Bis-phosphatidyl amine compounds containing multiple tertiary amino structures, and composition and use thereof

Abstract

Disclosed in the present application are bis-phosphatidyl amine compounds containing multiple tertiary amino structures, and a composition and the use thereof. The bis-phosphatidyl amine compounds are phospholipid compounds containing two phosphatidyl amine structures, the structures thereof being represented by following formula (I), and the definition of each substituent group being shown in the description. The compounds act together with other lipid components, such as cholesterol, DSPC / DOPE, DMG-PEG2000 and other assistant lipids so as to form lipid nanoparticles (LNPs). The LNPs can be used for high-efficiency delivery of drug molecules such as nucleic acids (siRNA, mRNA and pDNA), thereby achieving diagnosis and treatment of diseases such as cancers, fibrosis (of the liver, lung, kidney, etc.), and editing immune cells.

Description

Bisphosphatidyl amide compound containing multiple tertiary amino structures, composition and use thereof

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on December 6, 2022, with application number 202211559019.0 and invention name “Phosphatidyl compounds containing multiple tertiary amino structures, compositions and uses thereof”, the contents of which should be understood as incorporated into this application by reference. Technical Field

[0002] The present invention relates to but is not limited to pharmaceutical technology, and in particular to a bisphosphatidyl amide compound containing multiple tertiary amino structures, and its composition and use. Background Art

[0003] Nucleic acid therapy is developing into precision medicine that can target specific genes, placing higher demands on safe and effective delivery systems for target cells. Lipid nanoparticles (LNPs), primarily composed of cationic lipids, neutral lipids, steroid lipids, and polymer-conjugated lipids, have achieved these goals. However, due to differences in the type of action, formulation design, and biodistribution of therapeutic drugs, there remains a demand for highly effective, safe, and stable lipid compounds.

[0004] Summary of the Invention

[0005] In a first aspect, the present application provides a bisphosphatidyl amide compound containing multiple tertiary amino groups, wherein the bisphosphatidyl amide compound is a compound represented by formula (I) or a stereoisomer, prodrug, or pharmaceutically acceptable salt thereof:

[0006] In formula (I), n is an integer from 0 to 5;

[0007] L1 is C1-C 30 Alkyl, C2-C 40 alkenyl, or -R'-MR", and L2 is C5-C 30 Alkyl, C5-C 40 Alkenyl, or -R'-MR"; or, L1 and L2 are each independently C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR";

[0008] R1 and R2 are each independently -ZT, C4-C 30 Alkyl, C2-C 24wherein R1 and R2, together with the N to which they are attached and L4, form a 5- to 8-membered diazacycloalkylene group; wherein Z is an optionally substituted C1-C3 alkylene group, and T is H, hydroxy, C1-C4 alkoxy, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, an optionally substituted 4- to 8-membered azacycloalkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group, wherein the optionally substituted C1-C3 alkylene group, the optionally substituted 4- to 8-membered azacycloalkyl group, the optionally substituted aryl group, or the optionally substituted heteroaryl group is unsubstituted or substituted with one or more groups selected from the group consisting of C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy, and halogen; and wherein -ZT is not C4-C 30 alkyl;

[0009] R3 is H, C1-C 30 Alkyl, C2-C 24 alkenyl, or -R'-MR"; L 1’ C1-C 30 Alkyl, C2-C 40 alkenyl, or -R'-MR", and L 2’ C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR"; or, L 1’ and L 2’ Each independently is C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR";

[0010] R' is C1-C 12 Alkylene or C2-C 12 alkenylene;

[0011] M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -SS-, -C(O)N(R a )-、-N(R a )C(O)-、-OC(O)N(R a )-、-N(R a )C(O)O-、-N(R a )C(O)N(R a )-、-CH(OH)-、-C(OC(O)R a )-C 1-6 Alkylene-OC(O)-, -C(O)-C 1-6Alkylene-C(O)-O-, -P(O)(OR b )O-、C6-C 10 arylene and 5- to 10-membered heteroarylene;

[0012] R”、R a and R b Each independently selected from hydrogen, C1-C 30 Alkyl and C2-C 40 alkenyl;

[0013] L3, L4 and L5 are each independently C1-C 12 Alkylene, C2-C 12 Alkenylene, C3-C8 cycloalkylene, -AYB-, -AY- or -YB-, where A and B are each independently C1-C 12 Alkylene or C2-C 12 Alkenylene, Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene or heteroarylene.

[0014] In a second aspect, the present application provides a method for preparing the above-mentioned bisphosphatidyl amide compound.

[0015] In a third aspect, the present application provides a lipid composition comprising the above-mentioned bisphosphatidylcholine compound and a therapeutic agent or a preventive agent.

[0016] In a fourth aspect, the present application provides a lipid nanoparticle, wherein the lipid nanoparticle comprises the above-mentioned bisphosphatidylcholine compound or the above-mentioned lipid composition.

[0017] In a fifth aspect, the present application provides a pharmaceutical composition comprising the above-mentioned bisphosphatidylcholine compound, the above-mentioned lipid composition or the above-mentioned lipid nanoparticles, and a pharmaceutically acceptable diluent or excipient.

[0018] In a sixth aspect, the present application provides the use of the above-mentioned lipid composition, the above-mentioned lipid nanoparticles or the above-mentioned pharmaceutical composition as a drug for treating or preventing diseases in individuals in need thereof.

[0019] In a seventh aspect, the present application provides the use of the above-mentioned lipid composition, the above-mentioned lipid nanoparticles or the above-mentioned pharmaceutical composition in the preparation of a drug for treating or preventing a disease in an individual in need thereof.

[0020] In an eighth aspect, the present application provides the use of the above-mentioned lipid composition, the above-mentioned lipid nanoparticles or the above-mentioned pharmaceutical composition for vaccinating drugs against viral pathogens.

[0021] In a ninth aspect, the present application provides a method for treating or preventing a disease in an individual in need thereof, wherein the method comprises administering the above-mentioned lipid composition, the above-mentioned lipid nanoparticles or the above-mentioned pharmaceutical composition to the individual.

[0022] In a tenth aspect, the present application provides a method for vaccinating an individual in need against viral pathogens, wherein the method comprises administering the above-mentioned lipid composition, the above-mentioned lipid nanoparticles or the above-mentioned pharmaceutical composition to the individual.

[0023] Other features and advantages of the present application will be described in the following description, and in part will become apparent from the description, or will be understood by practicing the present application. Other advantages of the present application can be realized and obtained through the solutions described in the description and the drawings.

[0024] Summary of the Figures

[0025] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0026] FIG1 shows the in vivo transfection of different lipid nanoparticles in the present application (dosage: 3 μg Fluc-mRNA / mouse), wherein a commercial MC3DSPC preparation was used as a positive control;

[0027] Figure 2 compares the efficiency of expressing Fluc mRNA in activated human primary T cells using bisphosphamide LNPs in this application. 100,000 human T cells were plated in a 96-well plate and treated with approximately 100 ng of mRNA delivered by LNPs. After 24 hours of incubation, bioluminescence was measured using a Biotek microplate reader. Most of the phospholipid LNPs in this application showed an intensity of mRNA delivery to primary T cells comparable to that of the transfection reagent and ALC0315, while PL16 enhanced by 10 times, PL101 enhanced by 100 times, PL102 enhanced by 10 times, PL40 enhanced by 100 times, PL67 enhanced by 60 times, and PL82 enhanced by 5 times.

[0028] Figure 3 shows the expression of Fluc mRNA in activated human NK cells delivered by representative bisphosphamide lipid LNPs of the present invention. 30,000 human NK92 MI cells were plated in a 96-well plate and treated with approximately 100 ng of mRNA delivered by LNPs. After 24 hours of incubation, bioluminescence was measured using a Biotek platereader. Most of the present phospholipid LNPs demonstrated comparable mRNA delivery to NK cells as the transfection reagent and ALC0315, while PL16 enhanced mRNA delivery by 50-fold, PL101 by 35-fold, PL102 by 17-fold, and PL40 by 40-fold.

[0029] Figure 4 shows the physical characterization of the bisphosphoramide LNPs of the present invention. With the exception of PL71 (a single-phosphoramide control), the bisphosphoramide LNPs of the present invention were measured to have a size of approximately 150 nm and a polydispersity index (PDI) below 0.3. Zeta potential measurements showed that most lipid nanoparticles were close to -10 mV to 10 mV, indicating a neutral charge.

[0030] Figure 5 shows the expression of GFP mRNA delivered by representative bisphosphoramide lipid LNPs of the present application in activated human T cells and NK cells. Each well was treated with approximately 100 ng, 500 ng and 1000 ng of mRNA delivered by LNPs. After 40 hours of incubation, the expression of GFP was quantified by flow cytometry. Most bisphosphoramide lipid LNPs showed a dose-dependent increase in mRNA expression in both cell types. In T cells, PL101 and PL40 loaded with GFP mRNA achieved a transfection rate of more than 80% at a dose of 0.5 ug / well and 1.0 ug / well, and PL16 was about 55%. The transfection rate of PL16 lipid nanoparticles loaded with GFP mRNA in NK92MI reached more than 75% at a dose of 0.5 ug / well and 1.0 ug / well, while PL101 and PL40 were 40%;

[0031] Figure 6 shows the expression of CAR-CD19 mRNA in activated human T and NK cells. Each well was treated with approximately 100 ng, 500 ng and 1000 ng of mRNA delivered by LNPs. After 40 hours of incubation, the expression of GFP was quantified by flow cytometry. Most of the bisphosphoramide lipid LNPs in this application showed a dose-dependent increase in mRNA expression in both cell types. The expression of CAR-CD19 on the cell surface was slightly lower than that of GFP. In T cells, PL40 had the highest transfection efficiency, about 40%, followed by PL101, which reached 35% at 1ug / well, and PL32 reached 15%; in NK92MI cells, the transfection efficiency of the three lipid nanoparticles was low, PL101 increased by about 10% compared to the control lipid, and PL101 and PL32 were only about 6%;

[0032] Figure 7 shows cytotoxic CAR-CD19-expressing T cells targeting CD19-positive NALM6 B cells. a. Cytotoxic T cell activity was measured by fluorescence assay using GFP-expressing NALM6 cells overexpressing human CD19 as targets. E:T ratio, ratio of effector cells to target cells; CD19CAR, T cells transfected with mRNA encoding CD19-CAR delivered by LNPs. Data represent n = 3 independent experiments. Target and effector cells were co-cultured for 24 hours. Fluorescence microscopy revealed that as the number of CD19-CAR-positive effector cells increased, the number of NALM6 cells decreased significantly, while there was no significant change in the control group, indicating that CD19 CAR-positive T cells had a significant specific killing effect on NALM6 cells. b. The cytotoxicity of CD19 CAR T cells was determined using the CytoTox 96 non-radioactive cytotoxicity assay (Promega). GFP-expressing NALM6 cells were co-cultured with CAR-CD19, control CAR, or control T cells for 24 hours. n = 3. Cytotoxicity was measured by LDH assay. The results showed that target cells were killed when co-cultured with corresponding CAR T cells, while no obvious cytotoxicity was observed when co-cultured with irrelevant CAR T cells and control T cells;

[0033] Figure 8 shows the in vivo transfection of the present invention's bisphosphoramide lipids in mouse spleen cells. a. Evaluation of PL40 LNP distribution in loxP-GFP-luciferase mice. PL40 LNPs were used to transfect CRE mRNA. Two days later, expression of Cre recombinase and luciferase was assessed using IVIS. b. Cell populations expressing CRE mRNA delivered by PL40 LNPs in the spleen. Flow cytometric analysis of GFP demonstrated that PL40 LNPs can transfect both T cells and myeloid cells. Transfection intensity was similar for CD4+ T cells and CD8+ T cells.

[0034] Details

[0035] In a first aspect, the present application provides a bisphosphatidyl amide compound containing multiple tertiary amino groups, wherein the bisphosphatidyl amide compound is a compound represented by formula (I) or a stereoisomer, prodrug, or pharmaceutically acceptable salt thereof:

[0036] In formula (I), n is an integer from 0 to 5;

[0037] L1 is C1-C 30 Alkyl, C2-C 40 alkenyl, or -R'-MR", and L2 is C5-C 30 Alkyl, C5-C 40Alkenyl, or -R'-MR"; or, L1 and L2 are each independently C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR";

[0038] R1 and R2 are each independently -ZT, C4-C 30 Alkyl, C2-C 24 wherein R1 and R2, together with the N to which they are attached and L4, form a 5- to 8-membered diazacycloalkylene group; wherein Z is an optionally substituted C1-C3 alkylene group, and T is H, hydroxy, C1-C4 alkoxy, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, an optionally substituted 4- to 8-membered azacycloalkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group, wherein the optionally substituted C1-C3 alkylene group, the optionally substituted 4- to 8-membered azacycloalkyl group, the optionally substituted aryl group, or the optionally substituted heteroaryl group is unsubstituted or substituted with one or more groups selected from the group consisting of C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy, and halogen; and wherein -ZT is not C4-C 30 alkyl;

[0039] R3 is H, C1-C 30 Alkyl, C2-C 24 alkenyl, or -R'-MR"; L 1’ C1-C 30 Alkyl, C2-C 40 alkenyl, or -R'-MR", and L 2’ C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR"; or, L 1’ and L 2’ Each independently is C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR";

[0040] R' is C1-C 12 Alkylene or C2-C 12 alkenylene;

[0041] M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -SS-, -C(O)N(R a )-、-N(R a )C(O)-、-OC(O)N(Ra )-、-N(R a )C(O)O-、-N(R a )C(O)N(R a )-、-CH(OH)-、-C(OC(O)R a )-C 1-6 Alkylene-OC(O)-, -C(O)-C 1-6 Alkylene-C(O)-O-, -P(O)(OR b )O-、C6-C 10 arylene and 5- to 10-membered heteroarylene;

[0042] R”、R a and R b Each independently selected from hydrogen, C1-C 30 Alkyl and C2-C 40 alkenyl;

[0043] L3, L4 and L5 are each independently C1-C 12 Alkylene, C2-C 12 Alkenylene, C3-C8 cycloalkylene, -AYB-, -AY- or -YB-, where A and B are each independently C1-C 12 Alkylene or C2-C 12 Alkenylene, Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene or heteroarylene.

[0044] In some embodiments of the first aspect, in formula (I), n is an integer from 0 to 5;

[0045] L1 and L2 are each independently C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR";

[0046] R1 and R2 are each independently C4-C 30 Alkyl, C2-C 24 alkenyl, or -R'-MR"; or, R1 and R2 together with the N to which they are attached and L4 form a 5- to 8-membered diazacycloalkylene group;

[0047] R3 is H, C1-C 30 Alkyl, C2-C 24 alkenyl, or -R'-MR"; L 1’ and L 2’ Each independently is C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR";

[0048] R' is C1-C 12 Alkylene or C2-C 12 alkenylene;

[0049] M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -SS-, -C(O)N(R a )-、-N(R a )C(O)-、-OC(O)N(R a )-、-N(R a )C(O)O-、-N(R a )C(O)N(R a )-、-CH(OH)-、-C(OC(O)R a )-C 1-6 Alkylene-OC(O)-, -C(O)-C 1-6 Alkylene-C(O)-O-, -P(O)(OR b )O-、C6-C 10 arylene and 5- to 10-membered heteroarylene;

[0050] R”、R a and R b Each independently selected from hydrogen, C1-C 30 Alkyl and C2-C 40 alkenyl;

[0051] L3, L4 and L5 are each independently C1-C 12 Alkylene, C2-C 12 Alkenylene, C3-C8 cycloalkylene, -AYB-, -AY- or -YB-, where A and B are each independently C1-C 12 Alkylene or C2-C 12 Alkenylene, Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene or heteroarylene.

