used to deliver drugs, including but not limited to small molecule drugs, nucleic acid drugs, and protein / peptide drugs. The nucleic acid drugs include, but are not limited to, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomirs / antimirs), messenger RNA interfering with complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), circular DNA (ceDNA), and short interfering RNA (siRNA).

[0044] Based on this, the present invention also provides a drug delivery system comprising the lipid compound or lipid nanoparticles described herein. In some embodiments, the drug delivery system of the present invention includes the aforementioned small molecule drugs, nucleic acid drugs, protein and peptide drugs, etc.

[0045] Pharmaceutical Composition In one aspect, the present invention provides a pharmaceutical composition comprising the lipid compound, lipid nanoparticles, or drug delivery system described herein.

[0046] In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a carrier, excipient, or stabilizer that is non-toxic to cells or mammals exposed to the doses and concentrations used, or has an acceptable level of toxicity as determined by a person skilled in the art.

[0047] The administration method of the pharmaceutical compositions of the present invention is not particularly limited, and examples include, but are not limited to, parenteral administration (e.g., but not limited to intravenous, intramuscular, subcutaneous, or intradermal injection), mucosal administration (e.g., but not limited to nasal, oral, or intestinal administration), oral administration, and topical administration. Accordingly, the pharmaceutical compositions of the present invention can be formulated into various dosage forms, such as, but not limited to, tablets, capsules, granules, powders, solutions, injections, ointments, and nanoformulations.

[0048] application One aspect of the present invention provides the use of the lipid compounds or lipid nanoparticles described herein in the preparation of drug delivery systems.

[0049] This invention also provides the use of the lipid compounds, lipid nanoparticles, or drug delivery systems described herein in the preparation of pharmaceutical compositions. In some embodiments, the pharmaceutical composition includes small molecule drugs, nucleic acid drugs, and protein or peptide drugs, such as, but not limited to, mRNA vaccines, protein vaccines, in vivo CAR-T cell therapy drugs, in vitro CAR-T cell therapy drugs, nucleic acid aptamer-drug conjugates (ApDCs), etc.

[0050] method One aspect of the present invention provides a method for enhancing cellular immune responses in vitro, comprising the step of contacting cells in vitro with the lipid compounds, lipid nanoparticles, or drug delivery systems described in the present invention.

[0051] In a preferred embodiment, the method of the present invention includes: (1) culturing cells in vitro; (2) contacting the cells in vitro with the lipid compound, lipid nanoparticles, or drug delivery system described in the present invention; and (3) detecting the immune response level of the cells. The cells are not particularly limited, and examples include, but are not limited to, THP-1 cells, PBMCs, BMDCs, etc.; the detection of the immune response level of the cells can be performed using methods known in the art, such as, but not limited to, flow cytometry, QCM-D-based endosome affinity assays, OCR detection, etc.

[0052] Example 1 This embodiment exemplifies a method for preparing lipid compounds.

[0053] 1. Compound of Formula I (also known as 2100) 2-Hexyloctanoic acid (10.0 g, 43.8 mmol) was mixed with DMF (100 mL) and K₂CO₃ (12.1 g, 87.6 mmol). The mixture was stirred at room temperature for 15 min under nitrogen protection. After the system cooled and reached saturation, 1,6-dibromohexane (11.2 g, 46 mmol) was slowly added, and the mixture was stirred at room temperature for 2 h. After the reaction was complete, the mixture was extracted with ethyl acetate, washed, dried, and filtered to obtain 6-bromohexyl-2-hexyloctanoic acid ester.

[0054] 6-Bromohexyl-2-hexyloctanoate (5.00 g, 12.8 mmol) was mixed with DMF (50 mL), 4-aminobut-1-ol (1.37 g, 15.3 mmol), K₂CO₃ (3.53 g, 25.6 mmol), and KI (0.211 g, 1.28 mmol). The mixture was stirred and heated to 60 °C for 16 h under nitrogen protection. After the reaction was complete, the mixture was extracted with ethyl acetate, washed, dried, filtered, and purified to obtain the target product 2100.

