FAPα hydrolysis-induced charge reversal liposome and composition, preparation method therefor, and use thereof

Abstract

The present invention belongs to the technical field of biomedical polymer materials, and particularly relates to an FAPα hydrolysis-induced charge reversal liposome and composition, a preparation method therefor, and use thereof. The FAPα hydrolysis-induced charge reversal liposome has a structure represented by formula (1). The FAPα hydrolysis-induced charge reversal liposome provided by the present invention exhibits deep tumor penetration, utilizes the specific enzymatic cleavage effect of overexpressed FAPα in tumor tissues to achieve charge reversal, and has the characteristic of enhancing drug uptake and delivery efficiency in CAFs. In a hypoxic tumor microenvironment that affects the individualized therapeutic effect on most conventional tumors, the FAPα responsive liposome can effectively deliver a drug into a tumor, so that the tumor can be profoundly killed, thereby achieving a significant tumor inhibition effect.

Description

A FAPα hydrolysis-induced charge flip liposome and composition as well as preparation method and application thereof Technical Field

[0001] The present invention belongs to the technical field of biomedical polymer materials, and particularly relates to a fibroblast activation protein-α (FAPα) FAPα hydrolysis-induced charge reversal liposome and composition, as well as a preparation method and application thereof. Background Art

[0002] In recent years, the application of nanomedicines in tumor therapy has made great progress, and a variety of anti-tumor nanomedicines have been launched or entered clinical trials. Numerous studies have shown that the presence of a complex tumor microenvironment (TME) plays an important role in both tumor cell resistance and drug penetration within the tumor, and is a key factor limiting the efficacy of existing nanomedicines. Therefore, how to reshape the TME to improve tumor resistance and enhance the in vivo delivery efficiency of anti-tumor nanomedicines is of great significance for overcoming existing nanomedicine delivery barriers and improving the efficacy of tumor treatment. Tumor-associated fibroblasts (CAFs), as the main stromal cells in the TME, are widely present in a variety of solid tumors, including breast cancer, colorectal cancer, prostate cancer, non-small cell lung cancer, bladder cancer, pancreatic cancer, renal cancer, and hepatocellular carcinoma. Their biological functions include inducing tumorigenesis, immunosuppression, enhancing drug resistance, hindering the diffusive transport of nanomedicines (extravasation and diffusion), and competing for drug uptake. The widespread distribution of CAFs in solid tumors and their important physiological functions make them a very promising TME remodeling target in the development of anti-tumor nanomedicines. Summary of the Invention

[0003] Based on the overexpression characteristics of FAPα in CAFs and its specific hydrolase activity, the present invention designs and synthesizes liposomes with FAPα-specific responsiveness. On the one hand, it meets the application requirements of clear structure and long-term plasma stability. On the other hand, it utilizes the specific enzymatic cleavage of FAPα overexpressed in tumor tissue to induce charge reversal of the conjugate, and then combines passive diffusion with active transcytosis to achieve efficient tumor tissue penetration, thereby simultaneously delivering therapeutic drugs to CAFs and deep tumor cells, achieving the purpose of directly inhibiting tumor cell proliferation and efficiently regulating TME.

[0004] The first purpose of the present invention is to overcome the problem that existing nanotechnology cannot solve the problem of deep drug penetration and direct inhibition of tumor cell proliferation, and to provide a FAPα hydrolysis-induced charge-flipped liposome with good anti-tumor effect.

[0005] The second object of the present invention is to provide a method for preparing the above-mentioned FAPα hydrolysis-induced charge-flipped liposomes.

[0006] The third object of the present invention is to provide a lipid composition in which FAPα hydrolysis causes a charge reversal.

[0007] The fourth object of the present invention is to provide the use of the above-mentioned FAPα hydrolysis-induced charge-flipping lipid composition in tumor diagnosis, treatment and surgical navigation.

[0008] Specifically, the FAPα hydrolysis-induced charge-flipped liposome provided by the present invention has a structure shown in formula (1):

[0009] R1 is a lipid group or a lipid group;

[0010] R2 is C1-C1 bonded to R1 by carbon-carbon bond, phosphoester bond, ester bond, amide bond, urethane bond, ether bond, thioether bond or various chemical bonds formed by bioorthogonal chemical reaction. 10 Alkyl, C6-C 20 aromatic or hydrophilic groups;

[0011] R3 is C1-C2-1, C2-1, C3-1, C4-1, C5-1, C6-1, C7-1, C8-1, C9-1, C10-1, C11-1, C12-1, C13-1, C14-1, C15-1, C16-1, C17-1, C18-1, C19-2, C20-2, C21-2, C22- 10 Alkyl, C6-C 20 aromatic group, amino acid group, polypeptide group, polyether group, polyester group, polyamide group, polythioether group or polypolypeptide group;

[0012] R4 is a zwitterionic group;

[0013] n is 1 to 8, and specifically can be 1, 2, 3, 4, 5, 6, 7 or 8.

[0014] In a preferred embodiment, R1 is derived from at least one of sterols, sterols, enols, fatty acids, fatty alcohols, fatty amines, fatty acid glycerides, fatty alcohol glycidyl ethers, sphingolipids, phospholipids, polyketides, vitamins and fluorides.

[0015] In a preferred embodiment, R1 has at least one of the following structures:

[0016] m = 1 to 10, n = 1 to 10;

[0017] m=10~20,n=10~20;

[0018] m = 1 to 10, n = 1 to 10;

[0019] m=10~20,n=10~20;

[0020] m=5~15;

[0021] m=1~10, n=5~15, p=1~10;

[0022] m=1~10, n=1~10, p=1~10, q=1~10;

[0023] m=10~20, n=10~20, p=1~10;

[0024] m = 10-15, n = 10-20;

[0025] n = 10 to 20;

[0026] n = 10 to 20;

[0027] m=5~20,n=4~15,R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;

[0028] m=5~30,n=4~20,R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;

[0029] m=3~15,n=3~20,R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;

[0030] m=2~15,n=2~20,R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;

[0031] m=2~20,n=2~15,R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;

[0032] m=2~15,n=2~20,R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;

[0033] m=2~10,n=2~20,R11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;

[0034] m=2~10,n=2~20,R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;

[0035] m = 8 to 15, n = 10 to 20;

[0036] m=10~20,n=10~20;

[0037] m=1~15, n=10~20, p=1~5;

[0038] m=1~15, n=10~20, p=1~5.

[0039] In a preferred embodiment, the hydrophilic group in R2 is derived from at least one of polyethylene glycol, poly(N-(2-hydroxypropyl)methacrylamide), polyvinylpyrrolidone, poly(2-methacryloyloxyethyl phosphorylcholine), and poly(methacryloylethyl sulfobetaine). When R2 is a hydrophilic group bonded to R1 via a carbon-carbon bond, phosphoester bond, ester bond, amide bond, urethane bond, ether bond, thioether bond, or various chemical bonds formed by bioorthogonal chemical reactions, it can also enhance the stability of the liposomes and increase the duration of drug action, achieving prolonged action in the blood and achieving better anti-tumor efficacy.