[0052] In some embodiments of the first aspect, in formula (I), n is an integer from 0 to 5;

[0053] L1 is C1-C 30 Alkyl, C2-C 40 alkenyl, or -R'-MR", and L2 is C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR";

[0054] R1 and R2 are each independently -ZT, C4-C 30 Alkyl, C2-C 24 wherein R1 and R2, together with the N to which they are attached and L4, form a 5- to 8-membered diazacycloalkylene group; wherein Z is an optionally substituted C1-C3 alkylene group, and T is H, hydroxy, C1-C4 alkoxy, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, an optionally substituted 4- to 8-membered azacycloalkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group, wherein the optionally substituted C1-C3 alkylene group, the optionally substituted 4- to 8-membered azacycloalkyl group, the optionally substituted aryl group, or the optionally substituted heteroaryl group is unsubstituted or substituted with one or more groups selected from the group consisting of C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy, and halogen; and wherein -ZT is not C4-C 30 alkyl;

[0055] R3 is H, C1-C 30 Alkyl, C2-C 24 alkenyl, or -R'-MR"; L 1’ C1-C 30 Alkyl, C2-C 40 alkenyl, or -R'-MR", and L 2’ C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR";

[0056] R' is C1-C 12 Alkylene or C2-C 12 alkenylene;

[0057] M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -SS-, -C(O)N(R a )-、-N(R a )C(O)-、-OC(O)N(R a )-、-N(R a )C(O)O-、-N(R a )C(O)N(R a )-、-CH(OH)-、-C(OC(O)R a )-C 1-6 Alkylene-OC(O)-, -C(O)-C 1-6Alkylene-C(O)-O-, -P(O)(OR b )O-、C6-C 10 arylene and 5- to 10-membered heteroarylene;

[0058] R”、R a and R b Each independently selected from hydrogen, C1-C 30 Alkyl and C2-C 40 alkenyl;

[0059] L3, L4 and L5 are each independently C1-C 12 Alkylene, C2-C 12 Alkenylene, C3-C8 cycloalkylene, -AYB-, -AY- or -YB-, where A and B are each independently C1-C 12 Alkylene or C2-C 12 Alkenylene, Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene or heteroarylene.

[0060] In some embodiments of the first aspect, n is 0, and the compound of formula (I) is a compound of formula (I-1):

[0061] In formula (I-1), L1 and L2 are each independently C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR";

[0062] R1 is C4-C 30 Alkyl, C2-C 24 alkenyl, or -R'-MR";

[0063] R3 is H, C1-C 30 Alkyl, C2-C 24 alkenyl, or -R'-MR"; L 1’ and L 2’ Each independently is C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR": optionally, L 1’ is the same group as L1, and L 2’ is the same group as L2:

[0064] R' is C1-C 12 Alkylene or C2-C 12 alkenylene;

[0065] M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -SS-, -C(O)N(R a )-、-N(R a )C(O)-、-OC(O)N(R a )-、-N(R a )C(O)O-、-N(R a )C(O)N(R a )-、-CH(OH)-、-C(OC(O)R a )-C 1-6 Alkylene-OC(O)-, -C(O)-C 1-6 Alkylene-C(O)-O-, -P(O)(OR b )O-、C6-C 10 arylene and 5- to 10-membered heteroarylene;

[0066] R”、R a and R b Each independently selected from hydrogen, C1-C 30 Alkyl and C2-C 40 alkenyl;

[0067] L3 and L5 are each independently C1-C 12 Alkylene, C2-C 12 Alkenylene, C3-C8 cycloalkylene, -AYB-, -AY- or -YB-, where A and B are each independently C1-C 12 Alkylene or C2-C 12 Alkenylene, Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene or heteroarylene; or

[0068] n is 0, and the compound of formula (I) is the compound of formula (I-1-1):

[0069] Each substituent in formula (I-1-1) is as defined above.

[0070] In some embodiments of the first aspect, n is 0, and the compound of formula (I) is a compound of formula (I-1):

[0071] In formula (I-1), L1 is C1-C 30 Alkyl, C2-C 40 alkenyl, or -R'-MR", and L2 is C5-C30 Alkyl, C5-C 40 alkenyl, or -R'-MR";

[0072] R1 is -ZT, C4-C 30 Alkyl, C2-C 24 wherein Z is an optionally substituted C1-C3 alkylene group, and T is H, hydroxyl, C1-C4 alkoxyl, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, an optionally substituted 4- to 8-membered azacycloalkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group, wherein the optionally substituted C1-C3 alkylene group, the optionally substituted 4- to 8-membered azacycloalkyl group, the optionally substituted aryl group, or the optionally substituted heteroaryl group is unsubstituted or substituted with one or more groups selected from the group consisting of C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxyl, C1-C6 haloalkoxyl, hydroxyl, and halogen; and wherein -ZT is not C4-C 30 alkyl;

[0073] R3 is H; L 1’ C1-C 30 Alkyl, C2-C 40 alkenyl, or -R'-MR", and L 2’ C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR"; optionally, L 1’ is the same group as L1, and L 2’ is the same group as L2:

[0074] R' is C1-C 12 Alkylene or C2-C 12 alkenylene;

[0075] M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -SS-, -C(O)N(R a )-、-N(R a )C(O)-、-OC(O)N(R a )-、-N(R a )C(O)O-、-N(R a )C(O)N(R a )-、-CH(OH)-、-C(OC(O)R a )-C 1-6 Alkylene-OC(O)-, -C(O)-C 1-6Alkylene-C(O)-O-, -P(O)(OR b )O-、C6-C 10 arylene and 5- to 10-membered heteroarylene;

[0076] R”、R a and R b Each independently selected from hydrogen, C1-C 30 Alkyl and C2-C 40 alkenyl;

[0077] L3 and L5 are each independently C1-C 12 Alkylene, C2-C 12 Alkenylene, C3-C8 cycloalkylene, -AYB-, -AY- or -YB-, where A and B are each independently C1-C 12 Alkylene or C2-C 12 Alkenylene, Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene or heteroarylene; or

[0078] n is 0, and the compound of formula (I) is the compound of formula (I-1-1):

[0079] Each substituent in formula (I-1-1) is as defined above.

[0080] In some embodiments of the first aspect, n is 1, and the compound of formula (I) is a compound of formula (I-2):

[0081] L1 and L2 are each independently C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR";

[0082] R1 and R2 together with the N to which they are attached and L4 form a 5- to 8-membered diazacycloalkylene group; or, R1 and R2 are each independently C4-C 30 Alkyl, C2-C 24 Alkenyl, or -R'-MR", L4 is C1-C 12 Alkylene, C2-C 12 Alkenylene, C3-C8 cycloalkylene, -AYB-, -AY- or -YB-, where A and B are each independently C1-C 12 Alkylene or C2-C 12 alkenylene, Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene, or heteroarylene;

[0083] R3 is H, C1-C 30 Alkyl, C2-C 24 alkenyl, or -R'-MR"; L 1’ and L 2’ Each independently is C5-C 30 Alkyl, C5-C 40 , or -R'-MR"; optionally, L 1’ is the same group as L1, and L 2’ is the same group as L2;

[0084] R' is C1-C 12 Alkylene or C2-C 12 alkenylene;

[0085] M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -SS-, -C(O)N(R a )-、-N(R a )C(O)-、-OC(O)N(R a )-、-N(R a )C(O)O-、-N(R a )C(O)N(R a )-、-CH(OH)-、-C(OC(O)R a )-C 1-6 Alkylene-OC(O)-, -C(O)-C 1-6 Alkylene-C(O)-O-, -P(O)(OR b )O-、C6-C 10 arylene and 5- to 10-membered heteroarylene;

[0086] R”、R a and R b Each independently selected from hydrogen, C1-C 30 Alkyl and C2-C 40 alkenyl;

[0087] L3 and L5 are each independently C1-C 12 Alkylene, C2-C 12 Alkenylene, C3-C8 cycloalkylene, -AYB-, -AY- or -YB-, where A and B are each independently C1-C 12 Alkylene or C2-C 12Alkenylene, Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene or heteroarylene; or

[0088] n is 1, and the compound of formula (I) is a compound of formula (I-2-1):

[0089] Each substituent in formula (I-2-1) is as defined above.

[0090] In some embodiments of the first aspect, wherein n is 1, the compound of formula (I) is a compound of formula (I-2):

[0091] L1 is C1-C 30 Alkyl, C2-C 40 alkenyl, or -R'-MR", and L2 is C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR";

[0092] R1 and R2 together with the N to which they are attached and L4 form a 5- to 8-membered diazacycloalkylene group; or, R1 and R2 are each independently -ZT, C4-C 30 Alkyl, C2-C 24 -R'-MR", where Z is an optionally substituted C1-C3 alkylene, and T is H, hydroxy, C1-C4 alkoxy, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, an optionally substituted 4- to 8-membered azacycloalkyl, an optionally substituted aryl, or an optionally substituted heteroaryl, wherein the optionally substituted C1-C3 alkylene, the optionally substituted 4- to 8-membered azacycloalkyl, the optionally substituted aryl, or the optionally substituted heteroaryl is unsubstituted or substituted with one or more groups selected from the group consisting of C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy, and halogen; and -ZT is not C4-C 30 Alkyl; L4 is C1-C 12 Alkylene, C2-C 12 Alkenylene, C3-C8 cycloalkylene, -AYB-, -AY- or -YB-, where A and B are each independently C1-C 12 Alkylene or C2-C 12 alkenylene, Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene, or heteroarylene;

[0093] R3 is H, C1-C30 Alkyl, C2-C 24 alkenyl, or -R'-MR"; L 1’ C1-C 30 Alkyl, C2-C 40 alkenyl, or -R'-MR", and L 2’ C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR"; optionally, L 1’ is the same group as L1, and L 2’ is the same group as L2;

[0094] R' is C1-C 12 Alkylene or C2-C 12 alkenylene;

[0095] M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -SS-, -C(O)N(R a )-、-N(R a )C(O)-、-OC(O)N(R a )-、-N(R a )C(O)O-、-N(R a )C(O)N(R a )-、-CH(OH)-、-C(OC(O)R a )-C 1-6 Alkylene-OC(O)-, -C(O)-C 1-6 Alkylene-C(O)-O-, -P(O)(OR b )O-、C6-C 10 arylene and 5- to 10-membered heteroarylene;

[0096] R”、R a and R b Each independently selected from hydrogen, C1-C 30 Alkyl and C2-C 40 alkenyl;

[0097] L3 and L5 are each independently C1-C 12 Alkylene, C2-C 12 Alkenylene, C3-C8 cycloalkylene, -AYB-, -AY- or -YB-, where A and B are each independently C1-C 12 Alkylene or C2-C 12Alkenylene, Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene or heteroarylene; or

[0098] n is 1, and the compound of formula (I) is a compound of formula (I-2-1):

[0099] Each substituent in formula (I-2-1) is as defined above.

[0100] In some embodiments of the first aspect, L 1’ , L 2’ , L1 and L2 are each independently C6-C 40 Alkyl, C6-C 40 alkenyl, or -R'-MR"; where R' is C1-C 10 Alkylene or C2-C 10 Alkenylene; M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -SS-, -C(O)N(R a )-、-N(R a )C(O)-、-OC(O)N(R a )-、-N(R a )C(O)O-、-N(R a )C(O)N(R a )-、-CH(OH)-、-C(OC(O)R a )-C 1-6 Alkylene-OC(O)-, -C(O)-C 1-6 Alkylene-C(O)-O-, -P(O)(OR b )O-、C6-C 10 Arylene and 5- to 10-membered heteroarylene; R" is C1-C 30 Alkyl or C2-C 40 Alkenyl; R a and R b Each independently selected from hydrogen, C1-C 30 Alkyl and C2-C 40 Alkenyl.

[0101] In some embodiments of the first aspect, L 1’ , L 2’ , L1 and L2 are each independently C6-C 30 Alkyl, C6-C 40 alkenyl, or -R'-MR"; where R' is C1-C10 Alkylene or C2-C 10 Alkenylene; M is -C(O)-, -C(O)O-, -C(OC(O)R a )-C 1-6 Alkylene-OC(O)-, -C(O)-C 1-6 Alkylene-C(O)-O- or -CH(OH)-; R a and R" are each independently C1-C 30 Alkyl or C2-C 40 Alkenyl.

[0102] In some embodiments of the first aspect, L 1’ , L 2’ , L1 and L2 are each independently one of the following structures:

[0103] In some embodiments of the first aspect, L 1’ , L 2’ , L1 and L2 are each independently one of the following structures:

[0104] In some embodiments of the first aspect, L1 is C1-C 30 Alkyl, C2-C 40 alkenyl, or -R'-MR", and L2 is C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR"; L 1’ C1-C 30 Alkyl, C2-C 40 alkenyl, or -R'-MR", and L 2’ C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR"; where R' is C1-C 10 Alkylene or C2-C 10 Alkenylene; M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -SS-, -C(O)N(R a )-、-N(R a )C(O)-、-OC(O)N(R a )-、-N(R a )C(O)O-、-N(R a)C(O)N(R a )-、-CH(OH)-、-C(OC(O)R a )-C 1-6 Alkylene-OC(O)-, -C(O)-C 1-6 Alkylene-C(O)-O-, -P(O)(OR b )O-、C6-C 10 Arylene and 5- to 10-membered heteroarylene; R" is C1-C 30 Alkyl or C2-C 40 Alkenyl; R a and R b Each independently selected from hydrogen, C1-C 30 Alkyl and C2-C 40 Alkenyl.

[0105] In some embodiments of the first aspect, L1 is C1-C 30 Alkyl, C2-C 40 alkenyl, or -R'-MR", and L2 is C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR"; L 1’ C1-C 30 Alkyl, C2-C 40 alkenyl, or -R'-MR", and L 2’ C5-C 30 Alkyl, C5-C 40 alkenyl, or -R'-MR"; where R' is C1-C 10 Alkylene or C2-C 10 Alkenylene; M is -C(O)-, -C(O)O-, -C(OC(O)R a )-C 1-6 Alkylene-OC(O)-, -C(O)-C 1-6 Alkylene-C(O)-O-, or -CH(OH)-; R a and R" are each independently C1-C 30 Alkyl or C2-C 40 Alkenyl.

[0106] In some embodiments of the first aspect, L 1’ , L 2’ , L1 and L2 are each independently one of the following structures:

[0107] Or, L 1’and L1 are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, L 2’ and L2 are as defined above.

[0108] In some embodiments of the first aspect, L 1’ , L 2’ , L1 and L2 are each independently one of the following structures:

[0109] Or, L 1’ and L1 are each independently ethyl, L 2’ and L2 are as defined above.