[0055] 2. Compound of Formula II (also known as 2101) 6-Bromohexane-1-amine (10.0 g, 38.3 mmol) was dispersed in DCM (100 ml), and TEA (13.3 ml, 95.8 mmol) was slowly added under ice bath conditions, followed by the slow addition of 4-nitrophenyl chlorocarbamate (9.27 g, 45.9 mmol). The reaction was stirred at room temperature for 6 h. After the reaction was complete, the sample was washed, dried, filtered, and purified to obtain 4-nitrophenyl (6-bromobenzyl)carbamate.

[0056] The obtained 4-nitrophenyl(6-bromobenzyl)carbamate (5.80 g, 16.8 mmol) was dissolved in toluene, and dihexylamine (3.4 g) was added. The mixture was heated to 85 °C and stirred for 16 h. After the reaction was completed, the mixture was washed, dried, filtered, and purified to obtain 3-(6-bromohexyl)-1,1-dihexylurea.

[0057] 3-(6-bromohexyl)-1,1-dihexylurea (4.00 g, 10.2 mmol), ACN (50 ml), K₂CO₃ (2.82 g, 20.4 mmol), and KI (168 mg, 1.02 mmol) were mixed and stirred until homogeneous. 4-aminobutyric acid (1.09 g, 12.3 mmol) was added, and the mixture was heated to 70 °C and stirred for 16 h. After the reaction was complete, the product was washed, dried, filtered, and purified to obtain the target product 2101.

[0058] 3. Compound of Formula III (also known as 2102) 7-Tridecyl alcohol (2.00 g, 10.0 mmol) was dissolved in toluene (30 mL), and CDI (1.95 g, 12.0 mmol) was slowly added at room temperature. The mixture was heated to 60 °C and stirred for 6 h. After the reaction was complete, the mixture was washed, dried, filtered, and purified to obtain tridecyl-7-yl-1H-imidazol-1-carboxylic acid ester.

[0059] Tridecyl-7-yl-1H-imidazol-1-carboxylic acid ester (2.94 g, 10.0 mmol) was dissolved in toluene, and 5-bromopentan-1-ol (1.84 g, 11.0 mmol) and DBU (0.30 g, 2.0 mmol) were added. The mixture was stirred at 25 °C for 16 h. After the reaction was complete, the sample was washed, dried, filtered, and purified to obtain 5-(7-tridecyloxy)pentyl bromide.

[0060] 5-(7-Tridecyloxy)pentyl bromide (3.93 g, 10.0 mmol) was dissolved in MeCN, and K₂CO₃ (2.76 g, 20.0 mmol) and KI (0.17 g, 1.0 mmol) were added and stirred to disperse. 4-Aminobut-1-ol (1.07 g, 12.0 mmol) was added, and the mixture was heated to 80 °C and stirred for 16 h. After the reaction was complete, the product was washed, dried, filtered, and purified to obtain the target product 2102.

[0061] 4. Compound of Formula IV (also known as 2103) Di(6-aminohexyl)amine (25.8 g, 100 mmol) was completely dissolved in 500 mL of DCM. While stirring, 500 mL of saturated sodium bicarbonate aqueous solution was added, and the mixture was cooled to 0–5 °C in an ice-water bath. Di-tert-butyl carbonate (48.0 g, 220 mmol) was dissolved in 100 mL of DCM and slowly added dropwise to the mixture under low temperature and stirring. After the addition was complete, the ice bath was removed, and the reaction mixture was allowed to rise to room temperature and stirred overnight. After the reaction was complete, the mixture was washed, dried, filtered, and purified to obtain di-tert-butyl(azadiylbis(hexane-6,1-diyl))dicarbamate. 4-(N-tert-butoxycarbonylamino)-1-butanol was prepared using 4-amino-1-butanol (9.0 g, 100 mmol) in the same manner.

[0062] The prepared 4-(N-tert-butoxycarbonylamino)-1-butanol (18 g, 83 mmol) was dissolved in 150 mL of DCM and cooled to 0 °C in an ice bath. Triethylamine (13.9 mL, 100 mmol) was added, followed by p-toluenesulfonyl chloride (19.0 g, 100 mmol) in portions with stirring. The ice bath was removed, and the mixture was stirred at room temperature for 4 h. After the reaction was complete, the mixture was washed, dried, and filtered to obtain 4-(N-tert-butoxycarbonylamino)butyl p-toluenesulfonate.