[0040] In a preferred embodiment, R2 has at least one of the following structures:

[0041] n=1~6;

[0042] m=0~6,n=0~6;

[0043] m=0~6,n=0~6;

[0044] n = 1 to 115;

[0045] n = 1 to 115;

[0046] n = 1 to 115;

[0047] n=1~6;

[0048] m=0~6,n=0~6;

[0049] m=0~6,n=0~6;

[0050] n=1~115;

[0051] n=1~115;

[0052] n=1~115;

[0053] n=1~115;

[0054] n=1~115;

[0055] n=1~115;

[0056] n=1~115;

[0057] n=1~115;

[0058] n=1~6;

[0059] m=0~6,n=0~6;

[0060] m=0~6,n=0~6;

[0061] n=1~6;

[0062] m=0~6,n=0~6;

[0063] m=0~6,n=0~6;

[0064] n=1~115;

[0065] n=1~115;

[0066] n=1~115;

[0067] n=1~115;

[0068] n = 1 to 115;

[0069] n = 20 to 400;

[0070] n = 20 to 400;

[0071] n = 20 to 400;

[0072] n=20~400.

[0073] In a preferred embodiment, R3 has at least one of the following structures:

[0074] n=1~3;

[0075] n = 1 to 12;

[0076] m and n are each independently 1 to 5;

[0077] n=1~6;

[0078] n=1~6;

[0079] n = 1 to 12;

[0080] n=1~6;

[0081] In the above formulas, R 31 For nothing, n = 1 to 15;

[0082] In the above formulas, R 31 ` and R 32 ` are each independently O or -NH-; R 31 `` is H or C1-C5 alkyl;

[0083] In the above formulas, R 32 For nothing, n=1~15.

[0084] In a preferred embodiment, R4 has at least one of the following structures:

[0085] m=2~5;

[0086] m = 1 to 5;

[0087] m = 2 to 5, n = 1 to 4;

[0088] n = 1 to 5;

[0089] n = 1 to 5;

[0090] In a preferred embodiment, the FAPα hydrolysis-induced charge-flipped liposome has at least one of the following structures:

[0091] m is 1 to 10, n is 1 to 10, p is 1 to 10, q is 1 to 10, s is 1 to 5, and t is 1 to 5;

[0092] m is 10 to 20, n is 1 to 10, p is 1 to 5, and q is 1 to 5;

[0093] m is 10 to 15, n is 10 to 15, p is 1 to 10, q is 1 to 10, s is 1 to 10, t is 1 to 10, and r is 1 to 10;

[0094] m is 5 to 15, n is 5 to 15, p is 1 to 5, o is 1 to 50, r is 1 to 5, s is 1 to 5, and t is 1 to 5;

[0095] m is 40 to 50, n is 1 to 10;

[0096] m is 1 to 5, n is 1 to 50;

[0097] n is 1 to 10;

[0098] m is 1 to 5, n is 1 to 10, and p is 1 to 10;

[0099] m is 5 to 15, n is 5 to 15, p is 15 to 25, and q is 5 to 10;

[0100] m is 10 to 20, n is 10 to 20, p is 1 to 5, q is 1 to 5, r is 1 to 5, s is 1 to 5, and t is 1 to 5;

[0101] m is 1 to 5, n is 5 to 10, p is 5 to 15, q is 100 to 150, s is 1 to 5, and r is 1 to 5;

[0102] m is 1 to 5, n is 5 to 15, p is 5 to 15, q is 1 to 10, o is 1 to 5, r is 1 to 5, s is 1 to 5, and t is 1 to 5.

[0103] The preparation method of the FAPα hydrolysis-induced charge-flipped liposomes provided by the present invention is carried out according to the following steps:

[0104] Method (I):

[0105] S11, bonding the R2 source compound to the R1 source compound, wherein the R2 source compound is a substance that can increase the stability of the R1 source compound to obtain a stable lipid / lipid compound;

[0106] S12, bonding the R3 source compound to the stable lipid / lipid compound to obtain a branched lipid / lipid compound;

[0107] S13, conjugating a glycine-proline dipeptide to a branched lipid / lipid compound; when the glycine-proline dipeptide contains a zwitterionic group, the resulting conjugated product is a FAPα hydrolysis-induced charge-flipped liposome; when the glycine-proline dipeptide does not contain a zwitterionic group, a zwitterionic group is conjugated to the glycine-proline dipeptide end of the resulting conjugated product to obtain a FAPα hydrolysis-induced charge-flipped liposome; or,

[0108] Method (II):

[0109] S21, bonding the R3 source compound to the R1 source compound to obtain a branched lipid / lipid compound;

[0110] S22, conjugating a glycine-proline dipeptide to a branched lipid / lipid compound; when the glycine-proline dipeptide contains a zwitterionic group, the resulting conjugated product is a FAPα hydrolysis-induced charge-flipped liposome; when the glycine-proline dipeptide does not contain a zwitterionic group, a zwitterionic group is conjugated to the glycine-proline dipeptide end of the resulting conjugated product to obtain a FAPα hydrolysis-induced charge-flipped liposome; or,

[0111] Method (III):

[0112] S31, bonding the R2 source compound to the R1 source compound, wherein the R2 source compound is a substance capable of increasing the stability of the R1 source compound, to obtain a stable lipid / lipid compound;

[0113] S32. Conjugate the glycine-proline dipeptide to a stable lipid / lipid compound; when the glycine-proline dipeptide contains a zwitterionic group, the resulting conjugated product is a FAPα hydrolysis-induced charge-flipped liposome; when the glycine-proline dipeptide does not contain a zwitterionic group, a zwitterionic group is attached to the glycine-proline dipeptide end of the resulting conjugated product to obtain a FAPα hydrolysis-induced charge-flipped liposome.

[0114] In a preferred embodiment, the R1 source compound is selected from at least one of sterols, sterols, enols, fatty acids, fatty alcohols, fatty amines, fatty acid glycerides, fatty alcohol glycidyl ethers, sphingolipids, phospholipids, polyketides, vitamins and fluorides containing reactive groups.

[0115] In a preferred embodiment, after the reaction is completed, the remaining residue of the R1 source compound has a structure shown in formula (2) to formula (29).

[0116] In a preferred embodiment, after the reaction is completed, the remaining residue of the R2 source compound has a structure shown in formula (30) to formula (61).

[0117] In a preferred embodiment, after the reaction is completed, the remaining residue of the R3 source compound has a structure shown in formula (62) to formula (75).