[0110] In some embodiments of the first aspect, R1 and R2 are each independently one of the following structures:

[0111] In some embodiments of the first aspect, R1 and R2 are each independently one of the following structures:

[0112] In some embodiments of the first aspect, R1 and R2 are each independently one of the following structures or groups:

[0113] methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxy-n-propyl, benzyl, 2-phenylethyl, 3-phenylpropyl, dimethylaminomethyl, 2-(dimethylamino)ethyl, 3-(dimethylamino)propyl, diethylaminomethyl, 2-(diethylamino)ethyl, 3-(diethylamino)propyl, (pyrrolidin-1-yl)methyl, 2-(pyrrolidin-1-yl)ethyl, 3-(pyrrolidin-1-yl)propyl, 2-methyl-3-(pyrrolidin-1-yl)propyl, (piperidin-1-yl)methyl, 2-(piperidin-1-yl)ethyl, 3-(piperidin-1-yl)propyl, (2-ethylpiperidin-1-yl)methyl, 2-(2-ethylpiperidin-1-yl)ethyl , 3-(2-ethylpiperidin-1-yl)propyl, (3-ethyl-6-methylpiperidin-1-yl)methyl, 2-(3-ethyl-6-methylpiperidin-1-yl)ethyl, 3-(3-ethyl-6-methylpiperidin-1-yl)propyl, (azepan-1-yl)methyl, 2-(azepan-1-yl)ethyl, 3-(azepan-1-yl)propyl, (piperazin-1-yl)methyl, 2-(piperazin-1-yl)ethyl, 3-(piperazin-1-yl)propyl, (4-methylpiperazin-1-yl)methyl, 2-(4-methylpiperazin-1-yl)ethyl, 3-(4-methylpiperazin-1-yl)propyl, (morpholin-1-yl)methyl, 2-(morpholin-1-yl)ethyl, 3-(morpholin-1-yl)propyl,

[0114] In some embodiments of the first aspect, R1 and R2 are each independently one of the following structures or groups:

[0115] methyl, 2-hydroxyethyl, 3-hydroxy-n-propyl, 3-phenylpropyl, 3-(diethylamino)propyl, (pyrrolidin-1-yl)methyl, 2-(pyrrolidin-1-yl)ethyl, 3-(pyrrolidin-1-yl)propyl, 2-methyl-3-(pyrrolidin-1-yl)propyl, 2-(piperidin-1-yl)ethyl, (2-ethylpiperidin-1-yl)methyl, 2-(2-ethylpiperidin-1-yl)ethyl, 3-(2-ethylpiperidin-1-yl)propyl, (3 1-yl)ethyl, 2-(3-ethyl-6-methylpiperidin-1-yl)ethyl, 3-(3-ethyl-6-methylpiperidin-1-yl)propyl, 2-(azepan-1-yl)ethyl, 3-(azepan-1-yl)propyl, (piperazin-1-yl)methyl, 2-(piperazin-1-yl)ethyl, 3-(piperazin-1-yl)propyl, 3-(4-methylpiperazin-1-yl)propyl, 2-(morpholin-1-yl)ethyl,

[0116] In some embodiments of the first aspect, R1 and R2 together with the N to which they are respectively attached and L4 form a group selected from the group consisting of the optionally substituted:

[0117] In some embodiments of the first aspect, R1 and R2 together with the N to which they are respectively attached and L4 form the following group which is optionally substituted:

[0118] In some embodiments of the first aspect, L3 and L5, or when L4 is present, L3, L4 and L5 are each independently methylene, ethylene, propylene, butylene, pentylene, hexylene, vinylene, propenylene, butenylene, pentenylene, hexenylene, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, -AYB-, -AY- or -YB-, where A and B are each independently is methylene, ethylene, propylene, butylene, pentylene, hexylene, vinylene, propenylene, butenylene, pentenylene or hexenylene, and Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, phenylene, pyrrolylene, imidazolylene, thiazolylene, thienylene, furylene, pyridylene or pyrimidylene.

[0119] In some embodiments of the first aspect, L3 and L5, or when L4 is present, L3, L4 and L5 are each independently -CH2-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)CH2-, -CH2CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH H2CH2CH(CH3)-, -AYB-, -AY- or -YB-, where A and B are each independently -CH2-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2- or -CH2CH2CH(CH3)-, and Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)- or phenylene.

[0120] In some embodiments of the first aspect, L3 is -CH2-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)CH2-, -CH2CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH(CH3) )-, -AYB-, -AY- or -YB-, where A and B are each independently -CH2-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2- or -CH2CH2CH(CH3)-, and Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-.

[0121] In some embodiments of the first aspect, L3 is -CH2CH2- or -CH2CH2CH2-.

[0122] In some embodiments of the first aspect, L4 is -CH2-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)CH2-, -CH2CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH(CH3) )-, -AYB-, -AY- or -YB-, where A and B are each independently -CH2-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2- or -CH2CH2CH(CH3)-, and Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-.

[0123] In some embodiments of the first aspect, L4 is -CH2CH2- or -CH2CH2CH2-.

[0124] In some embodiments of the first aspect, L5 is -CH2-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH(CH3)- , -AYB-, -AY- or -YB-, where A and B are each independently -CH2-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2- or -CH2CH2CH(CH3)-, and Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)- or phenylene.

[0125] In some embodiments of the first aspect, L5 is -CH2CH2-, -CH2CH2CH2-, -CH2CH(OH)CH2-, or

[0126] In some embodiments of the first aspect, n is 0; R1 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxy-n-propyl, benzyl, 2-phenylethyl, 3-phenylpropyl, dimethylaminomethyl, 2-(dimethylamino)ethyl, 3-(dimethylamino)propyl, diethylaminomethyl, 2-(diethylamino)ethyl, 3-(diethylamino)propyl, (pyrrolidin-1-yl)methyl, 2-(pyrrolidin-1-yl)ethyl, 3-(pyrrolidin-1-yl)propyl, 2-methyl-3-(pyrrolidin-1-yl)propyl, (piperidin-1-yl)methyl, 2-(piperidin-1-yl)ethyl, 3-(piperidin-1-yl)propyl, (2-ethylpiperidin-1-yl)methyl, 2-(2- 1-yl)ethyl, (3-(2-ethylpiperidin-1-yl)propyl, (3-ethyl-6-methylpiperidin-1-yl)methyl, 2-(3-ethyl-6-methylpiperidin-1-yl)ethyl, 3-(3-ethyl-6-methylpiperidin-1-yl)propyl, (azepan-1-yl)methyl, 2-(azepan-1-yl)ethyl, 3-(azepan-1-yl)propyl, (piperazin-1-yl)methyl, 2-(piperazin-1-yl)ethyl, 3-(piperazin-1-yl)propyl, (4-methylpiperazin-1-yl)methyl, 2-(4-methylpiperazin-1-yl)ethyl, 3-(4-methylpiperazin-1-yl)propyl, (morpholin-1-yl)methyl, 2-(morpholin-1-yl)ethyl, or 3-(morpholin-1-yl)propyl;

[0127] R3 is H;

[0128] L 1’ , L 2’ , L1 and L2 are each independently one of the following structures:

[0129] Or, L 1’ and L1 are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, L 2’ and L2 is as defined above;

[0130] L3 is -CH2CH2- or -CH2CH2CH2-;

[0131] L5 is -CH2CH2-, -CH2CH2CH2-, -CH2CH(OH)CH2- or

[0132] In some embodiments of the first aspect, n is 1; R1 and R2 together with the N to which they are respectively attached and L4 form the following group which is optionally substituted:

[0133] L3 is -CH2CH2- or -CH2CH2CH2-;

[0134] L5 is -CH2CH2-, -CH2CH2CH2- or -CH2CH(OH)CH2-;

[0135] R3 is H;

[0136] L 1’ , L 2’ , L1 and L2 are each independently one of the following structures:

[0137] Or, L 1’ and L1 are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, L 2’ and L2 are as defined above.

[0138] In some embodiments of the first aspect, L1 and L2 are each independently C5-C 30 Alkyl, C5-C 24 wherein R', M and R" are as defined above.

[0139] In some embodiments of the first aspect, L 1’ and L 2’ Each independently is C5-C 30 Alkyl, C5-C 24 wherein R', M and R" are as defined above.

[0140] In some embodiments of the first aspect, R″, R a and R b Each independently selected from hydrogen, C1-C 12 Alkyl and C2-C 12 Alkenyl.

[0141] In some embodiments of the first aspect, the present application provides a bisphosphatidyl amide compound selected from one of the following compounds:

[0142] or a stereoisomer, prodrug, or pharmaceutically acceptable salt thereof.

[0143] In a second aspect, the present application provides a method for preparing the above-mentioned bisphosphatidyl amide compound, the preparation method comprising the following steps:

[0144] The compound of formula (II) is subjected to amidation reaction with the compound of formula (III) to obtain the compound of formula (I)

[0145] Here, X in formula (III) is a leaving group, such as chlorine, bromine or iodine; the definitions of other groups in the compounds of formula (II) and formula (III) are the same as those of the corresponding groups in the compounds of formula (I).

[0146] In a third aspect, the present application provides a lipid composition comprising the above-mentioned bisphosphatidylcholine compound and a therapeutic agent or a preventive agent.

[0147] In some embodiments of the third aspect, the lipid composition further comprises an additional lipid selected from one or more of a neutral lipid, a steroid, and a polymer-conjugated lipid.

[0148] In some embodiments of the third aspect, the neutral lipid is selected from one or more of DSPC, DPPC, DOPC, POPC, DOPE, DSPG, DOPG, DOPS, DGTS, DOPA and SM, preferably DOPE.

[0149] In some embodiments of the third aspect, the molar ratio of the neutral lipid to the bisphosphatidylcholine compound of the present application is 2:1 to 8:1.

[0150] In some embodiments of the third aspect, the steroid is cholesterol.

[0151] In some embodiments of the third aspect, the molar ratio of the steroid to the bisphosphatidylcholine compound of the present application is 5:1 to 1:1.

[0152] In some embodiments of the third aspect, the polymer-conjugated lipid is a PEGylated lipid selected from the group consisting of PEG-DAG, PEG-PE, PEG-S-DAG (diacylglycerol) and PEG-cer (ceramide).

[0153] In some embodiments of the third aspect, the therapeutic agent or preventive agent is selected from one or more of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides and protein drugs.

[0154] In some embodiments of the third aspect, the lipid composition is in the form of lipid nanoparticles.

[0155] In a fourth aspect, the present application provides a lipid nanoparticle, wherein the lipid nanoparticle comprises the above-mentioned bisphosphatidylcholine compound or the above-mentioned lipid composition.

[0156] In a fifth aspect, the present application provides a pharmaceutical composition comprising the above-mentioned bisphosphatidylcholine compound, the above-mentioned lipid composition or the above-mentioned lipid nanoparticles, and a pharmaceutically acceptable diluent or excipient.

[0157] In a sixth aspect, the present application provides the use of the above-mentioned lipid composition, the above-mentioned lipid nanoparticles or the above-mentioned pharmaceutical composition as a drug for treating or preventing diseases in individuals in need thereof.

[0158] In a seventh aspect, the present application provides the use of the above-mentioned lipid composition, the above-mentioned lipid nanoparticles or the above-mentioned pharmaceutical composition in the preparation of a drug for treating or preventing a disease in an individual in need thereof; optionally, the disease is fatty liver, obesity, tumor, arthritis, viral infection or autoimmune disease; or T cells, macrophages or NK cells are edited in vivo for the treatment of cancer or autoimmune diseases.

[0159] In an eighth aspect, the present application provides the use of the above-mentioned lipid composition, the above-mentioned lipid nanoparticles or the above-mentioned pharmaceutical composition for vaccinating drugs against viral pathogens.

[0160] In a ninth aspect, the present application provides a method for treating or preventing a disease in an individual in need thereof, the method comprising administering the above-mentioned lipid composition, the above-mentioned lipid nanoparticles or the above-mentioned pharmaceutical composition to the individual; optionally, the disease is fatty liver, obesity, tumor, arthritis, viral infection or autoimmune disease; or editing T cells, macrophages or NK cells in vivo for the treatment of cancer or autoimmune diseases.

[0161] In a tenth aspect, the present application provides a method for vaccinating an individual in need against viral pathogens, wherein the method comprises administering the above-mentioned lipid composition, the above-mentioned lipid nanoparticles or the above-mentioned pharmaceutical composition to the individual. DETAILED DESCRIPTION

[0162] To make the purpose, technical solutions and advantages of this application more clear, the embodiments of the present invention will be described in detail below. It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of this application can be combined with each other in any way.

[0163] abbreviation

[0164] DCM dichloromethane

[0165] EtOH

[0166] MeOH methanol

[0167] DMF N,N-dimethylformamide

[0168] DCC N,N′-dicyclohexylcarbodiimide

[0169] DMAP 4-dimethylaminopyridine

[0170] DDQ Dichlorodicyanobenzoquinone

[0171] NaH sodium hydride

[0172] Et3N triethylamine

[0173] TFA trifluoroacetic acid

[0174] (Boc)2O Di-tert-butyl dicarbonate

[0175] Tris-EDTA

[0176] DOPE Dioleoylphosphatidylethanolamine

[0177] DSPC Distearoylphosphatidylcholine

[0178] DMG-PEG 2000 1,2-Dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000

[0179] RT Room temperature

[0180] Experimental Materials

[0181] Linolenic alcohol, 4-methoxybenzyl chloride, cis-3-dodecanol, 5-bromovaleric acid, 4-hydroxybutyric acid, 1-undecanol, and DDQ were purchased from Shanghai Bidex Pharmaceutical Co., Ltd.; 9-heptadecanol, 3-undecanol, 1,8-octanediol, 2-hexyl-1-decanol, NaH, DCC, DMAP, di-tert-butyl dicarbonate, ultra-dry dichloromethane, and deuterated chloroform were purchased from Beijing Bailingwei Technology Co., Ltd.; triethylamine, phosphorus oxychloride, 1,4-bis(3-aminopropyl)piperazine, and N,N-dimethyl-1,3-propanediamine were purchased from Beijing Inokai Technology Co., Ltd.; 1-octadecanol was purchased from Alfa Aesar (China) Chemical Co., Ltd.; the dialysis bag was a regenerated cellulose membrane with a pore size of 10 kDa; D-fluorescein sodium salt was purchased from Shandong Kangside Biotechnology Co., Ltd.; and RiboGreen reagent was purchased from Invitrogen, USA. DMEM medium, penicillin-streptomycin double antibody, and 0.25% trypsin containing EDTA were purchased from Beijing Zhongke Maichen Technology Co., Ltd. Fetal bovine serum was purchased from PAN, Germany. Cell culture dishes and culture plates were purchased from Corning, USA.

[0182] Experimental instruments

[0183] Electrospray ionization mass spectrometer (Xevo G2 Q-TOF, Waters, USA), nuclear magnetic resonance spectrometer (AVANCE III HD400M, Bruker, Switzerland), pure water / ultrapure water integrated system (Milli-Q Intergral 10, Merck Millipore, USA), nanoparticle size and zeta potential analyzer (Zetasizer Nano ZSP, MalVERN, UK), microplate reader (Synergy H1, Biotek, USA), small animal in vivo imager (Lumina Series III, PerkinElmer, USA), and rotary evaporator (Hei-VAP Value Digital, Heidolph, Germany).

[0184] Reference example 1

[0185] Synthesis of PL32

[0186] 1,8-Octanediol (730 mg, 5 mmol) was first dissolved in 5 mL of THF. NaH (240 mg, 10 mmol) was then added under ice and allowed to react for 10 minutes. 4-Methoxybenzyl chloride (783 mg, 5 mmol) was then added dropwise to the system and allowed to react overnight at room temperature. Upon completion of the reaction, water was added to quench the reaction, followed by extraction with DCM, concentration under reduced pressure, and purification by column chromatography using a 5:1 ratio of petroleum ether to ethyl acetate to afford the product, 8-(4-methoxybenzyloxy)-octan-1-ol (PL30-1). PL30-1 was dissolved in acetone and Jones reagent was added dropwise under ice until the solution turned brownish yellow. The reaction was allowed to react at room temperature for 2 hours. After completion of the reaction, water was added to quench the reaction, followed by extraction with DCM, concentration under reduced pressure, and purification by column chromatography using a 5:1 ratio of petroleum ether to ethyl acetate to afford the liquid compound PL30-2.

[0187] PL30-2 (560 mg, 2 mmol) and 3-undecanol (344 mg, 2 mmol) were dissolved in 10 mL of DCM, followed by the addition of DCC (412 mg, 2 mmol) and DMAP (24 mg, 0.2 mmol) and allowed to react at room temperature overnight. After the reaction, the mixture was filtered and washed with DCM. The filtrate was concentrated under reduced pressure and purified by column chromatography using a 25:1 ratio of petroleum ether to ethyl acetate to afford the product PL32-1. A mixed solvent of DCM / H₂O (10 mL / 1 mL) was added to PL32-1 (434 mg, 1 mmol), followed by the addition of DDQ (227 mg, 1 mmol) and the reaction was allowed to react at room temperature for 4 h. After the reaction, water was added and the mixture was extracted with ethyl acetate, washed several times with water, and the organic phase was concentrated and purified by column chromatography using a 25:1 ratio of petroleum ether to ethyl acetate to afford the product PL32-2. 1 H NMR (400MHz, CDCl3): 4.86-4.80 (m, 1H), 3.98 (t, J=8Hz, 2H), 2.31 (t, J=8Hz, 2H), 1.69-1.51 (m, 8H), 1.36-1.28 (m, 18H), 0.91-0.87 (m, 6H).