[0063] Di-tert-butyl(azadiylbis(hexane-6,1-diyl))dicarbamate (47 g, 89 mmol) and anhydrous potassium carbonate (24.6 g, 178 mmol) were suspended in 400 mL of acetonitrile and refluxed at 85 °C under nitrogen protection. The 4-(N-tert-butoxycarbonylamino)butyl-p-toluenesulfonate obtained in the previous step was dissolved in 50 mL of acetonitrile and slowly added dropwise to the reflux solution. The mixture was then refluxed at 85 °C for 36–48 h. After the reaction was complete, the mixture was cooled to room temperature, washed, dried, filtered, and purified to obtain di-tert-butyl((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)dicarbamate.

[0064] The di-tert-butyl((4-hydroxybutyl)zazonyl)bis(hexane-6,1-diyl)dicarbamate (30.0 g, 45 mmol) obtained in the previous step was dissolved in 100 mL of DCM. The solution was cooled to 0°C in an ice-water bath, and trifluoroacetic acid (100 mL) was slowly added dropwise with stirring. After the addition was complete, the ice bath was removed, the reaction solution was allowed to rise to room temperature, and stirring was continued for 2-3 h. After the reaction was completed, the solution was washed, dried, filtered, and purified to obtain 4-(bis(6-aminohexyl)amino)but-1-ol.

[0065] 2-Hexyloctanoic acid (25.6 g, 100 mmol) and HOBt (15.3 g, 100 mmol) were dissolved in 400 mL of DCM, cooled to 0°C in an ice-water bath, and EDCl (19.2 g, 100 mmol) was added under a nitrogen stream. The mixture was stirred and activated for 30 min. 4-(bis(6-aminohexyl)amino)but-1-ol (18.0 g, 45.5 mmol) and triethylamine (19.0 mL, 136.5 mmol) synthesized in the previous step were dissolved in 100 mL of DCM. This mixture was slowly added to the activated acid solution under stirring at 0°C. After the addition was complete, the ice bath was removed, and the reaction mixture was slowly raised to room temperature and stirred overnight under nitrogen protection. After the reaction was complete, the mixture was washed, dried, filtered, and purified to obtain 2103.

[0066] 5. Compound of formula V (also known as 2104) 4-Amino-1-butanol (20.0 g, 224 mmol) was completely dissolved in 400 mL of DCM. A mild oxidant, Dess-Martin Periodinane (114 g, 269 mmol), was added in portions at 0 °C with stirring. After the addition was complete, the ice bath was removed, and the reaction was stirred at room temperature for 2 h. After the reaction was complete, the mixture was carefully quenched with 300 mL of a 1:1 saturated NaHCO3 / saturated Na2S2O3 mixture, and stirred vigorously for 15 min. The mixture was extracted, separated, and washed to obtain an organic aldehyde solution. Under nitrogen protection in an ice-water bath, di(6-aminohexyl)amine (28.9 g, 112 mmol) was added to the aldehyde solution, and the mixture was stirred at room temperature for 1 h. The reactants were then cooled to 0 °C, and sodium triacetoxyborohydride (71.2 g, 336 mmol) was added in portions with stirring. After the addition was complete, the ice bath was removed, and the mixture was stirred at room temperature overnight. After the reaction was completed, the reaction solution was cooled to 0°C, quenched with 2 M NaOH solution until pH > 10, extracted, washed, dried, filtered, and purified to obtain 4-(bis(6-aminohexyl)amino)but-1-ol.

[0067] 7-Tridecyl alcohol (20.0 g, 94 mmol) and triethylamine (13.1 mL, 94 mmol) were dissolved in 200 mL of anhydrous THF and cooled to -20 °C. Pentafluorophenyl chloroformate (19.4 g, 94 mmol) was dissolved in 30 mL of anhydrous THF and slowly added dropwise to the reaction solution. After the addition was complete, the mixture was stirred at -20 °C for 1 h, and then slowly heated to room temperature and stirred for 2 h. After the reaction was completed, the mixture was filtered and concentrated. The crude product was dissolved in n-hexane, washed, dried, and filtered to obtain pentafluorophenyl tridecyl carbonate.