[0118] In the preparation of FAPα hydrolysis-induced charge-flipped liposomes provided herein, the R1 source compound must contain an active group reactive with either the R2 source compound or the R3 source compound; the R2 source compound must contain an active group reactive with both the R1 source compound and a stabilizing lipid / lipid compound; the R3 source compound must contain an active group reactive with both a stabilizing lipid / lipid compound or the R1 source compound and a glycine-proline dipeptide; and the glycine-proline dipeptide must contain an active group reactive with both a branched lipid / lipid compound and, optionally, a zwitterionic compound. The type of the active group is not particularly limited and may be at least one of a carboxyl group, an amino group, a hydroxyl group, and the like.

[0119] In the preparation of FAPα hydrolysis-induced charge-flipped liposomes provided herein, the terms "bonding," "linking," "combining," and "joining" are used solely to distinguish the chemical reactions in different steps for ease of description and have no other special meanings. The conditions for bonding, linking, combining, and joining can be selected based on the reaction raw materials and reaction type, as known to those skilled in the art and will not be elaborated upon here.

[0120] The FAPα hydrolysis-induced charge-flipping lipid composition provided by the present invention contains the above-mentioned FAPα hydrolysis-induced charge-flipping liposome.

[0121] In a preferred embodiment, the FAPα hydrolysis-induced charge-flipping liposomes are loaded with at least one of chemotherapeutic drugs, molecular targeted drugs, protein drugs, gene drugs, ultrasound contrast agents, fluorescent dyes, nuclear magnetic resonance contrast agents, CT contrast agents, photosensitizers and radioactive nuclides.

[0122] In a preferred embodiment, the chemotherapy drug is selected from at least one of paclitaxel, docetaxel, doxorubicin, epirubicin, amphotericin, camptothecin, 7-ethyl-10-hydroxycamptothecin, irinotecan, gemcitabine, cisplatin, gambogic acid, sulfonic acid, methotrexate, maytansine, alpha-amanitin, MMAE, MMAF, DM4, calicheamicin, exitecan, mitomycin C and arsenic trioxide.

[0123] In a preferred embodiment, the molecular targeted drug is selected from at least one of imatinib, gefitinib, icotinib and sunitinib.

[0124] In a preferred embodiment, the protein drug is at least one selected from nimotuzumab, cetuximab, trastuzumab, bevacizumab, catalase, glucose oxidase and pegaspargase.

[0125] In a preferred embodiment, the gene drug is selected from at least one of therapeutic DNA, siRNA, microRNA and mRNA.

[0126] In a preferred embodiment, the ultrasound contrast agent is sulfur hexafluoride and / or propane octafluoride.

[0127] In a preferred embodiment, the fluorescent dye is at least one selected from rhodamine, indocyanine green, fluorescein and BODIPY.

[0128] In a preferred embodiment, the nuclear magnetic contrast agent is selected from at least one of gadolinium ions, manganese ions and iron ions.

[0129] In a preferred embodiment, the CT contrast agent is selected from at least one of iohexol, diatrizoate and gold nanoparticles.

[0130] In a preferred embodiment, the photosensitizer is at least one selected from porphyrin, porphin and indocyanine green.

[0131] In a preferred embodiment, the radioactive isotope is selected from at least one of fluorine-18, technetium-99m, iodine-123, iodine-125, iodine-124, iodine-131, cobalt-60, cesium-137, iridium-192, gold-198, tantalum-182, strontium-90, yttrium-90, gallium-68 and lutetium-177.

[0132] In a preferred embodiment, the FAPα hydrolysis-induced charge-flipping lipid composition further contains auxiliary phospholipids and optionally cholesterol, wherein the use of auxiliary phospholipids and cholesterol can enhance the stability of liposomes and prolong blood circulation time.

[0133] In a preferred embodiment, the molar ratio of the FAPα hydrolysis-induced charge-flipped liposomes, the auxiliary phospholipid, and the cholesterol is 1:(0.1-5):(0-2). Specifically, based on 1 mol of the FAPα hydrolysis-induced charge-flipped liposomes, the amount of the auxiliary phospholipid is preferably 0.1-5 mol, such as 0.1 mol, 0.5 mol, 1 mol, 1.5 mol, 2 mol, 2.5 mol, 3 mol, 3.5 mol, 4 mol, 4.5 mol, 5 mol, or any value therebetween; the amount of cholesterol is preferably 0-2 mol, such as 0, 0.2 mol, 0.4 mol, 0.6 mol, 0.8 mol, 1.0 mol, 1.2 mol, 1.4 mol, 1.6 mol, 1.8 mol, 2 mol, or any value therebetween.

[0134] In a preferred embodiment, the auxiliary phospholipid is selected from lysophosphatidylethanolamine, N-palmitoyl-D-erythro-sphingosine phosphocholine, 1,2-dilauroyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphate (sodium salt), N-(carbonyl-methoxypolyethylene glycol)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (sodium salt), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt), Sodium salt), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (sodium salt), distearoylphosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphoglycerol (sodium salt), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-stearoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, Dipalmitoylphosphatidylglycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (sodium salt), 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dierucoyl-sn-glycero-3-phosphocholine, L-α-dioleoylphosphatidyl-DL-glycerol (sodium salt), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1-myristoyl-2-lyso-sn-glycero-3-phosphocholine, 1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine, 1,2-dioleoylphosphatidyl-DL-glycerol (sodium salt), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine At least one of palmitoyl-sn-glycero-3-phospho-L-serine (sodium salt), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, distearoyl-rac-glycero-polyethylene glycol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol, N-(carbonyl-methoxypolyethylene glycol)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (sodium salt), and N-(carbonyl-methoxypolyethylene glycol)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (sodium salt).

[0135] The present invention also provides the use of the above-mentioned FAPα hydrolysis-induced charge-flipping liposomes and / or FAPα hydrolysis-induced charge-flipping lipid compositions in tumor diagnosis, treatment and surgical navigation.

[0136] The FAPα hydrolysis-induced charge-reversal liposomes provided by the present invention have deep tumor penetration, utilize the specific enzymatic cleavage of FAPα overexpressed in tumor tissue to cause charge reversal, and enhance the drug uptake and delivery efficiency of CAFs. In vitro cell experiments have verified that they have charge reversal, high endocytosis rate, high cell growth inhibition effect and FAPα responsiveness. At the same time, in the constructed subcutaneous tumor model, the FAPα-responsive liposomes can achieve significant tumor inhibition. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] FIG1 is a Zeta potential charge reversal curve of the FAPα hydrolysis-induced charge-flipping lipid composition obtained in Example 2 under FAPα enzyme catalysis;

[0138] FIG2 is a diagram showing an experiment of endocytosis of a charge-flipped lipid composition induced by hydrolysis of FAPα obtained in Example 2;

[0139] FIG3 is a diagram showing an in vitro tumor penetration experiment of the charge-flipped lipid composition induced by FAPα hydrolysis obtained in Example 2;

[0140] FIG4 is a graph showing the tumor inhibition effect of the FAPα hydrolysis-induced charge-flipped lipid composition obtained in Example 2 on subcutaneous 4T1 breast cancer tumor model mice;

[0141] FIG5 is a graph showing the tumor inhibition effect of the FAPα hydrolysis-induced charge-flipped lipid composition obtained in Example 2 in subcutaneous Hepg-2 liver cancer model mice. DETAILED DESCRIPTION

[0142] The present invention will be described in detail below through examples.