[0188] PL32-2 (628 mg, 2 mmol) was dissolved in 5 mL of DCM and 2 mmol of triethylamine was added to prepare a solution. This solution was then slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) in an ice bath (0°C) under nitrogen protection. The reaction was incubated at 5-20°C for 2 h. The reaction mixture was then added dropwise to a mixture of 1,4-bis(3-aminopropyl)piperazine (400 mg, 2 mmol) and triethylamine (2 eq) in dichloromethane and allowed to react at room temperature for 2 h. After the reaction was complete, the product was washed with saturated sodium bicarbonate and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography using a dichloromethane:methanol:ammonia solution ratio of 45:3:1 afforded the liquid compound PL32-3. PL32-3 (873 mg, 1 mmol) was dissolved in DMF, and 1-bromododecane (747 mg, 3 mmol) and KCO (414 mg, 3 mmol) were added, respectively, and the mixture was allowed to react at room temperature overnight. After completion, the reaction was quenched with water, extracted with dichloromethane, washed three times with water, and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography using dichloromethane:methanol = 20:1 as the eluent afforded liquid compound PL32 (665.43 mg, 55%). 1 H NMR (400MHz, CDCl3): 4.85-4.81 (m, 2H), 4.00-3.94 (m, 4H), 3.03-2.96 (m, 4H), 2 .47-2.29(m, 20H), 1.69-1.51(m, 20H), 1.36-1.28(m, 76H), 0.92-0.87(m, 18H).

[0189] Reference example 2

[0190] Synthesis of PL71

[0191] Linolenic alcohol (266 mg, 1 mmol) was dissolved in 5 mL of DCM and 1 mmol of triethylamine was added to prepare a solution. This solution was then slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) in an ice bath (0°C). Under nitrogen, the mixture was reacted at 0-5°C for 2 h. Subsequently, a dichloromethane mixture of PL101-4 (314 mg, 1 mmol) and triethylamine (1 eq) was added dropwise to the system and allowed to react from 5°C to room temperature for 2 h. This was followed by a dichloromethane mixture of 4-methyl-1-piperazinepropylamine (157 mg, 1 mmol) and triethylamine (1 eq) at room temperature for 2 h. After the reaction was complete, the mixture was washed with saturated sodium bicarbonate and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography using a dichloromethane:methanol ratio of 30:1 afforded the liquid compound PL71 (640.95 mg, 82%). 1H NMR (400MHz, CDCl3): 5.40-5.32 (m, 4H), 4.85-4.29 (m, 1H), 4.01-3.95 (m, 4H), 3.03-2.40 (m, 16 H), 2.36 (s, 3H), 2.09-2.04 (m, 4H), 1.79-1.52 (m, 12H), 1.36-1.27 (m, 34H), 0.92-0.87 (m, 9H).

[0192] Example 1

[0193] Synthesis of PL1

[0194] Linolenic alcohol (533 mg, 2 mmol) was dissolved in 5 mL of DCM and 2 mmol of triethylamine was added to prepare a solution. This solution was then slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) in an ice bath (5°C). Nitrogen was then added and the mixture was allowed to slowly return to room temperature for 4 hours. After 4 hours, a dichloromethane mixture of N,N-dimethyl-1,3-propanediamine (102 mg, 1 mmol) and triethylamine (1 eq) was added dropwise to the system and allowed to react at room temperature for 2 hours. After completion of the reaction, the product was washed with saturated sodium bicarbonate and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography using a dichloromethane:methanol ratio of 15:1 afforded liquid compound PL1 (394 mg, 58%). 1 H NMR (400MHz, CDCl3): 5.41-5.29 (m, 8H), 4.01-3.91 (m, 4H), 2.96-2.89 (m, 2H), 2.78 (t, J=8Hz, 4H), 2.51- 2.47 (m, 2H), 2.33 (s, 6H), 2.10-2.05 (m, 8H), 1.75-1.65 (m, 6H), 1.42-1.31 (m, 64H), 0.92 (t, J=8Hz, 6H). MS m / z(ESI): 679.59[M+H] + .

[0195] Example 2

[0196] Synthesis of PL15

[0197] 1-Undecanol (344 mg, 2 mmol) was dissolved in 5 mL of DCM and 2 mmol of triethylamine was added to prepare a solution. This solution was then slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) in an ice bath (0°C) under nitrogen protection. The reaction was incubated at 5-20°C for 2 h. A dichloromethane mixture of 1,4-bis(3-aminopropyl)piperazine (100 mg, 0.5 mmol) and triethylamine (1 eq) was then added dropwise to the system and allowed to react at room temperature for 2 h. After completion of the reaction, the product was washed with saturated sodium bicarbonate and concentrated under reduced pressure to afford the crude product. Purification by column chromatography using a dichloromethane:methanol ratio of 20:1 afforded liquid compound PL15 (425 mg, 87%). 1 H NMR (400MHz, CDCl3): 3.91-3.99(m, 8H), 3.64-3.61(m, 2H), 3.01-2.94(m, 4H), 2 .88-2.23 (m, 12H), 1.68-1.61 (m, 12H), 1.37-1.25 (m, 64H), 0.87 (t, J=8Hz, 12H). MS m / z(ESI):977.82[M+H] + .

[0198] Example 3

[0199] Synthesis of PL16

[0200] Linolenic alcohol (533 mg, 2 mmol) was dissolved in 5 mL of DCM and 2 mmol of triethylamine was added to prepare a solution. This solution was then slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) in an ice bath (0°C). Under nitrogen, the mixture was allowed to react at 5-20°C for 2 h. Subsequently, a dichloromethane mixture of 1,4-bis(3-aminopropyl)piperazine (100 mg, 0.5 mmol) and triethylamine (1 eq) was added dropwise to the reaction mixture and allowed to react at room temperature for 2 h. After completion of the reaction, the mixture was washed with saturated sodium bicarbonate and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography using a dichloromethane:methanol ratio of 25:1 afforded liquid compound PL16 (541.2 mg, 80%). 1H NMR (400MHz, CDCl3): 5.41-5.29 (m, 16H), 4.02-3.90 (m, 8H), 3.64 (s, 2H), 2.99-2.97 (m, 4H), 2.78 (t, J=4Hz , 8H), 2.61-2.43 (m, 12H), 2.07-2.02 (m, 16H), 1.69-1.62 (m, 12H), 1.39-1.26 (m, 64H), 0.89 (t, J=8Hz, 12H). MS m / z(ESI): 1354.16[M+H] + .

[0201] Example 4

[0202] Synthesis of PL37

[0203] 2-Hexyl-1-decanol (485 mg, 2 mmol) was dissolved in 5 mL of DCM and 2 mmol of triethylamine was added to prepare a solution. This solution was then slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) in an ice bath (0°C). Under nitrogen, the mixture was allowed to react at 5-20°C for 2 h. Subsequently, a dichloromethane mixture of 1,4-bis(3-aminopropyl)piperazine (100 mg, 0.5 mmol) and triethylamine (1 eq) was added dropwise to the system and allowed to react at room temperature for 2 h. After the reaction was complete, the product was washed with saturated sodium bicarbonate and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography using a dichloromethane:methanol ratio of 25:1 afforded liquid compound PL37 (502.8 mg, 80%). 1 H NMR (400MHz, CDCl3): 4.02-3.91 (m, 8H), 3.12-2.16 (m, 16H), 1.79-1.62 (m, 8H), 1.38-0.88 (m, 96H), 0.89 (t, J=8Hz, 24H).

[0204] Example 5

[0205] Synthesis of PL101

[0206] 1,8-Octanediol (730 mg, 5 mmol) was first dissolved in 5 mL of THF. NaH (240 mg, 10 mmol) was then added under ice and allowed to react for 10 minutes. 4-Methoxybenzyl chloride (783 mg, 5 mmol) was then added dropwise to the mixture and allowed to react overnight at room temperature. Upon completion, the reaction was quenched with water, extracted with DCM, concentrated under reduced pressure, and purified by column chromatography using a 5:1 ratio of petroleum ether to ethyl acetate to afford the product PL101-1. PL101-1 was dissolved in acetone and Jones reagent was added dropwise under ice until the solution turned brownish yellow. The mixture was allowed to react at room temperature for 2 hours. After completion, the reaction was quenched with water, extracted with DCM, concentrated under reduced pressure, and purified by column chromatography using a 5:1 ratio of petroleum ether to ethyl acetate to afford the liquid compound PL101-2. PL101-2 (560 mg, 2 mmol) and 3-undecanol (344 mg, 2 mmol) were dissolved in 10 mL of DCM, followed by the addition of DCC (412 mg, 2 mmol) and DMAP (24 mg, 0.2 mmol) and allowed to react at room temperature overnight. After the reaction, the mixture was filtered and washed with DCM. The filtrate was concentrated under reduced pressure and purified by column chromatography using a 25:1 ratio of petroleum ether to ethyl acetate to afford product PL101-3. A mixed solvent of DCM / H₂O (10 mL / 1 mL) was added to PL101-3 (434 mg, 1 mmol), followed by the addition of DDQ (227 mg, 1 mmol) and allowed to react at room temperature for 4 h. After the reaction, water was added and the mixture was extracted with ethyl acetate, washed several times with water, and the organic phase was concentrated and purified by column chromatography using a 25:1 ratio of petroleum ether to ethyl acetate to afford product PL101-4. 1 H NMR (400MHz, CDCl3): 4.86-4.80 (m, 1H), 3.98 (t, J=8Hz, 2H), 2.31 (t, J=8Hz, 2H), 1.69-1.51 (m, 8H), 1.36-1.28 (m, 18H), 0.91-0.87 (m, 6H).

[0207] Linolenic alcohol (266 mg, 1 mmol) was dissolved in 5 mL of DCM and 1 mmol of triethylamine was added to prepare a solution. This solution was then slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) in an ice bath (0°C). The mixture was reacted at 0-5°C for 2 h under nitrogen. A dichloromethane mixture of PL101-4 (314 mg, 1 mmol) and triethylamine (1 eq) was then added dropwise to the system and allowed to react from 5°C to room temperature for 2 h. This was followed by a dichloromethane mixture of 1,4-bis(3-aminopropyl)piperazine (100 mg, 0.5 mmol) and triethylamine (1 eq) at room temperature for 2 h. After completion of the reaction, the product was washed with saturated sodium bicarbonate and concentrated under reduced pressure to yield the crude product. Purification by column chromatography using a dichloromethane:methanol ratio of 15:1 afforded the liquid compound PL101 (899 mg, 62%). 1 H NMR (400MHz, MeOD): 5.44-5.31 (m, 8H), 4.86-4.80 (m, 2H), 4.02-3.94 (m, 8H), 3.00-2.29 (m, 20H), 2.10-2.04 (m, 8H), 1.71-1.50 (m, 24H), 1.38-1.28 (m, 68H), 0.93-0.87 (m, 18H).

[0208] Example 6

[0209] Synthesis of PL102

[0210] PL101-2 (560 mg, 2 mmol) and 9-heptadecanol (513 mg, 2 mmol) were first dissolved in 10 mL of DCM. DCC (412 mg, 2 mmol) and DMAP (24 mg, 0.2 mmol) were then added and allowed to react at room temperature overnight. After the reaction, the mixture was filtered and washed with DCM. The filtrate was concentrated under reduced pressure and purified by column chromatography using a 25:1 ratio of petroleum ether to ethyl acetate to obtain the product PL102-1. A mixed solvent of DCM / H₂O (10 mL / 1 mL) was added to PL102-1 (519 mg, 1 mmol), followed by DDQ (227 mg, 1 mmol) and allowed to react at room temperature for 4 h. After the reaction, water was added and the mixture was extracted with ethyl acetate. The mixture was washed with water several times, and the organic phase was concentrated and purified by column chromatography using a 25:1 ratio of petroleum ether to ethyl acetate to obtain the product PL102-2. 1 H NMR (400MHz, CDCl3): 4.91-4.85 (m, 1H), 3.98 (t, J=8Hz, 2H), 2.30 (t, J=8Hz, 2H), 1.69-1.50 (m, 8H), 1.36-1.28 (m, 18H), 0.90 (t, J=8Hz, 6H).

[0211] Linolenic alcohol (266 mg, 1 mmol) was dissolved in 5 mL of DCM and 1 mmol of triethylamine was added to prepare a solution. This solution was then slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) in an ice bath (0°C). The mixture was then reacted at 0-5°C for 2 h under nitrogen. A dichloromethane mixture of PL102-2 (398 mg, 1 mmol) and triethylamine (1 eq) was then added dropwise to the system and allowed to react from 5°C to room temperature for 2 h. This was followed by a dichloromethane mixture of 1,4-bis(3-aminopropyl)piperazine (100 mg, 0.5 mmol) and triethylamine (1 eq) at room temperature for 2 h. After completion of the reaction, the mixture was washed with saturated sodium bicarbonate and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography using a dichloromethane:methanol ratio of 15:1 afforded the liquid compound PL102 (906 mg, 56%). 1 H NMR (400MHz, MeOD): 5.44-5.32 (m, 8H), 4.90-4.85 (m, 2H), 4.04-3.86 (m, 8H), 3.00-2.28 (m, 20H), 2.10-2.05 (m, 8H), 1.75-1.50 (m, 24H), 1.40-1.28 (m, 92H), 0.93-0.88 (m, 18H).

[0212] Example 7

[0213] Synthesis of PL39

[0214] PL102-2 (796 mg, 2 mmol) was dissolved in 5 mL of DCM and 2 mmol of triethylamine was added to prepare a solution. This solution was then slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) in an ice bath (0°C). Under nitrogen, the mixture was allowed to react at 5-20°C for 2 h. Subsequently, a dichloromethane mixture of 1,4-bis(3-aminopropyl)piperazine (100 mg, 0.5 mmol) and triethylamine (1 eq) was added dropwise to the reaction mixture and allowed to react at room temperature for 2 h. After completion of the reaction, the mixture was washed with saturated sodium bicarbonate and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography using a dichloromethane:methanol ratio of 20:1 afforded the liquid compound PL39 (1119 mg, 59%). 1 H NMR (400MHz, CDCl3): 4.89-4.86 (m, 4H), 4.01-3.93 (m, 4H), 3.05-2.27 (m, 24H), 1.67-1.50 (m, 32H), 1.36-1.27 (m, 120H), 0.89 (t, J=8Hz, 24H).

[0215] Example 8

[0216] Synthesis of PL40

[0217] PL101-4 (628 mg, 2 mmol) was dissolved in 5 mL of DCM and 2 mmol of triethylamine was added to prepare a solution. This solution was then slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) in an ice bath (0°C) under nitrogen protection. The reaction was allowed to proceed at 5°C-20°C for 2 h. Subsequently, a dichloromethane mixture of 1,4-bis(3-aminopropyl)piperazine (100 mg, 0.5 mmol) and triethylamine (1 eq) was added dropwise to the system and allowed to react at room temperature for 2 h. After the reaction was complete, the product was washed with saturated sodium bicarbonate and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography using a dichloromethane:methanol ratio of 20:1 afforded liquid compound PL40 (749 mg, 46%). 1 H NMR (400MHz, CDCl3): 4.86-4.79 (m, 4H), 4.00-3.94 (m, 8H), 3.15-2.10 (m, 24H), 1.69-1.51 (m, 32H), 1.36-1.28 (m, 72H), 0.91-0.87 (m, 24H).