[0068] 10.0 g (28.2 mmol) of 4-(bis(6-aminohexyl)amino)but-1-ol was dissolved in 11.8 mL (84.6 mmol) of triethylamine in 300 mL of anhydrous THF, and the mixture was cooled to 0 °C in an ice-water bath. The active ester (63 mmol) obtained in the previous step was dissolved in 50 mL of anhydrous THF and slowly added dropwise to the amine solution. After the addition was complete, the ice bath was removed, and the mixture was stirred overnight at room temperature. After the reaction was completed, the solution was concentrated, washed, dried, and filtered to obtain 2104.

[0069] 6. Compound of formula VI (also known as 2105) Dissolve 25.0 g (105 mmol) of 8-bromooctanoic acid in 100 mL of DCM, cool to 0 °C in an ice-water bath, and slowly add 11.3 mL (131 mmol) of oxalyl chloride dropwise with stirring. After the addition is complete, remove the ice bath and stir at room temperature for 2-3 h. Remove excess oxalyl chloride and solvent by vacuum distillation to obtain 8-bromooctanoic acid chloride.

[0070] 7-Tridecylthiol (28.7 g, 115 mmol) and triethylamine (16.0 mL, 115 mmol) were dissolved in 200 mL of anhydrous tetrahydrofuran and cooled to 0°C in an ice-water bath. 8-Bromooctanoyl chloride (105 mmol) obtained in the previous step was dissolved in 50 mL of anhydrous tetrahydrofuran and slowly added dropwise to the thiol solution. After the addition was complete, the ice bath was removed, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was extracted, washed, dried, filtered, and purified to obtain S-(tetran-7-yl)-8-bromooctanoic acid sulfate.

[0071] 4-Amino-1-butanol (6.0 g, 67.4 mmol), S-(tetran-7-yl)-8-bromooctyl sulfate (25.0 g, 51.8 mmol), and anhydrous potassium carbonate (14.3 g, 103.6 mmol) were suspended in 250 mL of anhydrous acetonitrile and heated to 85 °C under nitrogen protection with stirring for 24–36 h. After the reaction was completed, the mixture was filtered, washed, concentrated, and purified to obtain S-(tetran-7-yl)-8-((4-hydroxybutyl)amino)octyl sulfate.

[0072] The S-(tetran-7-yl)-8-((4-hydroxybutyl)amino)octane thioester (15.0 g, 30.3 mmol), S-(tetran-7-yl)-8-bromooctyl sulfate (16.0 g, 33.3 mmol), and anhydrous potassium carbonate (8.4 g, 60.6 mmol) obtained in the previous step were suspended in 200 mL of anhydrous acetonitrile and heated to 85 °C under nitrogen protection with stirring for 36–48 h. After the reaction was completed, the mixture was filtered, washed, dried, concentrated, and purified to obtain 2105.

[0073] 7. Compound of formula VII (also known as 2108) 9-Heptadecanol (20.0 g, 73 mmol) and anhydrous pyridine (5.9 mL, 73 mmol) were dissolved in 200 mL of dichloromethane and cooled to -5°C with stirring. Triphosgene (7.2 g, 24 mmol) dissolved in 50 mL of dichloromethane was slowly added dropwise to the above cold solution, keeping the internal temperature below 5°C. After the addition was complete, the cooling bath was removed, and the reaction solution was slowly raised to room temperature with stirring for 3–4 h. After the reaction was completed, the solution was extracted, washed, dried, filtered, and concentrated under reduced pressure to obtain 9-heptadecyl chloroformate.

[0074] 5-Bromo-1-pentanol (25.0 g, 138 mmol) and anhydrous pyridine (11.1 mL, 138 mmol) were dissolved in 400 mL of dichloromethane and cooled to 0°C in an ice-water bath. Under stirring and nitrogen protection, 9-heptadecyl chloroformate (-125 mmol) prepared in the previous step was dissolved in 100 mL of dichloromethane and slowly added dropwise to the reaction mixture. After the addition was complete, the ice bath was removed, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was extracted, washed, dried, filtered, and purified to obtain 5-bromopentyl(9-heptadecyl) carbonate.