[0143] Preparation Example 1

[0144] (1a) Synthesis of intermediate c

[0145] Compound a (1 mmol), DCC (2 mmol), and DMAP (2 mmol) were added to DCM (10 mL), mixed, and stirred at room temperature for 4 h. Compound b (1 mmol) was then added and stirred for 48 h. During the reaction, a by-product, dicyclohexylurea (DCU), was generated, usually appearing as a white precipitate. After the reaction was completed, the generated dicyclohexylurea (DCU) precipitate was removed by filtration. The organic phase was washed with dilute hydrochloric acid (1 M) to remove unreacted DCC and DMAP. It was washed with saturated sodium bicarbonate solution to neutralize possible acidic impurities. Finally, the organic phase was washed with distilled water. The organic phase was then dried over anhydrous sodium sulfate and filtered to remove the desiccant. The organic phase was concentrated under reduced pressure using a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent). The target component was collected and concentrated to obtain pure compound c.

[0146] (1b) Synthesis of intermediate e

[0147] Compound d (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Product c (1 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound e.

[0148] (1c) Synthesis of intermediate g

[0149] Compound f (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Product e (0.5 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound g.

[0150] (1d) Synthesis of product i

[0151] Compound h (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Product g (0.5 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound i.

[0152] The chemical shifts of product i are as follows: 1HNMR (400MHz, CDCl3, δ): 7.99–7.92(m,2H),7.81(t,J=7.3Hz,2H),7.65(t,1H),5.27(ddd,4H) ,5.02(p,J=7.0Hz,1H),4.29–4.10(m,12H),4.07–3.93(m,4H),3.86–3.74(m,3H),3.73–3.50(m ,21H),3.49–3.32(m,6H),3.23(s,9H),2.72–2.65(m,1H),2.65–2.57(m,5H),2.35–2.14(m,8H ),2.02(dq,2H),1.98–1.78(m,8H),1.58–1.45(m,2H),1.31–1.16(m,28H),0.93–0.84(m,3H).

[0153] Preparation Example 2

[0154] (2a) Synthesis of intermediate DSPE-Lys

[0155] Boc-Lys(Boc)-OH (1 mmol), EDC (2 mmol), and NHS (2 mmol) were first added to DCM (10 mL) and stirred at room temperature for 4 h. DSPE (1 mmol) was then added and stirred for 12 h. After the reaction, 10 mL of trifluoroacetic acid (TFA) was added and stirred for 1 h. The mixture was concentrated by rotary evaporation and then dialyzed against methanol for 24 h. After dialysis, the mixture was dried to obtain Lys-modified DSPE (DSPE-Lys, approximately 0.65 g, 74.5% yield).

[0156] (2b) Synthesis of intermediate DSPE-Lys-diGP

[0157] Boc-Gly-pro-OH (Boc-glycine-L-proline, 1 mmol), EDC (2 mmol) and NHS (2 mol) were mixed in DCM (10 mL), stirred at room temperature for 4 h, and then DSPE-Lys (0.5 mmol) was added and stirred for 24 h. Then 10 mL of TFA was added, and the mixture was stirred for 1 h. After concentration, dialyzed and dried, DSPE-Lys-diGP (about 0.51 g, yield 68.9%) was obtained.

[0158] (2c) Synthetic product DSPE-Lys-diGPS

[0159] DSPE-Lys-diGP and propane sultone were added to DCM (10 mL), refluxed at 40° C. for 2 d, concentrated, and dialyzed in water to obtain the product DSPE-Lys-diGPS.

[0160] The chemical shifts of DSPE-Lys-diGPS are as follows: 1 H NMR (400MHz, CDCl3, δ): 4.91-4.86 (m, 1H), 4.51 (d, 2H), 4.46-4.30 (m, 5H) , 4.20-4.08(m, 4H), 3.57(td, 2H), 3.46-3.36(m, 7H), 3.34-3.26(m, 2H), 3 .22-3.14(m,7H),2.72(t,4H),2.68-2.59(m,2H),2.37-2.25(m,5H),1.98 -1.77(m, 14H), 1.66-1.50(m, 7H), 1.46-1.20(m, 65H), 0.91-0.85(m, 6H).

[0161] Preparation Example 3

[0162] (3a) Synthesis of intermediate c

[0163] Compound a (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound b (1 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound c.

[0164] (3b) Synthesis of intermediate e

[0165] Compound d (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound c (1 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound i.

[0166] (3c) Synthetic product g

[0167] Compound f (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound e (1 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound i.

[0168] The chemical shift of product g is as follows: 1 H NMR (400MHz, CDCl3, δ): 7.98–7.86 (m, 1H), 7.45 (t, J = 6.8Hz, 1H), 7.03–6.96 (m, 3H), 6. 62–6.51(m,3H),6.42(t,J=6.9Hz,1H),5.49–5.33(m,3H),4.26–4.12(m,2H),4.01–3.86 (m,2H),3.78–3.51(m,25H),3.46–3.30(m,3H),3.24–3.08(m,2H),2.96(s,6H),2.87–2 .65(m,5H),2.21–2.12(m,1H),2.05–1.91(m,2H),1.89–1.76(m,6H),1.11–1.05(m,3H).

[0169] Preparation Example 4

[0170] (4a) Synthesis of intermediate c

[0171] Compound a (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound b (1 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound c.

[0172] (4b) Synthesis of intermediate e

[0173] Compound d (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound c (1 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target fraction and concentrate to obtain pure compound e.

[0174] (4c) Synthesis of intermediate g

[0175] Compound f (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound e (0.5 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure using a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound g.

[0176] (4d) Synthesis of product i

[0177] Product g and propane sultone were added to DCM (10 mL) and refluxed at 40° C. for 2 d. After concentration, the product i was dialyzed in water to give the product i.

[0178] The chemical shifts of product i are as follows: 1H NMR (400MHz, CDCL3, δ): 5.45 (s, 12H), 4.60-4.47 (m, 4H), 4.46-4.34 (m, 4H), 4.27-4.14 (m, 6H), 4.07 (q, 3H), 3.95-3.85 (m, 7 H), 3.69-3.51(m, 234H), 3.44(td, 2H), 2.76-2.58(m, 8H), 2.44(q, 20H), 2.05-1.88(m, 5H), 1.84-1.74(m, 1H), 1.11(t, 3H).