[0218] Example 9

[0219] Synthesis of PL48

[0220] Linolenic alcohol (533 mg, 2 mmol) was dissolved in 5 mL of DCM and 2 mmol of triethylamine was added to prepare a solution. This solution was then slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) under nitrogen for 4 hours. After 4 hours, a mixture of N′,N-bis(3-aminopropyl)methylamine (145.25 mg, 1 mmol) and triethylamine (1 eq) in dichloromethane was added dropwise to the system and allowed to react at room temperature for 2 hours. After completion of the reaction, the product was washed with saturated sodium bicarbonate and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography using a dichloromethane:methanol ratio of 30:1 afforded liquid compound PL48 (636.51 mg, 49%). 1 H NMR (400MHz, CDCl3): 5.44-5.28 (m, 16H), 4.01-3.94 (m, 8H), 3.12-3.00 (m, 4H), 2.78 (t, J=8Hz, 8H), 2.58-2.45 (m, 4H), 2.09-2.04 (m, 11H), 1.69-1.64 (m, 12H), 1.39-1.27 (m, 64H), 0.92 (t, J=8Hz, 12H).

[0221] Example 10

[0222] Synthesis of PL49

[0223] Linolenic alcohol (533 mg, 2 mmol) was dissolved in 5 mL of DCM and 2 mmol of triethylamine was added to prepare a solution. This solution was then slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) under nitrogen for 4 hours. After 4 hours, a mixture of 3-bromopropylamine hydrobromide (218.92 mg, 1 mmol) and triethylamine (2 eq) in dichloromethane was added dropwise to the system and allowed to react at room temperature for 1 hour. After completion of the reaction, the product was washed with saturated sodium bicarbonate and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography using a dichloromethane:methanol ratio of 30:1 afforded liquid compound PL49-1 (650.55 mg, 91%).

[0224] PL49-1 (713.45 mg, 1 mmol) was dissolved in 10 mL of DMF, and ethanolamine (30.5 mg, 0.5 mmol) and KCO (138 mg, 1 mmol) were added and allowed to react at room temperature overnight. After the reaction was complete, the product was washed with saturated sodium chloride and concentrated under reduced pressure to obtain a crude product. Purification by column chromatography using dichloromethane:methanol = 30:1 as the eluent afforded liquid compound PL49 (385.12 mg, 58%). 1 H NMR (400MHz, CDCl3): 5.42-5.32(m, 16H), 4.01-3.97(m, 8H), 3.76-3.67(m, 2H), 3.51-3.41(m, 2H), 3.04-2.91(m, 4H), 2 .79 (t, J=4Hz, 12H), 2.10-2.05 (m, 16H), 1.92-1.81 (m, 4H), 1.72-1.65 (m, 8H), 1.40-1.28 (m, 64H), 0.91 (t, J=4Hz, 12H).

[0225] Example 11

[0226] Synthesis of PL50

[0227] PL49-1 (713.45 mg, 1 mmol) was dissolved in 10 mL of DMF, and 3-phenyl-1-propylamine (67.5 mg, 0.5 mmol) and KCO (138 mg, 1 mmol) were added and allowed to react at room temperature overnight. After the reaction was complete, the product was washed with saturated sodium chloride and concentrated under reduced pressure to obtain a crude product. Purification by column chromatography using dichloromethane:methanol = 30:1 as the eluent afforded liquid compound PL50 (441.67 mg, 63%). 1H NMR (400MHz, CDCl3): 7.31-7.27(m, 2H), 7.21-7.18(m, 3H), 5.42-5.31(m, 16H), 4.01-3.92(m, 8H), 3.43(s, 2H), 3.00-2.93(m, 4H), 2.79 (t, J=4Hz, 8H), 2.64-2.48 (m, 8H), 2.09-2.04 (m, 16H), 1.90-1.80 (m, 2H), 1.68-1.63 (m, 12H), 1.39-1.28 (m, 64H), 0.91 (t, J=4Hz, 12H).

[0228] Example 12

[0229] Synthesis of PL51

[0230] PL49-1 (713.45 mg, 1 mmol) was dissolved in 10 mL of DMF, and (2-methyl-3-pyrrolidin-1-ylpropyl)amine (71 mg, 0.5 mmol) and KCO (138 mg, 1 mmol) were added and allowed to react at room temperature overnight. After the reaction was complete, the product was washed with saturated sodium chloride and concentrated under reduced pressure to obtain a crude product. Purification by column chromatography using dichloromethane:methanol = 30:1 as the eluent afforded compound PL51 (472.08 mg, 67%) as a liquid. 1 H NMR (400MHz, CDCl3): 5.42-5.31 (m, 16H), 4.04-3.92 (m, 8H), 3.08-2.94 (m, 10H), 2.84-2.63 (m, 12H), 2.09-2.04 (m, 18H), 1.92-1.57 (m, 16H), 1.39-1.28 (m, 65H), 1.02 (t, J=8Hz, 3H), 0.91 (t, J=4Hz, 12H).

[0231] Example 13

[0232] Synthesis of PL52

[0233] PL49-1 (713.45 mg, 1 mmol) was dissolved in 10 mL of DMF, and N₁,N₁-dipropyl-1,3-propanediamine (79 mg, 0.5 mmol) and K₂CO₃ (138 mg, 1 mmol) were added and allowed to react at room temperature overnight. After the reaction was complete, the product was washed with saturated sodium chloride and concentrated under reduced pressure to obtain a crude product. Purification by column chromatography using dichloromethane:methanol = 30:1 as the eluent afforded liquid compound PL52 (505.95 mg, 71%). 1H NMR (400MHz, CDCl3): 5.42-5.30 (m, 16H), 4.02-3.94 (m, 8H), 3.42-3.03 (m, 12H), 2.78 (t, J =4Hz, 8H), 2.23-2.02 (m, 22H), 1.70-1.55 (m, 8H), 1.39-1.20 (m, 70H), 0.91 (t, J = 4Hz, 12H).

[0234] Example 14

[0235] Synthesis of PL63

[0236] PL49-1 (713.45 mg, 1 mmol) was dissolved in 10 mL of DMF, and 4-methyl-1-piperazinepropylamine (78.5 mg, 0.5 mmol) and KCO (138 mg, 1 mmol) were added and allowed to react at room temperature overnight. After the reaction was complete, the product was washed with saturated sodium chloride and concentrated under reduced pressure to obtain a crude product. Purification by column chromatography using dichloromethane:methanol = 30:1 as the eluent afforded liquid compound PL63 (541.20 mg, 76%). 1 H NMR (400MHz, CDCl3): 5.41-5.31(m, 16H), 4.02-3.93(m, 8H), 3.28-3.24(m, 4H), 3.05-2.91(m, 6H), 2 .80-2.54(m, 21H), 2.09-2.04(m, 16H), 1.82-1.64(m, 14H), 1.39-1.27(m, 64H), 0.91(t, J=4Hz, 12H).

[0237] Example 15

[0238] Synthesis of PL64

[0239] PL49-1 (713.45 mg, 1 mmol) was dissolved in 10 mL of DMF, and 1-(2-aminoethyl)piperidine (64.11 mg, 0.5 mmol) and KCO (138 mg, 1 mmol) were added and allowed to react at room temperature overnight. After the reaction was complete, the product was washed with saturated sodium chloride and concentrated under reduced pressure to obtain a crude product. Purification by column chromatography using dichloromethane:methanol = 30:1 as the eluent afforded liquid compound PL64 (509.17 mg, 73%). 1H NMR (400MHz, CDCl3): 5.44-5.31 (m, 16H), 4.02-3.94 (m, 8H), 3.65-3.61 (m, 2H), 3.04-2.97 (m, 4H), 2.79 (t, J=4 Hz, 8H), 2.52-2.46 (m, 18H), 2.09-2.04 (m, 16H), 1.72-1.64 (m, 12H), 1.41-1.27 (m, 64H), 0.91 (t, J=4Hz, 12H).

[0240] Example 16

[0241] Synthesis of PL65

[0242] Linolenic alcohol (266 mg, 1 mmol) was dissolved in 5 mL of DCM and 1 mmol of triethylamine was added to prepare a solution. This solution was then slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) in an ice bath (0°C). Under nitrogen, the reaction was incubated at 0-5°C for 2 h. Subsequently, a dichloromethane mixture of PL101-4 (314 mg, 1 mmol) and triethylamine (1 eq) was added dropwise to the system and allowed to react from 5°C to room temperature for 2 h. This was followed by a dichloromethane mixture of 3-bromopropylamine hydrobromide (218.92 mg, 1 mmol) and triethylamine (1 eq) at room temperature for 2 h. After completion of the reaction, the product was washed with saturated sodium bicarbonate and concentrated under reduced pressure to yield the crude product. Purification by column chromatography using a dichloromethane:methanol ratio of 30:1 afforded the liquid compound PL 65-1 (899 mg, 62%).

[0243] PL65-1 (761.47 mg, 1 mmol) was dissolved in 10 mL of DMF, and 3-(4-methyl-1-piperazine)propylamine (78.5 mg, 0.5 mmol) and KCO (138 mg, 1 mmol) were added and allowed to react at room temperature overnight. After the reaction was complete, the product was washed with saturated sodium chloride and concentrated under reduced pressure to obtain a crude product. Purification by column chromatography using dichloromethane:methanol = 30:1 as the eluent afforded liquid compound PL65 (364.80 mg, 48%). 1H NMR (400MHz, CDCl3): 5.41-5.32 (m, 8H), 4.86-4.29 (m, 2H), 4.02-3.93 (m, 8H), 3.38-3.25 (m, 4H), 3.03-2.97 (m, 4H), 2.79 (t, J=4Hz, 4H ), 2.59-2.49 (m, 12H), 2.38 (s, 3H), 2.30 (t, J=4Hz, 4H), 2.09-2.04 (m, 8H), 1.80-1.52 (m, 26H), 1.36-1.27 (m, 68H), 0.92-0.87 (m, 18H).

[0244] Example 17

[0245] Synthesis of PL66

[0246] Linolenic alcohol (266 mg, 1 mmol) was dissolved in 5 mL of DCM and 1 mmol of triethylamine was added to prepare a solution. This solution was then slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) in an ice bath (0°C) under nitrogen protection. The reaction was incubated at 0-5°C for 2 h. A dichloromethane mixture of PL101-4 (314 mg, 1 mmol) and triethylamine (1 eq) was then added dropwise to the system and allowed to react at 5°C-RT for 2 h. This mixture was then added to a dichloromethane mixture of N′,N-bis(3-aminopropyl)methylamine (72.6 mg, 0.5 mmol) and triethylamine (1 eq) at room temperature for 2 h. After the reaction was complete, the mixture was washed with saturated sodium bicarbonate and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography using dichloromethane:methanol=15:1 as the eluent gave liquid compound PL 66 (439.11 mg, 63%). 1 H NMR (400MHz, CDCl3): 5.44-5.31 (m, 16H), 4.88-4.79 (m, 2H), 4.02-3.83 (m, 8H), 3.24-2.75 (m, 12H), 2.32-2.27 (m, 4H), 2.09-2.02 (m, 11H), 1.70-1.50 (m, 24H), 1.38-1.28 (m, 64H), 0.93-0.87 (m, 18H).

[0247] Example 18

[0248] Synthesis of PL67

[0249] PL65-1 (761.47 mg, 1 mmol) was dissolved in 10 mL of DMF, and ethanolamine (30.5 mg, 0.5 mmol) and KCO (138 mg, 1 mmol) were added and allowed to react at room temperature overnight. After the reaction was complete, the product was washed with saturated sodium chloride and concentrated under reduced pressure to obtain a crude product. Purification by column chromatography using dichloromethane:methanol = 30:1 as the eluent afforded liquid compound PL67 (491.28 mg, 69%). 1 H NMR (400MHz, MeOD): 5.42-5.31 (m, 8H), 4.84-4.80 (m, 2H), 4.01-3.96 (m, 8H), 3.28-3.08 (m, 8H), 2.79 (t, J=4 Hz, 4H), 2.31 (t, J=8Hz, 4H), 2.10-2.05 (m, 10H), 1.72-1.51 (m, 24H), 1.38-1.28 (m, 68H), 0.93-0.87 (m, 18H).

[0250] Example 19

[0251] Synthesis of PL68

[0252] Hexylene glycol (236 mg, 2 mmol) and 2-hexylundecanoic acid (540 mg, 2 mmol) were dissolved in 5 mL of DCM. DCC (412 mg, 2 mmol) and DMAP (24 mg, 0.2 mmol) were then added and allowed to react overnight at room temperature. After completion of the reaction, the product was filtered and washed with DCM. The filtrate was concentrated under reduced pressure and purified by column chromatography using petroleum ether:ethyl acetate = 20:1 as the eluent to obtain the product PL68-1 (614 mg, 83%).

[0253] Linolenic alcohol (266 mg, 1 mmol) was dissolved in 5 mL of DCM and 1 mmol of triethylamine was added to prepare a solution. This solution was then slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) in an ice bath (0°C). The mixture was then reacted at 0-5°C for 2 h under nitrogen. A dichloromethane mixture of PL68-1 (370 mg, 1 mmol) and triethylamine (1 eq) was then added dropwise to the system and allowed to react from 5°C to room temperature for 2 h. This was followed by a dichloromethane mixture of N'N-bis(3-aminopropyl)methylamine (72.6 mg, 0.5 mmol) and triethylamine (1 eq) at room temperature for 2 h. After completion of the reaction, the product was washed with saturated sodium bicarbonate and concentrated under reduced pressure to yield the crude product. Purification by column chromatography using a dichloromethane:methanol ratio of 20:1 afforded the liquid compound PL68 (462.52 mg, 62%). 1H NMR (400MHz, CDCl3): 5.43-5.31 (m, 8H), 4.09-3.96 (m, 10H), 3.28-2.77 (m, 12H), 2.09-2.04 (m, 11H), 1.70-1.57 (m, 16H), 1.43-1.24 (m, 64H), 0.92-0.89 (m, 18H).

[0254] Example 20

[0255] Synthesis of PL69

[0256] Linolenic alcohol (266 mg, 1 mmol) was dissolved in 5 mL of DCM and 1 mmol of triethylamine was added to prepare a solution. This solution was then slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) in an ice bath (0°C). Under nitrogen, the reaction was incubated at 0-5°C for 2 h. A dichloromethane mixture of PL 68-1 (370 mg, 1 mmol) and triethylamine (1 eq) was then added dropwise to the system and allowed to react from 5°C to room temperature for 2 h. This was followed by a dichloromethane mixture of 3-bromopropylamine hydrobromide (218.92 mg, 1 mmol) and triethylamine (1 eq) at room temperature for 2 h. After completion of the reaction, the product was washed with saturated sodium bicarbonate and concentrated under reduced pressure to yield the crude product. Purification by column chromatography using a dichloromethane:methanol ratio of 30:1 afforded the liquid compound PL 69-1 (743.95 mg, 91%).

[0257] PL69-1 (817.53 mg, 1 mmol) was dissolved in 10 mL of DMF, and 3-(4-methyl-1-piperazine)propylamine (78.6 mg, 0.5 mmol) and KCO (138 mg, 1 mmol) were added and allowed to react at room temperature overnight. After the reaction was complete, the product was washed with saturated sodium chloride and concentrated under reduced pressure to obtain a crude product. Purification by column chromatography using dichloromethane:methanol = 30:1 as the eluent afforded liquid compound PL69 (590.70 mg, 73%). 1 H NMR (400MHz, CDCl3): 5.44-5.31 (m, 8H), 4.08 (t, J=8Hz, 4H), 4.01-3.95 (m, 8H), 3.30-3.25 (m, 4H), 3.05-2.97 (m, 4H), 2.79 (t, J=8Hz, 4 H), 2.62-2.52(m, 12H), 2.40(s, 3H), 2.35-2.30(m, 2H), 2.09-2.04(m, 8H), 1.82-1.55(m, 24H), 1.42-1.27(m, 78H), 0.93-0.88(m, 18H).