[0075] 4-Amino-1-butanol (6.0 g, 67 mmol), 5-bromopentyl(9-heptadecyl) carbonate (70.0 g, 147 mmol), and anhydrous potassium carbonate (23.3 g, 168 mmol) were suspended in 500 mL of anhydrous acetonitrile. Under nitrogen protection, the mixture was heated to 85 °C and stirred under reflux for 36–48 h. After the reaction was complete, the mixture was cooled to room temperature, filtered, washed, concentrated under reduced pressure, and purified to obtain 2108.

[0076] 8. Compound of formula VIII (also known as 2110) 11-Ceicosyl alcohol (30.0 g, 85 mmol) and anhydrous pyridine (6.9 mL, 85 mmol) were dissolved in 300 mL of dichloromethane and cooled to -5°C with stirring. Triphosgene (8.4 g, 28 mmol) dissolved in 50 mL of dichloromethane was slowly added dropwise to the above cold solution, keeping the internal temperature below 5°C. After the addition was complete, the cooling bath was removed, and the reaction solution was slowly raised to room temperature with stirring for 3–4 h. After the reaction was completed, the solution was extracted, washed, dried, filtered, and concentrated under reduced pressure to obtain 11-ceicosyl chloroformate.

[0077] 5-Bromo-1-pentanol (20.0 g, 110 mmol) and anhydrous pyridine (8.9 mL, 110 mmol) were dissolved in 500 mL of dichloromethane and cooled to 0°C in an ice-water bath. Under stirring and nitrogen protection, 11-cuicoalkyl chloroformate (-100 mmol) prepared in the previous step was dissolved in 100 mL of dichloromethane and slowly added dropwise to the reaction mixture. After the addition was complete, the ice bath was removed, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was extracted, washed, dried, filtered, and purified to obtain 5-bromopentyl(11-cuicoalkyl) carbonate.

[0078] 4-Amino-1-butanol (5.0 g, 56 mmol), 5-bromopentyl(11-tetradecyl) carbonate (70.0 g, 123 mmol), and anhydrous potassium carbonate (19.4 g, 140 mmol) were suspended in 600 mL of anhydrous acetonitrile. Under nitrogen protection, the mixture was heated to 85 °C and stirred under reflux for 48–60 h. After the reaction was complete, the mixture was cooled to room temperature, filtered, washed, concentrated under reduced pressure, and purified to obtain 2110.

[0079] 9. Compound of formula IX (also known as 2112) 13-Coctanol (10.0 g, 25 mmol) and anhydrous pyridine (2.0 mL, 25 mmol) were dissolved in 150 mL of dichloromethane and cooled to -5°C with stirring. Triphosgene (2.5 g, 8.3 mmol) dissolved in 30 mL of dichloromethane was slowly added dropwise to the above cold solution, keeping the internal temperature below 5°C. After the addition was complete, the cold bath was removed, and the reaction solution was slowly raised to room temperature while stirring continued for 4 h. After the reaction was completed, the solution was extracted, washed, dried, filtered, and concentrated under reduced pressure to obtain 13-octanyl chloroformate.

[0080] 5-Bromo-1-pentanol (6.1 g, 33 mmol) and anhydrous pyridine (2.7 mL, 33 mmol) were dissolved in 200 mL of dichloromethane and cooled to 0°C in an ice-water bath. Under stirring and nitrogen protection, 13-tetradecyl chloroformate (-30 mmol) prepared in the previous step was dissolved in 100 mL of dichloromethane and slowly added dropwise to the reaction mixture. After the addition was complete, the ice bath was removed, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was extracted, washed, dried, filtered, and purified to obtain 5-bromopentyl(13-tetradecyl) carbonate.

[0081] 4-Amino-1-butanol (0.9 g, 10 mmol), 5-bromopentyl(13-tetrapentane) carbonate (12.0 g, 22 mmol), and anhydrous potassium carbonate (3.5 g, 25 mmol) were suspended in 150 mL of anhydrous acetonitrile. The mixture was heated to 85 °C and stirred under nitrogen protection for 60 h. After the reaction was complete, the mixture was cooled to room temperature, filtered, washed, concentrated under reduced pressure, and purified to obtain 2112.