[0179] Preparation Example 5

[0180] (5a) Synthesis of intermediate c

[0181] Compound a (1 mmol), DCC (2 mmol), and DMAP (2 mmol) were added to DCM (10 mL), mixed, and stirred at room temperature for 4 h. Compound b (1 mmol) was then added and stirred for 48 h. During the reaction, a by-product, dicyclohexylurea (DCU), was generated, usually appearing as a white precipitate. After the reaction was completed, the generated dicyclohexylurea (DCU) precipitate was removed by filtration. The organic phase was washed with dilute hydrochloric acid (1 M) to remove unreacted DCC and DMAP. It was washed with saturated sodium bicarbonate solution to neutralize possible acidic impurities. Finally, the organic phase was washed with distilled water. The organic phase was then dried over anhydrous sodium sulfate and filtered to remove the desiccant. The organic phase was concentrated under reduced pressure using a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent). The target component was collected and concentrated to obtain pure compound c.

[0182] (5b) Synthesis of intermediate e

[0183] Compound d (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound c (1 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound e.

[0184] (5c) Synthesis of intermediate g

[0185] Compound f (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound e (0.5 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure using a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound g.

[0186] (5d) Synthesis of product i

[0187] Compound h (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound g (0.25 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound i.

[0188] (5d) Synthesis of product k

[0189] Compound j (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound i (0.25 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound k.

[0190] The chemical shifts of product i are as follows: 1H NMR (400MHz, CDCL3, δ): 4.57-4.46 (m, 6H), 4.43 (t, 2H), 4.40-4.28 (m, 16H), 4 .28-4.15(m, 8H), 3.93-3.85(m, 6H), 3.74(t, 2H), 3.57(d, 2H), 3.51(p, 1H), 3. 19(tdd, 6H), 2.91(dddd, 4H), 2.86-2.77(m, 12H), 2.68-2.53(m, 8H), 2.44(q, 64H), 1.98-1.77(m, 16H), 1.71-1.59(m, 3H), 1.47-1.19(m, 14H), 1.11(t, 6H).

[0191] Preparation Example 6

[0192] (6a) Synthesis of intermediate c

[0193] Compound b (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound a (1 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound c.

[0194] (6b) Synthesis of intermediate e

[0195] Compound d (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound c (0.5 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target fraction and concentrate to obtain pure compound e.

[0196] (6c) Synthesis of intermediate g

[0197] Compound f (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound e (0.5 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure using a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound g.

[0198] (6d) Synthesis of product i

[0199] Products g and h were added to DCM (10 mL) and refluxed at 40° C. for 2 d. After concentration, the product was purified by column chromatography to obtain the product.

[0200] The chemical shifts of product i are as follows: 1 H NMR(400MHz, CDCL3, δ)8.68(d,4H),7.98(s,2H),7.83(s,1H),6.69(s,2H),5.34(dtd ,4H),5.02(d,2H),4.93(d,2H),4.44(p,1H),4.26-4.16(m,3H),4.18-4.09(m,4H),4. 10(d,1H),4.09(s,3H),4.11-4.02(m,9H),3.98-3.92(m,2H),3.88(d,2H),3.82-3.74 (m,2H),3.77-3.71(m,2H),3.68-3.51(m,19H),3.47(dt,1H),3.25-3.16(m,2H),3.05 (dt,J=12.7,2H),2.91-2.81(m,2H),2.77-2.29(m,17H),2.27-2.21(m,1H),2.24-2.1 8(m,1H),2.21-2.11(m,1H),2.06(dddd,2H),2.02-1.93(m,1H),1.93-1.83(m,1H),1. 82(p,5H),1.71(tddd,1H),1.56-1.46(m,4H),1.42-1.33(m,2H),1.36-1.29(m,2H),1 .32-1.26(m,5H),1.27(s,1H),1.26(ddd,5H),1.26-1.16(m,10H),0.94-0.84(m,6H).

[0201] Preparation Example 7

[0202] (7a) Synthesis of intermediate c

[0203] Compound b (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound a (0.5 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound c.

[0204] (7b) Synthesis of intermediate e

[0205] Compound d (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound c (0.5 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target fraction and concentrate to obtain pure compound e.

[0206] (7c) Synthesis of intermediate g

[0207] Compound f (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound e (0.5 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure using a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound g.

[0208] (7d) Synthesis of product i

[0209] Product g and compound h were added to MeOH (10 mL), and the reaction mixture was stirred for 4 hours. After the reaction was completed, the product i was separated and purified by column chromatography.

[0210] The chemical shifts of product i are as follows: 1 H NMR (400MHz, CDCL3, δ): 8.03(s,1H),7.89(s,2H),7.46(d,2H),7.34(s,1H),7.24–7.18(m,8 H),4.72(t,1H),4.44(q,1H),4.37–3.91(m,27H),3.81–3.65(m,6H),3.66–3.40(m,26H),3.3 2(dd,1H),3.25–3.03(m,8H),2.99–2.78(m,4H),2.69–2.49(m,6H),2.43–2.30(m,4H),2.26– 2.06(m,6H),1.92–1.67(m,12H),1.60–1.34(m,16H),1.32–1.18(m,43H),0.91–0.85(m,6H).

[0211] Preparation Example 8

[0212] (8a) Synthesis of intermediate c

[0213] Compound a (1 mol) was dissolved in an anhydrous solvent and placed in an ice bath and cooled to 0 ° C. Compound b (1.2 mol) was slowly added dropwise and a base (triethylamine) was added dropwise to neutralize the generated HBr. After the addition was complete, the ice bath was removed, the reaction mixture was warmed to room temperature, and the stirring reaction was continued for 3 hours. After the reaction was completed, the reaction mixture was poured into ice water to quench the reaction. The product was extracted with an organic solvent (dichloromethane). The organic phase was washed with saturated sodium bicarbonate solution to remove unreacted acid and alkali. The organic phase was then dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation to obtain a crude product. The product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as eluent) to obtain compound c.

[0214] (8b) Synthesis of intermediate e

[0215] Compound c (1 mol) was dissolved in water, and compound d (30 mol), a catalyst (CuBr), and a ligand (Me4cyclam) were added to the reaction flask at a molar ratio of 1:1. The reaction flask was sealed, nitrogen was introduced to exclude oxygen, and the reaction system was heated to 70°C. The reaction mixture was stirred to ensure uniform polymerization. The reaction time was 24 hours. After the reaction was completed, the reaction flask was cooled to room temperature. The reaction flask was opened and exposed to air to terminate the polymerization reaction. The catalyst (CuBr / ligand) was removed by passing through a neutral alumina column. The reaction mixture was dialyzed to remove unreacted monomers and small molecule byproducts, and freeze-dried to obtain pure compound e.