[0258] Example 21

[0259] Synthesis of PL70

[0260] PL65-1 (761.47 mg, 1 mmol) was dissolved in 10 mL of DMF, and propanolamine (37.5 mg, 0.5 mmol) and KCO (138 mg, 1 mmol) were added and allowed to react at room temperature overnight. After the reaction was complete, the product was washed with saturated sodium chloride and concentrated under reduced pressure to obtain a crude product. Purification by column chromatography using dichloromethane:methanol = 30:1 as the eluent afforded liquid compound PL70 (409.83 mg, 57%). 1 H NMR (400MHz, CDCl3): 5.44-5.29 (m, 8H), 4.86-4.79 (m, 2H), 4.01-3.94 (m, 8H), 3.78 (t, J=8Hz, 2H), 3.50-2.90 (m, 10H), 2.30 (t, J=8Hz, 4H), 2.09-2.04(m, 8H), 1.87-1.51(m, 26H), 1.37-1.28(m, 68H), 0.93-0.87(m, 18H).

[0261] Example 22

[0262] Synthesis of PL80

[0263] PL65-1 (761.47 mg, 1 mmol) was dissolved in 10 mL of DMF, and 2-aminoethylmorpholine (65 mg, 0.5 mmol) and KCO (138 mg, 1 mmol) were added and allowed to react at room temperature overnight. After the reaction was complete, the product was washed with saturated sodium chloride and concentrated under reduced pressure to obtain a crude product. Purification by column chromatography using dichloromethane:methanol = 30:1 as the eluent afforded liquid compound PL80 (574.80 mg, 77%). 1 H NMR (400MHz, CDCl3): 5.44-5.31 (m, 8H), 4.84-4.81 (m, 2H), 4.02-3.93 (m, 8H), 3.77-3.71 (m, 4H), 3.39-2.93 (m, 8H), 2.79 (t, J= 4Hz, 4H), 2.43-2.68 (m, 8H), 2.30 (t, J=8Hz, 4H), 2.09-2.04 (m, 8H), 1.84-1.49 (m, 24H), 1.36-1.27 (m, 68H), 0.93-0.87 (m, 18H).

[0264] Example 23

[0265] Synthesis of PL82

[0266] 3-Allyloxy-1,2-propanediol (132 mg, 1 mmol) and linoleic acid (560 mg, 2 mmol) were dissolved in 5 mL of DCM. DCC (412 mg, 2 mmol) and DMAP (24 mg, 0.2 mmol) were then added and allowed to react overnight at room temperature. After completion of the reaction, the mixture was filtered and washed with DCM. The filtrate was concentrated under reduced pressure and purified by column chromatography using petroleum ether:ethyl acetate = 25:1 as the eluent to obtain the product PL82-1 (590 mg, 90%). 1 H NMR (400MHz, CDCl3): 5.94-5.84(m, 1H), 5.44-5.19(m, 10H), 4.38-4.34(m, 1H), 4.22-4.17(m, 1H), 4.03-4.00(m, 2H), 3.58(d, J =4Hz, 2H), 2.79 (t, J = 4Hz, 4H), 2.37-2.30 (m, 4H), 2.10-2.04 (m, 8H), 1.67-1.60 (m, 4H), 1.41-1.27 (m, 28H), 0.89 (t, J = 8Hz, 6H).

[0267] PL82-1 (656.54 mg, 1 mmol) was dissolved in a MeOH / DCM mixture, and Pd(PPh3)4 (1155.56 mg, 1 mmol) and barbituric acid (256 mg, 2 mmol) were added. The mixture was allowed to react at room temperature overnight. After completion of the reaction, saturated NaCl solution was added, and the mixture was extracted with DCM. The mixture was washed with saturated NaCl solution, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography using petroleum ether:ethyl acetate = 10:1 afforded the liquid compound PL82-2 (505.12 g, 82%). 1 H NMR (400MHz, CDCl3): 5.44-5.31 (m, 8H), 5.13-5.08 (m, 2H), 4.36-4.13(m, 2H), 3.76-3.74(m, 2H), 2.79(t, J=4Hz, 4H), 2.39-2.33(m, 4H), 2.10-2.05 (m, 8H), 1.67-1.62 (m, 4H), 1.40-1.27 (m, 31H), 0.92 (t, J=8Hz, 6H).

[0268] PL82-2 (616 mg, 1 mmol) was dissolved in 5 mL of DCM and 1 mmol of triethylamine was added to prepare a solution. This solution was then slowly added dropwise to phosphorus oxychloride (153 mg, 1 mmol) in an ice bath (0°C). Under nitrogen, the reaction was incubated at 0-5°C for 2 h. Subsequently, a dichloromethane mixture of EtOH (46 mg, 1 mmol) and triethylamine (1 eq) was added dropwise to the system and allowed to react from 5°C to room temperature for 2 h. This was followed by a dichloromethane mixture of 3-bromopropylamine hydrobromide (218.92 mg, 1 mmol) and triethylamine (1 eq) at room temperature for 2 h. After completion of the reaction, the product was washed with saturated sodium bicarbonate and concentrated under reduced pressure to obtain the crude product. Purification by column chromatography using a dichloromethane:methanol ratio of 30:1 afforded the liquid compound PL 82-3 (657.54 mg, 68%). 1 H NMR (400MHz, CDCl3): 5.44-5.45 (m, 10H), 4.38-4.06 (m, 6H), 3.51-3.48 (m, 2H), 3.15-3.07 (m, 2H), 2.79 (t, J =4Hz, 4H), 2.38-2.32 (m, 4H), 2.10-2.03 (m, 10H), 1.65-1.61 (m, 4H), 1.40-1.27 (m, 32H), 0.91 (t, J = 4Hz, 6H).

[0269] PL82-3 (843.48 mg, 1 mmol) was dissolved in 10 mL of DMF, and ethanolamine (30.5 mg, 0.5 mmol) and KCO (138 mg, 1 mmol) were added and allowed to react at room temperature overnight. After the reaction was complete, the product was washed with saturated sodium chloride and concentrated under reduced pressure to obtain a crude product. Purification by column chromatography using dichloromethane:methanol = 30:1 as the eluent afforded liquid compound PL82 (373.18 mg, 47%). 1 H NMR (400MHz, CDCl3): 5.44-5.24(m, 18H), 4.38-4.04(m, 12H), 3.94-3.65(m, 4H), 3.11-3.07(m, 8H), 2.81-2.77(m, 8H), 2.37-2.31 (m, 8H), 2.09-2.04 (m, 16H), 1.92-1.85 (m, 4H), 1.65-1.59 (m, 8H), 1.40-1.28 (m, 62H), 0.91 (t, J=4Hz, 12H).

[0270] Example 24

[0271] Synthesis of PL86

[0272] PL82-3 (843.48 mg, 1 mmol) was dissolved in 10 mL of DMF, and 2-aminoethylmorpholine (65 mg, 0.5 mmol) and KCO (138 mg, 1 mmol) were added and allowed to react at room temperature overnight. After the reaction was complete, the product was washed with saturated sodium chloride and concentrated under reduced pressure to obtain a crude product. Purification by column chromatography using dichloromethane:methanol = 30:1 as the eluent afforded liquid compound PL86 (613.46 mg, 74%). 1 H NMR (400MHz, MeOD): 5.44-5.24 (m, 18H), 4.36-4.04 (m, 12H), 3.75 (s, 4H), 3.33 (s, 4H), 3.05-2.99 (m, 4H), 0.91 (t, J=4Hz, 8H), 2. 52(s, 8H), 2.37-2.31(m, 8H), 2.09-2.04(m, 16H), 1.83-1.80(m, 4H), 1.65-1.60(m, 8H), 1.41-1.26(m, 62H), 0.93-0.87(m, 12H).

[0273] Example 25

[0274] Preparation of lipid nanoparticles

[0275] The prepared cationic compounds (PL1, PL15, PL16, PL37, PL101, PL102, PL39, PL40, PL48, PL49, PL50, PL51, PL52, PL63, PL64, PL65, PL66, PL67, PL68, PL69, PL70, PL80, PL82, PL86, PL71 (asymmetric lipid control), DOPE / DSPC, cholesterol, and DMG-PEG2000 were prepared into an ethanol phase with ethanol at a molar ratio of 35:16:46.5:2.5, and RNA was prepared into an aqueous phase with a 50 mM citric acid buffer solution at pH 4. The lipid nanoparticles were mixed by microfluidic technology at a volume ratio of 3:1 for the aqueous phase and ethanol phase, and then dialyzed in PBS for purification to remove the ethanol.

[0276] Example 26

[0277] Determination of lipid nanoparticle size

[0278] The particle size and distribution (PDI) of lipid nanoparticles were determined using a Malvern Zetasizer Nano ZS laser particle size analyzer using dynamic light scattering (DLS). Samples were diluted 100-fold with PBS before measurement, and 1 ml was used for measurement. Each sample was measured in triplicate.

[0279] Example 27

[0280] Determination of cell transfection efficiency of lipid nanoparticles

[0281] Prepare a 96-well plate, seed cells (100,000 human T or 30,000 NK92MI cells / well), 100 μL of culture medium, dilute lipid nanoparticles loaded with GFP mRNA or CAR-CD19 mRNA with culture medium, and finally add 50 μL of diluted LNP to each well. Set the dose gradient to 0.1 μg mRNA / well, 0.5 μg mRNA / well, and 1 μg mRNA / well. Incubate at 37°C for 48 hours, and then use flow cytometry to determine the positive rate of GFP and CAR-CD19 in the cells.

[0282] Example 28

[0283] Isolation and culture of primary human T cells

[0284] Thawed human PBMCs were used to isolate human CD3+ T cells using the EasySep Human T Cell Isolation Kit (Stemcell). The cells were then cultured in RPMI 1640 medium supplemented with 10% FBS (Gibco), 1% penicillin / streptomycin (P / S), and IL-2 (20 U / ml). In addition, human CD3 / CD28 T cell activator (Stemcell) was added at a concentration of 25 μl / ml for 3 days.

[0285] Example 29

[0286] In vitro cytotoxicity

[0287] On the first day of activation, T cells were transfected with PL LNP at 5 μg CAR mRNA per million T cells. Two days later, the activated T cells were used for co-culture. NALM6 cells overexpressing GFP and HEK 293T cells overexpressing Claudin18.2 were used as target cells. Each well of a round-bottom 96-well plate contained 20,000 target cells. Subsequently, T cells were added at a ratio of target cells: effector cells = 1:0.8, 1:4, and 1:8 using T cell culture medium containing 25 μl / ml CD3 / CD28 T cell activator. After 24 hours of co-culture, GFP fluorescence in NALM6 cells was observed under a fluorescence microscope. The cells were then centrifuged at 500 g for 3 minutes, and 50 μl of supernatant was collected. After 5-fold dilution, LDH release was measured using the CytoTox 96 non-radioactive cytotoxicity assay (Promega) to determine the level of cellular cytotoxicity.

[0288] Example 30

[0289] Gene Editing (Cre mRNA) and Flow Cytometry in the Loxp-GFP-Luciferase Mouse Model

[0290] PL Cre mRNA formulations were prepared as described above. The mice were then dialyzed overnight and administered intravenously at a dose of 0.3 mg / kg. Two days later, the mice were sacrificed, and the liver and spleen were imaged using the IVIS Lumina system (Perkin Elmer).

[0291] Mouse spleens were then ground into a single-cell suspension using a 70 μm cell strainer. ACK lysis buffer was used to remove red blood cells, followed by a single wash with PBS. The cells were then transferred to staining buffer and incubated at 4°C in the dark for 30 minutes. After an additional wash with ice-cold PBS, the cells were resuspended in PBS and analyzed by flow cytometry to assess the expression of PL LNPs on T cells and other immune cells in vivo. The following flow cytometry antibodies were used: APC anti-mouse CD3 (Biolegend), PE anti-mouse CD8a (Biolegend), PerCP Cy5.5 anti-mouse CD4 (Biolegend), and BV605 anti-human / mouse CD11b (Biolegend).

[0292] Experimental results

[0293] DOPE-based LNPs (PL15 and PL16) were prepared at a 10:1 bisphosphoramide lipid / RNA weight ratio. Microfluidics-based LNP preparation demonstrated excellent in vivo transfection efficiency, demonstrating expression of both PL15 and PL16. The DOPE-based LNPs exhibited superior in vivo expression compared to the MC3 control group, with PL16 showing an order of magnitude increase in expression compared to the MC3 control group. This demonstrates for the first time that bisphosphoramide lipids can be used for in vivo RNA delivery, with higher delivery efficiency compared to MC3.

[0294] The constructed bisphosphoramide lipids were used to construct lipid nanoparticles and their transfection efficiency in T cells and NK92MI cells was evaluated. The transfection efficiency in T cells is shown in Figure 2 . Compared to the transfection reagent and ALC0315, PL16 increased transfection efficiency by 10-fold, PL101 by 100-fold, PL102 by 10-fold, PL40 by 100-fold, PL67 by 60-fold, and PL82 by 5-fold. The transfection efficiency in the NK92MI cell line is shown in Figure 3 . Compared to the transfection reagent and ALC0315, PL16 increased transfection efficiency by 50-fold, PL101 by 35-fold, PL102 by 17-fold, and PL40 by 40-fold. The remaining bisphosphoramide lipids exhibited similar transfection efficiencies to the control lipid. Furthermore, we characterized the physicochemical properties of the selected bisphosphoramide lipid formulations (Figure 4). The results showed that most bisphosphoramide lipids had a particle size of approximately 150 nm, were uniformly distributed, and were electrically neutral. These properties are suitable for subsequent process scale-up and clinical translation.

[0295] To ultimately achieve chimeric antibody and gene editing in T and NK cells using LNPs, we further investigated the transfection efficiency of GFP mRNA and CAR mRNA in T and NK92MI cells using PL16, PL101, and PL40 lipid nanoparticles, and compared these results with the monophosphoramide lipid PL32. As shown in Figures 5 and 6, PL101 and PL40 lipid nanoparticles encapsulating GFP mRNA exhibited higher transfection efficiency in T cells, reaching over 80% at doses of 0.5 μg / well and 1.0 μg / well, respectively. PL16 lipid nanoparticles encapsulating GFP mRNA exhibited even higher transfection efficiency in NK92MI cells, reaching over 75% at doses of 0.5 μg / well and 1.0 μg / well. Transfection efficiency saturated in both cell lines at a concentration of 0.5 μg / well. In addition, PL101, PL40, and PL32 lipid nanoparticles (monophosphoramide PL32 as a control) loaded with chimeric antibody CAR CD19 mRNA were used to transfect T cells and NK92MI cells. In T cells and NK92MI cells, PL40 had the highest transfection rate, reaching saturation at a concentration of 0.5ug / well, which was approximately 40% and 10%, respectively. In addition, the transfection efficiency of bisphosphoramide lipids PL101 and PL40 was higher than that of the control monophosphoramide PL32. In summary, we found that PL40 and PL101 were better at delivering mRNA to T cells, and PL40 and PL16 were better at delivering mRNA to NK92MI. The bisphosphoramide lipid material can efficiently edit T cells and NK cells in vitro.

[0296] We further investigated the activity of LNPs containing CAR CD19 mRNA encapsulated with bisphosphamide lipids. We transfected these LNPs into human T cells and, after transfection, co-incubated them with B cells expressing high levels of CD19. GFP fluorescence microscopy revealed that the killing effect on B cells increased with the increase in the number of T cells expressing CAR CD19, while no killing was observed in the control group. LDH assays demonstrated that the killing intensity increased with the increase in T cells, reaching a maximum of 80% killing at an E:T ratio of 10, further demonstrating that LNP-transfected CAR T cells possessed strong specific killing activity (Figure 7). Furthermore, we evaluated the efficiency of direct T cell transfection with bisphosphamide lipids in vivo. Using LoxP-GFP-Fluc mice, we delivered mRNA encoding Cre recombinase, and cells expressing Cre protein produced GFP and Fluc. Our results showed that non-antibody-modified bisphosphoramide lipid LNPs can effectively transfect T cells in the spleen, with ~20% of T cells being efficiently edited, thus laying the experimental foundation for subsequent LNP editing of T cells in vivo (Figure 8).