[0082] 10. Compound X (also known as 2114) 15-Nicosanol (5.0 g, 10 mmol) and anhydrous pyridine (0.81 mL, 10 mmol) were dissolved in 100 mL of dichloromethane and cooled to -5 °C with stirring. Triphosgene (1.0 g, 3.3 mmol) dissolved in 20 mL of dichloromethane was slowly added dropwise to the above cold solution, keeping the internal temperature below 5 °C. After the addition was complete, the cooling bath was removed, and the reaction solution was slowly raised to room temperature with stirring for 4 h. After the reaction was completed, the solution was extracted, washed, dried, filtered, and concentrated under reduced pressure to obtain 15-nonacyl chloroformate.

[0083] 5-Bromo-1-pentanol (2.2 g, 12 mmol) and anhydrous pyridine (0.97 mL, 12 mmol) were dissolved in 150 mL of dichloromethane and cooled to 0°C in an ice-water bath. Under stirring and nitrogen protection, the chloroformate-15-nonadecanoyl ester prepared in the previous step was dissolved in 100 mL of dichloromethane and slowly added dropwise to the reaction mixture. After the addition was complete, the ice bath was removed, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was extracted, washed, dried, filtered, and purified to obtain 5-bromopentyl(15-nonadecanoyl) carbonate.

[0084] 4-Amino-1-butanol (0.18 g, 2 mmol), 5-bromopentyl(15-nonadecanoyl) carbonate (2.5 g, 4.4 mmol), and anhydrous potassium carbonate (0.69 g, 5 mmol) were suspended in 50 mL of anhydrous acetonitrile. Under nitrogen protection, the mixture was heated to 85 °C and stirred under reflux for 60–72 h. After the reaction was complete, the mixture was cooled to room temperature, filtered, washed, concentrated under reduced pressure, and purified to obtain 2114.

[0085] Example 2 This embodiment exemplifies the application of the lipid compounds of the present invention.

[0086] 1.1 Preparation of lipid nanoparticles LNPs were prepared by microfluidic mixing of organic and aqueous phases to achieve efficient embedding of nucleic acids. The organic phase consisted of ionizable lipids, cholesterol, phospholipids, and PEG lipids dissolved in anhydrous ethanol at a specific molar ratio; the aqueous phase consisted of nucleic acids (such as mRNA) dissolved in citrate buffer (pH=4.5±0.5), with a nucleic acid content of 1-50% (w / v).

[0087] 1.2 Characterization of lipid nanoparticles The particle size and polydispersity index (PDI) of LNPs were measured using a Malvern laser particle size analyzer; Quant-iT was used according to the manufacturer's instructions. TMThe RiboGreen® RNA Quantitative Kit was used to detect the nucleic acid encapsulation efficiency of LNPs. An optimal LNP formulation with superior particle size, PDI, and encapsulation efficiency was obtained by adjusting the molar ratio of ionizable lipids:cholesterol:phospholipids:PEG lipids and the N / P ratio (the molar ratio of elemental N to elemental P in the LNP / mRNA system).

[0088] Table 1. Test results of particle size, PDI and encapsulation efficiency for different LNP formulations. 1.3 Screening for LNP formulations with good immunizing effects The above 10 formulations were used to prepare LNPs containing mRNA of the herpes zoster antigen gE protein. Subsequently, on days 0 and 28, the LNP formulations containing 5 μg of mRNA were injected intramuscularly into C57BL / 6 mice (6-8 weeks old, female). On day 56, spleen cells were harvested to detect antigen-specific IFN-γ and IL-2 secreting cells in the spleen cells to characterize the cellular immune response.

[0089] The results are as follows Figure 1 and Figure 2 As shown, formulation 4 exhibits better immune efficacy compared to other formulations. Subsequent experiments were conducted using formulation 4 (ionizable lipids: cholesterol: DSPC: DMG-PEG2000 = 45:44:9:2) as a basis to prepare LNPs containing different ionizable lipids for comparison.