[0216] (8c) Synthesis of intermediate g

[0217] Compound e (1 mol) and sodium azide (4 mol) were dissolved in DMF and heated to 80°C to promote the reaction. The reaction mixture was stirred to ensure a uniform reaction. The reaction time was 24 hours. After the reaction was completed, the reaction mixture was cooled to room temperature. Unreacted sodium azide and generated sodium bromide were removed by filtration, and the reaction mixture was dialyzed to remove small molecule byproducts. Freeze-dried to obtain pure compound g.

[0218] (8d) Synthesis of Intermediate i

[0219] Mix CuSO4 pentahydrate and sodium ascorbate in water. Add Compounds G and H to a mixture of water and tert-butyl alcohol and stir until uniform. Add the prepared Cu(I) catalyst solution to the reaction mixture. Stir the reaction mixture at 50°C for 12 hours, then cool the reaction mixture to room temperature. Dialyze the reaction mixture to remove small molecule byproducts. This yields pure Compound I.

[0220] (8e) Synthesis of intermediate k

[0221] Compound i (1 mmol), EDC (4 mmol) and NHS (4 mol) were mixed evenly in DMF (100 mL) and stirred at room temperature for 4 h. Compound j (4 mmol) was then added and stirred for 48 h. After the reaction was completed, 10 mL of TFA was added and stirred for 12 h. The reaction mixture was dialyzed to remove small molecule byproducts and concentrated to obtain pure compound k.

[0222] (8f) Synthesis of intermediate m

[0223] Compound k and compound l were dissolved in DMF at a molar ratio of 1:4. A small amount of triethylamine (a basic catalyst) was added, and the reaction mixture was stirred at 40°C. After completion of the reaction, the solvent was evaporated to obtain a crude intermediate product. 10 mL of TFA was then added and stirred for 12 hours. The reaction mixture was dialyzed to remove small molecule byproducts and concentrated to yield pure compound m.

[0224] (8g) Synthetic product o

[0225] Compound m and compound n were dissolved in DMF at a molar ratio of 1:4. A small amount of triethylamine, a basic catalyst, was added, and the reaction mixture was stirred at room temperature. After completion of the reaction, the solvent was evaporated to obtain a crude intermediate product. 10 mL of TFA was then added and stirred for 12 hours. The reaction mixture was dialyzed to remove small molecule byproducts and concentrated to yield pure compound o.

[0226] (8h) Synthetic product q

[0227] Compound p (1 mmol), EDC (4 mmol) and NHS (4 mol) were mixed evenly in DMF (100 mL) and stirred at room temperature for 4 h. Then, compound o (0.25 mmol) was added and stirred for 48 h. After the reaction was completed, 10 mL of TFA was added and stirred for 12 h. The reaction mixture was dialyzed to remove small molecule byproducts and concentrated to obtain pure compound q.

[0228] (8i) Synthetic product s

[0229] Compound r (1 mmol), EDC (4 mmol) and NHS (4 mol) were mixed in DMF (100 mL) and stirred at room temperature for 4 h. Then, compound q (0.25 mmol) was added and stirred for 48 h. After the reaction was completed, 10 mL of TFA was added and stirred for 12 h. The reaction mixture was dialyzed to remove small molecule byproducts and concentrated to obtain pure compound s.

[0230] The chemical shifts of product s are as follows: 1H NMR (400MHz, CDCL3, δ)8.30(s,1H),8.14(s,1H),7.94(d,4H),7.68(s,4H),7.61–7. 52(m,24H),7.44(d,8H),7.29(s,4H),4.63(d,20H),4.57(d,5H),4.40(d,1H),4.31 (s,4H),4.13–3.99(m,8H),3.87(d,21H),3.75–3.70(m,10H),3.67(s,3H),3.62–3. 48(m,15H),3.42–3.29(m,28H),3.27(d,20H),3.19–3.07(m,35H),2.96(d,2H),2.81 (d,6H),2.75–2.67(m,12H),2.50–2.41(m,4H),2.26(d,5H),2.22–2.15(m,8H),2.1 0(d,5H),2.02–1.92(m,18H),1.91–1.83(m,16H),1.84–1.79(m,49H),1.78–1.68(m, 46H),1.64–1.58(m,13H),1.55(d,8H),1.53–1.46(m,31H),1.43–1.35(m,13H),1.3 5–1.27(m,90H),1.25(d,72H),1.18(s,15H),1.08(s,3H),1.03(s,3H),0.89(d,6H).

[0231] Preparation Example 9

[0232] (9a) Synthesis of intermediate c

[0233] Compound a (1 mmol), DCC (2 mmol), and DMAP (2 mmol) were added to DCM (10 mL), mixed, and stirred at room temperature for 4 h. Compound b (1 mmol) was then added and stirred for 48 h. During the reaction, a by-product, dicyclohexylurea (DCU), was generated, usually appearing as a white precipitate. After the reaction was completed, the generated dicyclohexylurea (DCU) precipitate was removed by filtration. The organic phase was washed with dilute hydrochloric acid (1 M) to remove unreacted DCC and DMAP. It was washed with saturated sodium bicarbonate solution to neutralize possible acidic impurities. Finally, the organic phase was washed with distilled water. The organic phase was then dried over anhydrous sodium sulfate and filtered to remove the desiccant. The organic phase was concentrated under reduced pressure using a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent). The target component was collected and concentrated to obtain pure compound c.

[0234] (9b) Synthesis of intermediate e

[0235] Compound d (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Product c (1 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound e.

[0236] (9c) Synthesis of intermediate g

[0237] Products e and f were added to DCM (10 mL) and refluxed at 40° C. for 2 d. After concentration, the product was separated and purified by column chromatography to obtain the product.

[0238] The chemical shift of product g is as follows: 1 H NMR(400MHz, CDCL3, δ)7.83(s,1H),4.36–4.23(m,3H),3.86–3.62(m,161H),3.63–3 .44(m,11H),2.95(dt,1H),2.81–2.59(m,7H),2.58–2.43(m,2H),2.36–2.26(m,2H) ,2.25–2.15(m,9H),1.91(dt,J=12.4,7.1Hz,1H),1.85–1.78(m,2H),1.78–1.66(m, 2H),1.66–1.47(m,8H),1.47–1.34(m,5H),1.32–1.10(m,16H),0.93–0.80(m,12H).