[0297] The above results indicate that bisphosphamide lipid materials (such as the most effective PL16, PL40, PL101, etc.) can achieve efficient delivery of CAR mRNA in vivo and in vitro, and can be used to construct CAR-T cells and CAR-NK cells in vivo, which is expected to achieve simpler and more controllable cell immunotherapy, reduce the cost of existing CAR-T technology and the potential immune toxicity induced by over-activation of T cells.

[0298] Summary of bisphosphoramide lipid expression results

[0299] Description of expression intensity level:

[0300] NA = Not tested

[0301] A: ≥2

[0302] B: ≥1 and <2

[0303] C: ≥0.1 and <1

[0304] D: <0.1

[0305] Although the embodiments disclosed in this application are as described above, the contents described are merely embodiments adopted to facilitate understanding of this application and are not intended to limit this application. Any person skilled in the art to which this application belongs may make any modifications and changes in the form and details of the implementation without departing from the spirit and scope disclosed in this application, but the scope of protection of this application shall still be based on the scope defined by the attached claims.

Claims

1. A bisphospholipid amide compound containing multiple tertiary amino structures, wherein the bisphospholipid amide compound is a compound represented by formula (I) or its stereoisomers, prodrugs, pharmaceutically acceptable salts: In formula (I), n is an integer from 0 to 5; L1 is C1-C 30 alkyl, C2-C 40 alkenyl, or -R'-M-R", and L2 is C5-C 30 alkyl, C5-C 40 alkenyl, or -R’-M-R”; or, L1 and L2 are each independently C5-C 30 alkyl, C5-C 40 alkenyl, or -R'-M-R"; R1 and R2 are each independently -Z-T, C4-C 30 alkyl, C2-C 24 alkenyl, or -R'-M-R''; alternatively, R1 and R2 together with the N and L4 to which they are each attached form a 5- to 8-membered diazacycloalkyl; herein, Z is an optionally substituted C1-C3 alkylene, and T is H, hydroxy, C1-C4 alkoxy, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, an optionally substituted 4- to 8-membered azacycloalkyl, an optionally substituted aryl or an optionally substituted heteroaryl, and the optionally substituted C1-C3 alkylene, optionally substituted 4- to 8-membered azacycloalkyl, optionally substituted aryl or optionally substituted heteroaryl is unsubstituted or substituted by one or more groups selected from: C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy and halogen; provided that -Z-T is not C4-C 30 alkyl; R3 is H, C1-C 30 alkyl, C2-C 24 alkenyl, or -R'-M-R"; L 1’ is C1-C 30 alkyl, C2-C 40 alkenyl, or -R’-M-R”, and L 2' is C5-C 30 alkyl, C5-C 40 alkenyl, or -R’-M-R”, or, L 1’ and L 2' are each independently C5-C 30 alkyl, C5-C 40 alkenyl, or -R'-M-R”; R’ is C1-C 12 alkylene or C2-C 12 alkenylene; M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -S-S-, -C(O)N(R a )-, -N(R a )C(O)-, -OC(O)N(R a )-, -N(R a )C(O)O-, -N(R a )C(O)N(R a )-, -C(OC(O)R a )-C 1-6 alkylene - O - C(O)-, -C(O)-C 1-6 alkylene - C(O)-O-, -CH(OH)-, -P(O)(OR b )O-, C6 - C 10 arylene and 5 - to 10 - membered heteroarylene; "R”, R a and R b each independently selected from hydrogen, C1-C 30 alkyl, and C2-C 40 alkenyl; L3, L4 and L5 are each independently C1-C 12 alkylene, C2-C 12 alkenylene, C3-C8 cycloalkylene, -A-Y-B-, -A-Y- or -Y-B-, where A and B are each independently C1-C 12 alkylene or C2-C 12 alkenylene, and Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene or heteroarylene.

2. The bisphosphatide amide compound according to claim 1, wherein In formula (I), n is an integer from 0 to 5; L1 and L2 are each independently C5-C 30 alkyl, C5-C 40 alkenyl, or -R'-M-R''; R1 and R2 are each independently C4-C 30 alkyl, C2-C 24 alkenyl, or -R'-M-R"; alternatively, R1 and R2 together with the N and L4 to which they are each attached form a 5- to 8-membered diazacycloalkylidene; R3 is H, C1-C 30 alkyl, C2-C 24 alkenyl, or -R’-M-R”; L 1’ and L 2’ each independently is C5-C 30 alkyl, C5-C 40 alkenyl, or -R’-M-R” R’ is C1-C 12 alkylene or C2-C 12 alkenylene; M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -S-S-, -C(O)N(R a )-, -N(R a )C(O)-, -OC(O)N(R a )-, -N(R a )C(O)O-, -N(R a )C(O)N(R a )-, -C(OC(O)R a )-C 1-6 alkylene - O - C(O)-, -C(O)-C 1-6 alkylene - C(O)-O-, -CH(OH)-, -P(O)(OR b )O-, C6 - C 10 arylene and 5 - to 10 - membered heteroarylene; R”, R a and R b each independently selected from hydrogen, C1-C 30 alkyl and C2-C 40 alkenyl; L3, L4 and L5 are each independently C1-C 12 alkylene, C2-C 12 alkenylene, C3-C8 cycloalkylene, -A-Y-B-, -A-Y- or -Y-B-, where A and B are each independently C1-C 12 alkylene or C2-C 12 alkenylene, and Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene or heteroarylene.

3. The bisphosphatamide compound according to claim 1, wherein, In formula (I), n is an integer from 0 to 5; L1 is C1-C 30 alkyl, C2-C 40 alkenyl, or -R'-M-R", and L2 is C5-C 30 alkyl, C5-C 40 alkenyl, or -R'-M-R"; R1 and R2 are each independently -Z-T, C4-C 30 alkyl, C2-C 24 alkenyl, or -R'-M-R''; alternatively, R1 and R2 together with the N and L4 to which they are each attached form a 5- to 8-membered diazacycloalkyl; herein, Z is an optionally substituted C1-C3 alkylene, and T is H, hydroxy, C1-C4 alkoxy, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, an optionally substituted 4- to 8-membered azacycloalkyl, an optionally substituted aryl or an optionally substituted heteroaryl, and the optionally substituted C1-C3 alkylene, optionally substituted 4- to 8-membered azacycloalkyl, optionally substituted aryl or optionally substituted heteroaryl is unsubstituted or substituted with one or more groups selected from the following: C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy and halogen; provided that -Z-T is not C4-C 30 alkyl; R3 is H, C1-C 30 alkyl, C2-C 24 alkenyl, or -R'-M-R”; L 1’ is C1-C 30 alkyl, C2-C 40 alkenyl, or -R'-M-R”, and L 2' is C5-C 30 alkyl, C5-C 40 alkenyl, or -R'-M-R”; R’ is C1-C 12 alkylene or C2-C 12 alkenylene; M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -S-S-, -C(O)N(R a )-, -N(R a )C(O)-, -OC(O)N(R a )-, -N(R a )C(O)O-, -N(R a )C(O)N(R a )-, -C(OC(O)R a )-C 1-6 alkylene - O - C(O)-, -C(O)-C 1-6 alkylene - C(O)-O-, -CH(OH)-, -P(O)(OR b )O-, C6 - C 10 arylene and 5 - to 10 - membered heteroarylene; "R”, R a and R b each independently selected from hydrogen, C1-C 30 alkyl and C2-C 40 alkenyl; L3, L4, and L5 are each independently C1-C 12 alkylene, C2-C 12 alkenylene, C3-C8 cycloalkylene, -A-Y-B-, -A-Y-, or -Y-B-, where A and B are each independently C1-C 12 alkylene or C2-C 12 alkenylene, and Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene, or heteroarylene.

4. The bisphosphonamide compound according to claim 1, wherein n is 0, and the compound of formula (I) is the compound of formula (I-1): In formula (1-1), L1 and L2 are each independently a C5-C 30 alkyl group, a C5-C 40 alkenyl group, or -R'-M-R''; R1 is C4-C 30 alkyl, C2-C 24 alkenyl, or -R'-M-R''; R3 is H, C1-C 30 alkyl, C2-C 24 alkenyl, or -R'-M-R"; L 1’ and L 2’ are each independently C5-C 30 alkyl, C5-C 40 alkenyl, or -R'-M-R"; optionally, L 1’ is the same group as L1, and L 2’ is the same group as L2; R’ is C1-C 12 alkylene or C2-C 12 alkenylene; M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -S-S-, -C(O)N(R a )-, -N(R a )C(O)-, -OC(O)N(R a )-, -N(R a )C(O)O-, -N(R a )C(O)N(R a )-, -CH(OH)-, -C(OC(O)R a )-C 1-6 alkylene -O-C(O)-, -C(O)-C 1-6 alkylene -C(O)-O-, -P(O)(OR b )O-, C6-C 10 arylene and 5- to 10-membered heteroarylene; R”, R a and R b each independently selected from hydrogen, C1-C 30 alkyl and C2-C 40 alkenyl; L3 and L5 are each independently C1-C 12 alkylene, C2-C 12 alkenylene, C3-C8 cycloalkylene, -A-Y-B-, -A-Y- or -Y-B-, wherein A and B are each independently C1-C 12 alkylene or C2-C 12 alkenylene, Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene or heteroarylene; or, When n is 0, the compound of formula (I) is the compound of formula (I-1-1): The definitions of the substituents in formula (I-1-1) are as defined above.

5. The bisphosphonamide compound according to claim 1, wherein n is 0, and the compound of formula (I) is the compound of formula (I-1): In formula (I-1), L1 is C1-C 30 alkyl, C2-C 40 alkenyl, or -R'-M-R", and L2 is C5-C 30 alkyl, C5-C 40 alkenyl, or -R'-M-R"; R1 is -Z-T, C4-C 30 alkyl, C2-C 24 alkenyl, or -R'-M-R''; wherein, Z is an optionally substituted C1-C3 alkylene, and T is H, hydroxy, C1-C4 alkoxy, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, an optionally substituted 4- to 8-membered azacycloalkyl, an optionally substituted aryl or an optionally substituted heteroaryl, and the optionally substituted C1-C3 alkylene, the optionally substituted 4- to 8-membered azacycloalkyl, the optionally substituted aryl or the optionally substituted heteroaryl is unsubstituted or substituted by one or more groups selected from the following: C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy and halogen; provided that, -Z-T is not C4-C 30 alkyl; R3 is H; L 1’ is C1-C 30 alkyl, C2-C 40 alkenyl, or -R'-M-R", and L 2' is C5-C 30 alkyl, C5-C 40 alkenyl, or -R'-M-R"; optionally, L 1’ is the same group as L1, and L 2' is the same group as L2; R’ is C1-C 12 alkylene or C2-C 12 alkenylene; M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -S-S-, -C(O)N(R a )-, -N(R a )C(O)-, -OC(O)N(R a )-, -N(R a )C(O)O-, -N(R a )C(O)N(R a )-, -CH(OH)-, -C(OC(O)R a )-C 1-6 alkylene - O - C(O)-, -C(O)-C 1-6 alkylene - C(O)-O-, -P(O)(OR b )O-, C6 - C 10 arylene and 5 - to 10 - membered heteroarylene; R”, R a and R b each independently selected from hydrogen, C1-C 30 alkyl, and C2-C 40 alkenyl; L3 and L5 are each independently C1-C 12 alkylene, C2-C 12 alkenylene, C3-C8 cycloalkylene, -A-Y-B-, -A-Y- or -Y-B-, wherein A and B are each independently C1-C 12 alkylene or C2-C 12 alkenylene, Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene or heteroarylene; or, When n is 0, the compound of formula (I) is the compound of formula (I-1-1): The definitions of the substituents in formula (I-1-1) are as defined above.

6. The bisphosphonamide compound according to claim 1 or 2, wherein, When n is 1, the compound of formula (I) is a compound of formula (I-2): L1 and L2 are each independently C5-C 30 alkyl, C5-C 40 alkenyl, or -R'-M-R''; R1 and R2, together with the N and L4 to which they are each attached, form a 5- to 8-membered diazacycloalkyl; or, R1 and R2 are each independently C4-C 30 alkyl, C2-C 24 alkenyl, or -R'-M-R", L4 is C1-C 12 alkylene, C2-C 12 alkenylene, C3-C8 cycloalkylene, -A-Y-B-, -A-Y- or -Y-B-, where A and B are each independently C1-C 12 alkylene or C2-C 12 alkenylene, Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene or heteroarylene; R3 is H, C1-C 30 alkyl, C2-C 24 alkenyl, or -R'-M-R''; L 1’ and L 2' are each independently C5-C 30 alkyl, C5-C 40 alkenyl, or -R'-M-R''; optionally, L 1’ is the same group as L1, and L 2’ is the same group as L2; R’ is C1-C 12 alkylene or C2-C 12 alkenylene; M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -S-S-, -C(O)N(R a )-, -N(R a )C(O)-, -OC(O)N(R a )-, -N(R a )C(O)O-, -N(R a )C(O)N(R a )-, -CH(OH)-, -C(OC(O)R a )-C 1-6 alkylene - O - C(O)-, -C(O)-C 1-6 alkylene - C(O)-O-, -P(O)(OR b )O-, C6 - C 10 arylene and 5 - to 10 - membered heteroarylene; "R”, R a and R b are each independently selected from hydrogen, C1-C 30 alkyl and C2-C 40 alkenyl; L3 and L5 are each independently C1-C 12 alkylene, C2-C 12 alkenylene, C3-C8 cycloalkylene, -A-Y-B-, -A-Y- or -Y-B-, wherein A and B are each independently C1-C 12 alkylene or C2-C 12 alkenylene, Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene or heteroarylene; or When n is 1, the compound of formula (I) is the compound of formula (I-2-1): The definitions of the substituents in formula (I-2-1) are as defined above.

7. The bisphosphonamide compound according to claim 1 or 3, wherein When n is 1, the compound of formula (I) is the compound of formula (I-2): L1 is C1-C 30 alkyl, C2-C 40 alkenyl, or -R'-M-R", and L2 is C5-C 30 alkyl, C5-C 40 alkenyl, or -R'-M-R"; R1 and R2, together with their respective attached N and L4, form a 5- to 8-membered diazacycloalkyl; or, R1 and R2 are each independently -Z-T, C4-C 30 alkyl, C2-C 24 alkenyl, or -R'-M-R", where Z is an optionally substituted C1-C3 alkylene, and T is H, hydroxy, C1-C4 alkoxy, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, an optionally substituted 4- to 8-membered azacycloalkyl, an optionally substituted aryl or an optionally substituted heteroaryl, and the optionally substituted C1-C3 alkylene, optionally substituted 4- to 8-membered azacycloalkyl, optionally substituted aryl or optionally substituted heteroaryl is unsubstituted or substituted with one or more groups selected from: C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy and halogen; provided that -Z-T is not C4-C 30 alkyl; L4 is C1-C 12 alkylene, C2-C 12 alkenylene, C3-C8 cycloalkylene, -A-Y-B-, -A-Y- or -Y-B-, where A and B are each independently C1-C 12 alkylene or C2-C 12 alkenylene, and Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene or heteroarylene; R3 is H, C1-C 30 alkyl, C2-C 24 alkenyl, or -R'-M-R''; L 1’ is C1-C 30 alkyl, C2-C 40 alkenyl, or -R'-M-R''; and L 2’ is C5-C 30 alkyl, C5-C 40 alkenyl, or -R'-M-R''; optionally, L 1’ is the same group as L1, and L 2’ is the same group as L2; R’ is C1-C 12 alkylene or C2-C 12 alkenylene; M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -S-S-, -C(O)N(R a )-, -N(R a )C(O)-, -OC(O)N(R a )-, -N(R a )C(O)O-, -N(R a )C(O)N(R a )-, -CH(OH)-, -C(OC(O)R a )-C 1-6 alkylene - O - C(O)-, -C(O)-C 1-6 alkylene - C(O)-O-, -P(O)(OR b )O-, C6 - C 10 arylene and 5 - to 10 - membered heteroarylene; R”, R a and R b each independently selected from hydrogen, C1-C 30 alkyl and C2-C 40 alkenyl; L3 and L5 are each independently C1-C 12 alkylene, C2-C 12 alkenylene, C3-C8 cycloalkylene, -A-Y-B-, -A-Y- or -Y-B-, wherein A and B are each independently C1-C 12 alkylene or C2-C 12 alkenylene, Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, arylene or heteroarylene; or, When n is 1, the compound of formula (I) is the compound of formula (I-2-1): The definitions of the substituents in formula (I-2-1) are as defined above.