[0090] 1.4 In vitro transfection of LNP cells LNPs containing eGFP-mRNA encapsulated with different ionizable lipids were prepared using the above method. Jurkat cells were revived, passaged to the required cell number in culture flasks, and then cultured at 5 × 10⁶ cells per well. 5 Cells were seeded in 48-well plates, and LNP formulations containing 0.5 μg mRNA per million cells were added. After overnight transfection, the proportion of eGFP-positive cells and the average fluorescence intensity were detected by flow cytometry to assess the transfection efficiency of the cells.

[0091] The results are as follows Figure 3 As shown, 2100, 2101 and 2102 achieved significantly higher cell transfection rates than their similar asymmetric counterparts. The average fluorescence intensity of 2100 and 2102 was also significantly higher than that of their control molecules, indicating that symmetrical structures are more conducive to cell transfection.

[0092] In this embodiment, ALC-0315 is a control compound of compound 2100, and its only difference from compound 2100 is that the carbon chain at the tail is asymmetrical; 2101-X is a control compound of compound 2101, and its only difference from compound 2101 is that the carbon chain at the tail is asymmetrical; 2102-X is a control compound of compound 2102, and its only difference from compound 2102 is that the carbon chain at the tail is asymmetrical.

[0093] The structural formula of ALC-0315 is shown in Formula I-1: Formula I-1; The structural formula of 2101-X is shown in Formula II-1: Formula II-1; The structural formula of 2102-X is shown in Formula III-1: III-1.

[0094] 1.5 Endosome Affinity Study Based on QCM-D QCM-D was used to detect the interaction between simulated endosomes and LNPs. First, simulated endosomes were introduced, and after equilibration, each LNP formulation was introduced, and the change in ΔF was detected.

[0095] Table 2. Endosome Affinity Test Results The results showed that molecules 2100, 2101, and 2102 exhibited significantly higher endosome affinity than their similar asymmetric counterparts. During processing via the endosome pathway into the cell membrane, protons entering the endosome more readily bound to tertiary amines and amides of ionizable lipids, thereby reducing the acidification effect of the endosome. This hindered the conversion of endosomes to lysosomes and enhanced their interaction with mitochondria, potentially promoting cell metabolism and improving the efficiency of T-cell immune responses.

[0096] 1.6 Immune Response Cells were spaced at 5 × 10⁶ cells per well. 5 Cells were seeded in 48-well plates, and LNP preparations were added at a dosage of 5 μg mRNA per million cells per well. After co-incubation with the cells for 6 h, the cells were washed off and then seeded onto cell culture plates for immediate analysis.

[0097] Table 3 OCR Level Detection Results The results showed that compared with the control molecules, 2100, 2101, and 2102 achieved better OCR levels, indicating that T cell mitochondria were significantly activated, thereby promoting cellular metabolism. Furthermore, the synergistic effect of 2102 was more pronounced, possibly due to its more planar molecular configuration, which further enhances the affinity of ionizable lipids for the endosome membrane, reduces endosome acidification, improves interaction with mitochondria, and enhances mitochondrial response efficiency.

[0098] 1.7 In vivo delivery performance of LNP Six- to eight-week-old female C57BL / 6 mice were used. An LNP preparation containing firefly luciferase mRNA was injected into the mice via the tail vein. Six hours later, a fluorescence imaging substrate was injected into the mice via intraperitoneal injection. The mice were then anesthetized with gas, and the fluorescence intensity within the mice was detected using a small animal in vivo imaging system at 5 minutes. The mice were subsequently euthanized by cervical dislocation, and the heart, liver, spleen, lungs, kidneys, and lymph nodes were rapidly separated and their fluorescence intensity was measured.

[0099] The results are as follows Figure 4 As shown, through systemic injection, each LNP formulation was able to deliver mRNA into mice and successfully express it. Among them, the expression of molecule 2102 was relatively high.

[0100] 1.8 Delivery and Immunization Efficacy of gE Vaccine for Herpes Zoster The LNP containing mRNA of herpes zoster antigen gE protein was prepared using the aforementioned preparation method. Subsequently, C57BL / 6 mice (6-8 weeks old, female) were injected intramuscularly with the LNP preparation containing 5 μg of mRNA on days 0 and 28. On day 56, spleen cells were harvested to detect antigen-specific IFN-γ and IL-2 secreting cells in the spleen cells to characterize the cellular immune response.