[0239] Preparation Example 10

[0240] (10a) Synthesis of intermediate c

[0241] Compound b (1 mol) was dissolved in an anhydrous solvent and placed in an ice bath and cooled to 0 ° C. Compound a (1.2 mol) was slowly added dropwise and a base (triethylamine) was added dropwise to neutralize the generated HBr. After the addition was complete, the ice bath was removed, the reaction mixture was warmed to room temperature, and the stirring reaction was continued for 3 hours. After the reaction was completed, the reaction mixture was poured into ice water to quench the reaction. The product was extracted with an organic solvent (dichloromethane). The organic phase was washed with saturated sodium bicarbonate solution to remove unreacted acid and alkali. The organic phase was then dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation to obtain a crude product. Compound c was further purified by column chromatography (using a silica gel column and an eluent of petroleum ether / ethyl acetate mixed solvent).

[0242] (10b) Synthesis of intermediate e

[0243] Dissolve compound c (1 mol) in water, add compound d (40 mol), catalyst (CuBr), and ligand (PMDETA) to the reaction flask, with a molar ratio of catalyst to ligand of 1:1. Seal the reaction flask, introduce nitrogen, exclude oxygen, and heat the reaction system to 70°C. Stir the reaction mixture to ensure uniform polymerization. The reaction time is 24 hours. After the reaction is completed, cool the reaction flask to room temperature. Open the reaction flask and expose it to air to terminate the polymerization reaction. Remove the catalyst (CuBr / ligand) by filtration or centrifugation. Dialyze the reaction mixture (using a dialysis bag with an appropriate molecular weight cutoff) to remove unreacted monomers and small molecule byproducts, and freeze-dry to obtain pure compound e.

[0244] (10c) Synthesis of intermediate g

[0245] Compound e and sodium azide were dissolved in DMF and heated to 80°C to promote the reaction. The reaction mixture was stirred to ensure a uniform reaction. The reaction time was 24 hours. After the reaction was completed, the reaction mixture was cooled to room temperature. Unreacted sodium azide and generated sodium bromide were removed by filtration, and the reaction mixture was dialyzed (using a dialysis bag with an appropriate molecular weight cutoff) to remove small molecule byproducts. Freeze-dried to obtain pure compound g.

[0246] (10d) Synthesis of Intermediate i

[0247] CuSO4 is mixed with sodium ascorbate in water, and the sodium ascorbate reduces Cu(II) to Cu(I) to form an active catalyst.

[0248] Compound g and compound h were added to a mixed solvent of water and tert-butyl alcohol and stirred until uniform. The prepared Cu(I) catalyst solution was added to the reaction mixture. The reaction mixture was stirred at 50°C and allowed to react for 12 hours. After completion of the reaction, the reaction mixture was dialyzed to remove small molecule byproducts, yielding pure compound i.

[0249] (10e) Synthesis of intermediate k

[0250] Compound j (1 mmol), EDC (4 mmol) and NHS (4 mol) were mixed evenly in DMF (100 mL) and stirred at room temperature for 4 h. Compound i (4 mmol) was then added and stirred for 48 h. After the reaction was completed, 10 mL of TFA was added and stirred for 12 h. The reaction mixture was then dialyzed to remove small molecule byproducts and concentrated to obtain pure compound k.

[0251] (10f) Synthesis of intermediate m

[0252] Compound k (1 mmol) and triethylamine were dissolved in 100 mL of DMF, mixed well, and stirred at room temperature for 4 h. Compound l (1.5 mmol) was slowly added dropwise. The reaction mixture was dialyzed to remove small molecule byproducts and concentrated to obtain pure compound m.

[0253] (10g) Synthetic product o

[0254] Compound m (1 mmol) and compound n (1.5 mmol) were mixed in DMF (10 mL). A small amount of triethylamine was added to the reaction system, which was then heated to 70°C. After completion of the reaction, the reaction mixture was dialyzed to remove small molecule byproducts, and the desired product was isolated and purified by vacuum distillation.

[0255] The chemical shift of product o is as follows: 1H NMR (400MHz, CDCL3, δ): 8.10(s,1H),7.99(s,1H),7.83(s,1H),7.62(d,10H),7.55(d,J=7.0Hz,18H),6.09(dq,2H),5.16–5.05(m,2H), 4.90(s,1H),4.67(p,1H),4.57–4.45(m,32H),4.33–4.26(m,2H),3.90–3.71(m,32H),3.69–3.39(m,29H),3.38–3.23(m,58H),2.96(td ,2H),2.85(ddt,1H),2.71(ddt,1H),2.50–2.32(m,3H),2.16(td,2H),2.12–2.02(m,4H),1.99–1.85(m,9H),1.84–1.75(m,12H),1.73– 1.54(m,118H),1.53–1.38(m,82H),1.35–1.24(m,91H),1.24–1.11(m,110H),0.94(d,3H),0.85(d,J=6.8Hz,3H),0.80(d,J=6.8Hz,3H).

[0256] Preparation Example 11

[0257] (11a) Synthesis of intermediate c

[0258] Compound a (1mol) and compound b (1.5mol) are added to the reaction flask of drying, anhydrous dichloromethane is added as solvent, and stirring is until completely dissolved. In the mixture, DCC (1.2 equivalents) is added, and the DMAP (1.2 equivalents) of catalytic amount is subsequently added. System gradually generates white precipitate (by-product dicyclohexylurea, DCU). After the reaction is complete, the DCU precipitation generated is removed by suction filtration, and filtrate is retained for subsequent treatment. Organic phase is washed with dilute hydrochloric acid (1M) to remove unreacted DCC and DMAP, then washed with saturated sodium bicarbonate solution and distilled water to neutrality. Anhydrous sodium sulfate drying organic phase is added again, desiccant is removed by filtration. Organic phase is concentrated under reduced pressure using rotary evaporator to obtain crude product. Purified by recrystallization (petroleum ether / ethyl acetate), crystalline product is obtained, which is compound c.

[0259] (11b) Synthesis of intermediate e

[0260] Compound d (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound c (1 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by recrystallization or column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent). The target fraction was collected and concentrated to obtain pure compound e.

[0261] (11c) Synthesis of intermediate g

[0262] Compound f (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound e (1 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by recrystallization or column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound g.

[0263] (11d) Synthesis of product i

[0264] Compound h (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound g (1 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound i.

[0265] The chemical shifts of product i are as follows: 1H NMR (400MHz, CDCL3, δ): 7.94(t,J=8.6Hz,1H),7.82(t,1H),7.75(t,1H),7.43(dd,1H),7.33(dd,1H),7.20(dt, 2H),7.14(dt,2H),5.34(tq,1H),5.09(ddddt,2H),4.69–4.58(m,2H),4.37(dt,2H),4.27(t,1H),4.05–3.93(m, 2H),3.86–3.39(m,29H),2.76–2.69(m,1H),2.60(dtt,1H),2.54–2.37(m,4H),2.25–2.16(m,3H),2.16–2.01(m, 6H),2.01–1.90(m,4H),1.88–1.81(m,3H),1.81–1.75(m,1H),1.75–1.68(m,1H),1.63(dddd,9H),1.57(dt,1H).