8. The bisphosphatidic acid amide compound according to any one of claims 1-7, wherein, L 1’ 、L 2’ 、L1 and L2 are each independently a C6-C 30 alkyl group, a C6-C 40 alkenyl group, or -R’-M-R”; wherein, R’ is a C1-C 10 alkylene group or a C2-C 10 alkenylene group; M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -S-S-, -C(O)N(R a ), -, N(R a )C(O)-, -OC(O)N(R a ), -, N(R a )C(O)O-, -N(R a )C(O)N(R a ), -, -CH(OH)-, -C(OC(O)R a )-C 1-6 alkylene -O-C(O)-, -C(O)-C 1-6 alkylene -C(O)-O-, -P(O)(OR b )O-, C6-C 10 arylene group and a 5- to 10-membered heteroarylene group; R” is a C1-C 30 alkyl group or a C2-C 40 alkenyl group; R a and R b are each independently selected from hydrogen, a C1-C 30 alkyl group and a C2-C 40 alkenyl group; or, L 1’ 、L 2’ 、L1 and L2 are each independently C6-C 30 alkyl, C6-C 40 alkenyl, or -R’-M-R”; wherein, R’ is C1-C 10 alkylene or C2-C 10 alkenylene; M is -C(O)-, -C(O)O-, -C(OC(O)R a )-C 1-6 alkylene-O-C(O)-, -C(O)-C 1-6 alkylene-C(O)-O- or -CH(OH)-; R a and R” are each independently C1-C 30 alkyl or C2-C 40 alkenyl; or, L 1' 、L 2’ , L1 and L2 are each independently one of the following structures: Or, L 1’ 、L 2’ 、L1 and L2 are each independently one of the following structures:

9. The bis(phosphatidic acid amide) compound according to any one of claims 1, 3, 5 and 7, wherein, L1 is C1-C 30 alkyl, C2-C 40 alkenyl, or -R'-M-R", and L2 is C5-C 30 alkyl, C5-C 40 alkenyl, or -R'-M-R"; L 1’ is C1-C 30 alkyl, C2-C 40 alkenyl, or -R'-M-R", and L 2’ is C5-C 30 alkyl, C5-C 40 alkenyl, or -R'-M-R"; herein, R' is C1-C 10 alkylene or C2-C 10 alkenylene; M is selected from -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(O)S-, -SC(O)-, -C(S)S-, -SC(S)-, -S(O)2-, -S-S-, -C(O)N(R a ), -, N(R a )C(O)-, -OC(O)N(R a ), -, N(R a )C(O)O-, -N(R a )C(O)N(R a ), -, -CH(OH)-, -C(OC(O)R a )-, -C 1-6 alkylene-O-C(O)-, -C(O)-C 1-6 alkylene-C(O)-O-, -P(O)(OR b )O-, C6-C 10 arylene and 5- to 10-membered heteroarylene; R" is C1-C 30 alkyl or C2-C 40 alkenyl; R a and R b are each independently selected from hydrogen, C1-C 30 alkyl and C2-C 40 alkenyl; or, L1 is C1-C 30 alkyl, C2-C 40 alkenyl, or -R’-M-R”, and L2 is C5-C 30 alkyl, C5-C 40 alkenyl, or -R’-M-R”; L 1’ is C1-C 30 alkyl, C2-C 40 alkenyl, or -R'-M-R”, and L 2’ is C5-C 30 alkyl, C5-C 40 alkenyl, or -R'-M-R”; herein, R’ is C1-C 10 alkylene or C2-C 10 alkenylene; M is -C(O)-, -C(O)O-, -C(OC(O)R a )-C 1-6 alkylene-O-C(O)-, -C(O)-C 1-6 alkylene-C(O)-O- or -CH(OH)-; R a and R” are each independently C1-C 30 alkyl or C2-C 40 alkenyl; or, L 1’ 、L 2’ 、 L1 and L2 are each independently one of the following structures: Alternatively, L 1’ and L1 are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, and L 2’ and L2 are as defined above; or L 1’ 、L 2’ 、 L1 and L2 are each independently one of the following structures: Alternatively, L 1’ and L1 are each independently ethyl, and L 2’ and L2 are as defined above.

10. The bisphosphonamide compound according to any one of claims 1 to 3 and 6 to 8, wherein, R1 and R2 are each independently one of the following structures: Or, R1 and R2 are each independently one of the following structures:

11. The bisphosphonamide compound according to any one of claims 1, 3 and 7, wherein, R1 and R2 are each independently one of the following structures or groups: Methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxy-n-propyl, benzyl, 2-phenylethyl, 3-phenylpropyl, dimethylaminomethyl, 2-(dimethylamino)ethyl, 3-(dimethylamino)propyl, diethylaminomethyl, 2-(diethylamino)ethyl, 3-(diethylamino)propyl, (pyrrolidin-1-yl)methyl, 2-(pyrrolidin-1-yl)ethyl, 3-(pyrrolidin-1-yl)propyl, 2-methyl-3-(pyrrolidin-1-yl)propyl, (piperidin-1-yl)methyl, 2-(piperidin-1-yl)ethyl, 3-(piperidin-1-yl)propyl, (2-ethylpiperidin-1-yl)methyl, 2-(2-ethylpiperidin-1-yl)ethyl, 3-(2-ethylpiperidin-1-yl)propyl, (3-ethyl-6-methylpiperidin-1-yl)methyl, 2-(3-ethyl-6-methylpiperidin-1-yl)ethyl, 3-(3-ethyl-6-methylpiperidin-1-yl)propyl, (azepan-1-yl)methyl, 2-(azepan-1-yl)ethyl, 3-(azepan-1-yl)propyl, (piperazin-1-yl)methyl, 2-(piperazin-1-yl)ethyl, 3-(piperazin-1-yl)propyl, (4-methylpiperazin-1-yl)methyl, 2-(4-methylpiperazin-1-yl)ethyl, 3-(4-methylpiperazin-1-yl)propyl, (morpholin-1-yl)methyl, 2-(morpholin-1-yl)ethyl, 3-(morpholin-1-yl)propyl, Or, R1 and R2 are each independently one of the following structures or groups: methyl, 2-hydroxyethyl, 3-hydroxypropyl, 3-phenylpropyl, 3-(diethylamino)propyl, (pyrrolidin-1-yl)methyl, 2-(pyrrolidin-1-yl)ethyl, 3-(pyrrolidin-1-yl)propyl, 2-methyl-3-(pyrrolidin-1-yl)propyl, 2-(piperidin-1-yl)ethyl, (2-ethylpiperidin-1-yl)methyl, 2-(2-ethylpiperidin-1-yl)ethyl, 3-(2-ethylpiperidin-1-yl)propyl, (3-ethyl-6-methylpiperidin-1-yl)methyl, 2-(3-ethyl-6-methylpiperidin-1-yl)ethyl, 3-(3-ethyl-6-methylpiperidin-1-yl)propyl, 2-(azepan-1-yl)ethyl, 3-(azepan-1-yl)propyl, (piperazin-1-yl)methyl, 2-(piperazin-1-yl)ethyl, 3-(piperazin-1-yl)propyl, 3-(4-methylpiperazin-1-yl)propyl, 2-(morpholin-1-yl)ethyl 12. The bisphosphatide amide compound according to any one of claims 1 to 3, 6 to 9, wherein, R1 and R2, together with the N and L4 to which they are respectively attached, form a group selected from the following optionally substituted groups: Or, R1 and R2, together with their respective attached N and L4, form an optionally substituted group selected from the following:

13. The bisphosphonamide compound according to any one of claims 1-12, wherein, L3 and L5, or when L4 is present, L3, L4 and L5 are each independently methylene, ethylene, propylene, butylene, pentylene, hexylene, vinyl, propenyl, butenyl, pentenyl, hexenyl, cyclopropylidene, cyclobutylidene, cyclopentylidene, cyclohexylidene, cycloheptylidene, -A-Y-B-, -A-Y- or -Y-B-, where A and B are each independently methylene, ethylene, propylene, butylene, pentylene, hexylene, vinyl, propenyl, butenyl, pentenyl or hexenyl, and Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, phenylene, pyrrolylidene, imidazolylidene, thiazolylidene, thienylidene, furylidene, pyridinylidene or pyrimidinyl; or, L3 and L5, or when L4 is present, L3, L4 and L5 are each independently -CH2-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH(CH3)-, -A-Y-B-, -A-Y- or -Y-B-, where A and B are each independently -CH2-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2- or -CH2CH2CH(CH3)-, and Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)- or phenylene; or, L3 is -CH2-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH(CH3)-, -A-Y-B-, -A-Y- or -Y-B-, where A and B are each independently -CH2-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2- or -CH2CH2CH(CH3)-, and Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-; or, L3 is -CH2CH2- or -CH2CH2CH2-; or, L4 is -CH2-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH(CH3)-, -A-Y-B-, -A-Y- or -Y-B-, where A and B are each independently -CH2-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2- or -CH2CH2CH(CH3)-, and Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-; or, L4 is -CH2CH2- or -CH2CH2CH2-; or, L5 is -CH2-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH(CH3)-, -A-Y-B-, -A-Y- or -Y-B-, where A and B are each independently -CH2-, -CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2- or -CH2CH2CH(CH3)-, and Y is -C(OH)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)- or phenylene; or, L5 is -CH2CH2-, -CH2CH2CH2-, -CH2CH(OH)CH2- or 14. The bisphosphonamide compound according to claim 1, 3 or 5, wherein, n is 0; R1 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxy-n-propyl, benzyl, 2-phenylethyl, 3-phenylpropyl, dimethylaminomethyl, 2-(dimethylamino)ethyl, 3-(dimethylamino)propyl, diethylaminomethyl, 2-(diethylamino)ethyl, 3-(diethylamino)propyl, (pyrrolidin-1-yl)methyl, 2-(pyrrolidin-1-yl)ethyl, 3-(pyrrolidin-1-yl)propyl, 2-methyl-3-(pyrrolidin-1-yl)propyl, (piperidin-1-yl)methyl, 2-(piperidin-1-yl)ethyl, 3-(piperidin-1-yl)propyl, (2-ethylpiperidin-1-yl)methyl, 2-(2-ethylpiperidin-1-yl)ethyl, 3-(2-ethylpiperidin-1-yl)propyl, (3-ethyl-6-methylpiperidin-1-yl)methyl, 2-(3-ethyl-6-methylpiperidin-1-yl)ethyl, 3-(3-ethyl-6-methylpiperidin-1-yl)propyl, (azepan-1-yl)methyl, 2-(azepan-1-yl)ethyl, 3-(azepan-1-yl)propyl, (piperazin-1-yl)methyl, 2-(piperazin-1-yl)ethyl, 3-(piperazin-1-yl)propyl, (4-methylpiperazin-1-yl)methyl, 2-(4-methylpiperazin-1-yl)ethyl, 3-(4-methylpiperazin-1-yl)propyl, (morpholin-1-yl)methyl, 2-(morpholin-1-yl)ethyl or 3-(morpholin-1-yl)propyl; R3 is H; L 1’ 、L 2’ , L1 and L2 are each independently one of the following structures: Alternatively, L 1’ and L1 are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, and L 2’ and L2 are as defined above; L3 is -CH2CH2- or -CH2CH2CH2-; L5 is -CH2CH2-, -CH2CH2CH2-, -CH2CH(OH)CH2- or 15. The bisphosphatide amide compound according to any one of claims 1 to 3, 6 to 9, wherein, n is 1; R1 and R2 together with the N and L4 to which they are each attached form an optionally substituted group selected from the following: L3 is -CH2CH2- or -CH2CH2CH2-; L5 is -CH2CH2-, -CH2CH2CH2- or -CH2CH(OH)CH2-; R3 is H; L 1’ 、L 2’ , L, 2’ , L1 and L2 are each independently one of the following structures: Alternatively, L 1’ and L1 are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, and L 2’ and L2 are as defined above.

16. The bisphosphonamide compound according to any one of claims 1 to 15, being one selected from the following compounds: or a stereoisomer, prodrug, pharmaceutically acceptable salt thereof.

17. A lipid composition, the lipid composition comprising a bisphosphatidic amide compound as described in any one of claims 1-16; and a therapeutic or prophylactic agent.

18. The lipid composition according to claim 17, wherein, The lipid composition further comprises additional lipids, the additional lipids being selected from one or more of neutral lipids, steroids, and polymer-conjugated lipids; Optionally, the neutral lipid is selected from one or more of DSPC, DPPC, DOPC, POPC, DOPE, DSPG, DOPG, DOPS, DGTS, DOPA, and SM, and preferably, it is DOPE; Optionally, the molar ratio of the neutral lipid to the bisphosphatidic amide compound is from 2:1 to 8:1; Optionally, the steroid is cholesterol; Optionally, the molar ratio of the steroid to the bisphosphatidic amide compound is from 5:1 to 1:1; Optionally, the polymer-conjugated lipid is a PEGylated lipid selected from PEG-DAG, PEG-PE, PEG-S-DAG, and PEG-cer.

19. The lipid composition according to claim 17, wherein, The therapeutic or prophylactic agent is selected from one or more of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides, and protein drugs.

20. The lipid composition according to any one of claims 17 to 19, wherein The lipid composition is in the form of lipid nanoparticles.

21. A lipid nanoparticle, the lipid nanoparticle comprising a bisphosphatidic amide compound as described in any one of claims 1-16 or a lipid composition as described in any one of claims 17-20.

22. A pharmaceutical composition, the pharmaceutical composition comprising a bisphosphatidic amide compound as described in any one of claims 1-16, a lipid composition as described in any one of claims 17-20, or a lipid nanoparticle as described in claim 21, and a pharmaceutically acceptable diluent or excipient.

23. Use of the lipid composition as described in any one of claims 17-20, the lipid nanoparticle as described in claim 21, or the pharmaceutical composition as described in claim 22 for the treatment or prevention of a disease in an individual in need thereof; or, use of the lipid composition as described in any one of claims 17-20, the lipid nanoparticle as described in claim 21, or the pharmaceutical composition as described in claim 22 in the preparation of a drug for the treatment or prevention of a disease in an individual in need thereof; optionally, the disease is fatty liver, obesity, tumor, arthritis, viral infection, or an autoimmune disease; or for in vivo editing of T cells, macrophages, NK cells, etc. for the treatment of cancer or autoimmune diseases.

24. A method for treating or preventing a disease in an individual in need thereof, the method comprising administering to the individual a lipid composition as described in any one of claims 17-20, a lipid nanoparticle as described in claim 21, or a pharmaceutical composition as described in claim 22; optionally, the disease is fatty liver, obesity, tumor, arthritis, viral infection, or an autoimmune disease; or for in vivo editing of T cells, macrophages, NK cells, etc. for the treatment of cancer or autoimmune diseases.

25. A method for inoculating an individual in need thereof against an antiviral pathogen, the method comprising administering to the individual the lipid composition according to any one of claims 17-20, the lipid nanoparticle according to claim 21, or the pharmaceutical composition according to claim 22.

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