[0101] The results are as follows Figure 5 As shown, the cellular immune responses of 2100 and 2102 were significantly higher than those of their similar asymmetric molecules, with 2100 stimulating more IFN-γ secretory cells and 2102 stimulating more IL-2 secretory cells.

[0102] 1.9 Application of LNP in in vitro CAR-T LNPs containing CD19 CAR mRNA were prepared using the aforementioned method. Human PBMC cells were resuscitated and cultured at 5 × 10⁶ cells per well. 5 Cells were seeded in 48-well plates, and LNP formulations containing 5 μg CD19 CAR mRNA per million cells were added. After 12 h of transfection, the medium was changed, and the proportion of CAR-positive cells at different time points was detected by flow cytometry to examine the transfection efficiency and expression time of the cells.

[0103] The results are as follows Figures 6-8 As shown, the cell transfection efficiency and expression time of 2100, 2101 and 2102 were significantly higher than those of their similar asymmetric molecules, with 2102 being significantly higher than the other groups.

[0104] Nalm-6-Luc cells were loaded at 5 × 10 4 / The cells were seeded in 96-well plates. LNPs containing CD19CAR mRNA were prepared using the method in Example 1. PBMCs were transfected with LNPs. The required number of CAR-T cells was calculated according to the effector-target ratios of 1:1, 1:2, 1:4, 1:8, and 1:16. The cells were added to the corresponding target cell wells, mixed, and incubated for 24 h. Cells were then lysed to extract luciferase. Luciferase substrate was then added, and the cell lysis rate was calculated by detecting chemiluminescence using a microplate reader.

[0105] Table 4 Cell lysis rate The results showed that 2100, 2101 and 2102 had higher cell lysis rates than their similar asymmetric molecules and were more effective at killing tumor cells.

[0106] 2×10⁻⁶ mice were injected via tail vein. 6 Nalm-6-Luc cells. Two days later, mice were injected via tail vein with 2×10⁶ cells transfected with different LNP formulations. 6 CAR-T cells. Subsequently, in vivo imaging was performed on mice to assess tumor burden.

[0107] Table 5 Results of in vivo imaging assessment in mice The results showed that the tumor burden of 2100, 2101 and 2102 was lower than that of their similar asymmetric molecules, and they had better in vitro CAR-T therapy effects.

[0108] 1.10 Application of LNP in in vivo CAR-T LNP was used to embed CD19 CAR mRNA, conjugated with CD5 antibody, and injected into mice via tail vein. Spleen, PBMCs, and lymph nodes were collected from the mice, and CAR counts were analyzed using flow cytometry. + T cells.

[0109] Table 6 CAR + T cell ratio The results showed that, after conjugation with the antibodies, 2100, 2101, and 2102 could achieve highly efficient T cell transfection in mice via systemic injection, exceeding that of similar asymmetric molecules.

[0110] 2×10⁻⁶ mice were injected via tail vein. 6 Nalm6-Luc cells. Two days later, mice were injected via tail vein with different LNPs containing CD19 CAR mRNA conjugated with CD5 antibodies. In vivo imaging was then performed on the mice to assess tumor burden.

[0111] Table 7 Results of in vivo imaging assessment of mice The results showed that the tumor burden of 2100, 2101 and 2102 was lower than that of their similar asymmetric molecules, and they had better in vivo CAR-T therapy efficacy.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of lipid compounds in the preparation of drug delivery systems, characterized in that, The lipid compound is the compound shown in Formula I: Equation I.

2. The application according to claim 1, characterized in that, The compound shown in Formula I is prepared by the following method: (I-1) React a carboxylic acid compound and a bromoalkane in an organic solvent at 10-30°C for 1-5 h to obtain intermediate I-1; (I-2) In the presence of a catalyst, the intermediate I-1 and the amino alcohol are reacted in an organic solvent at 50-70°C for 10-20 h to obtain compound I.

3. The application according to claim 2, characterized in that, The organic solvent includes at least one of dichloromethane, N,N-dimethylformamide, toluene, acetonitrile, n-hexane, and tetrahydrofuran; the catalyst includes at least one of alkali metal halides and organic bases.

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