[0266] Preparation Example 12

[0267] (12a) Synthesis of intermediate c

[0268] Compound b (1 mol) was dissolved in an anhydrous solvent and placed in an ice bath and cooled to 0 ° C. Compound a (1.2 mol) was slowly added dropwise and a base (triethylamine) was added dropwise to neutralize the generated HBr. After the addition was complete, the ice bath was removed, the reaction mixture was warmed to room temperature, and the stirring reaction was continued for 3 hours. After the reaction was completed, the reaction mixture was poured into ice water to quench the reaction. The product was extracted with an organic solvent (dichloromethane). The organic phase was washed with saturated sodium bicarbonate solution to remove unreacted acid and base. The organic phase was then dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation to obtain a crude product. The product was further purified by recrystallization (petroleum ether / ethyl acetate) to obtain compound c.

[0269] (12b) Synthesis of intermediate e

[0270] Dissolve compound c (1 mol) in water, add compound d (20 mol), catalyst (CuBr) and ligand (PMDETA) to the reaction flask, with a molar ratio of catalyst to ligand of 1:1. Seal the reaction flask, introduce nitrogen, exclude oxygen, and heat the reaction system to 70°C. Stir the reaction mixture to ensure uniform polymerization. The reaction time is 24 hours. After the reaction is completed, cool the reaction flask to room temperature. Open the reaction flask and expose it to air to terminate the polymerization reaction. Remove the catalyst (CuBr / ligand) by filtration. The reaction mixture is dialyzed (using a dialysis bag with an appropriate molecular weight cutoff) to remove unreacted monomers and small molecule byproducts, and freeze-dried to obtain pure compound e.

[0271] (12c) Synthesis of intermediate g

[0272] Compound e and sodium azide were dissolved in DMF and heated to 80°C to promote the reaction. The reaction mixture was stirred to ensure a uniform reaction. The reaction time was 24 hours. After the reaction was completed, the reaction mixture was cooled to room temperature. Unreacted sodium azide and generated sodium bromide were removed by filtration, and the reaction mixture was dialyzed (using a dialysis bag with an appropriate molecular weight cutoff) to remove small molecule byproducts. Freeze-dried to obtain pure compound g.

[0273] (12d) Synthesis of Intermediate i

[0274] CuSO4 pentahydrate and sodium ascorbate are mixed in water. Sodium ascorbate reduces Cu(II) to Cu(I), forming an active catalyst. Compounds g and h are added to a mixed solvent of water and tert-butyl alcohol and stirred until uniform. The prepared Cu(I) catalyst solution is added to the reaction mixture. The reaction mixture is stirred at 50°C and allowed to react for 12 hours. After completion of the reaction, the reaction mixture is dialyzed to remove small molecule byproducts, yielding pure Compound i.

[0275] (12e) Synthesis of intermediate k

[0276] Compound j (4 mmol), EDC (4 mmol) and NHS (4 mol) were mixed evenly in a DMSO / methanol mixture (100 mL) and stirred at room temperature for 4 h. Then, compound i (1 mmol) was added and stirred for 48 h. After the reaction was completed, 10 mL of TFA was added and stirred for 12 h. The reaction mixture was then dialyzed to remove small molecule byproducts and concentrated to obtain pure compound k.

[0277] (12f) Synthesis of intermediate m

[0278] Compound k (1 mmol) and triethylamine were mixed evenly in DMSO (100 mL) and stirred at room temperature for 4 h. Compound l (1 mmol) was slowly added dropwise. After the reaction was completed, the reaction mixture was dialyzed to remove small molecule byproducts and concentrated to obtain pure compound m.

[0279] (12g) Synthetic product o

[0280] Compound m (1 mmol) and compound n (1.5 mmol) were mixed in methanol (100 mL). A small amount of triethylamine was added to the reaction system, which was then heated to 70°C. After completion of the reaction, the reaction mixture was dialyzed to remove small molecule byproducts, and the desired product o was isolated and purified by vacuum distillation.

[0281] The chemical shift of product o is as follows: 1 H NMR (400MHz, CDCL3, δ): 7.91–7.74(m,2H),5.69(s,1H),5.60–5.44(m,1H),4.39–4.27(m,20H),4.27–4.16(m,11H),4.15–4.0 1(m,17H),4.01–3.87(m,1H),3.79–3.70(m,1H),3.70–3.61(m,23H),3.61–3.46(m,5H),3.31–3.20(m,66H),3.12–2.84(m,3H ),2.62–2.50(m,1H),2.47–2.33(m,1H),2.28–2.14(m,2H),2.05–1.94(m,1H),1.91–1.85(m,1H),1.84–1.76(m,4H),1.75–1. 66(m,22H),1.65–1.57(m,2H),1.50(dtd,1H),1.40–1.34(m,1H),1.31–1.22(m,11H),1.21–1.14(m,24H),0.90–0.86(m,3H).

[0282] Preparation Example 13

[0283] (13a) Synthesis of intermediate c

[0284] Compound a (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound b (1 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound c.

[0285] (13b) Synthesis of intermediate e

[0286] Compound c (1 mmol), EDC (2 mmol), and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound d (1 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by recrystallization or column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent). The target fraction was collected and concentrated to obtain pure compound e.

[0287] (13c) Synthesis of intermediate g

[0288] Compound e and compound f were dissolved in water in a 1:2 molar ratio. A small amount of triethylamine (a basic catalyst) was added, and the reaction mixture was stirred at 40°C. After completion of the reaction, the solvent was evaporated to obtain a crude intermediate product. 10 mL of TFA was then added and stirred for 12 hours. The crude product was further purified by column chromatography using a silica gel column and a methanol / dichloromethane mixture as the eluent. The target fraction was collected and concentrated to yield pure compound g.

[0289] (13d) Synthesis of Intermediate i

[0290] Compound g and compound h were dissolved in water in a molar ratio of 1:2, a small amount of alkaline catalyst triethylamine was added, and the reaction mixture was stirred at room temperature. After the reaction was completed, the intermediate crude product was obtained by evaporating the solvent, and then 10 mL of TFA was added and stirred for 12 hours. The crude product was finally further purified by column chromatography (using a silica gel column and a methanol / dichloromethane mixed solvent as the eluent). The target component was collected and concentrated to obtain pure compound i.

[0291] (13e) Synthesis of intermediate k

[0292] Compound j (1 mmol), EDC (2 mmol), and NHS (2 mmol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h. Compound i (0.25 mmol) was then added and stirred for 48 h. After completion of the reaction, 10 mL of TFA was added and stirred for 12 h. The organic phase was then washed with water to remove unreacted EDC, NHS, and by-products. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and then concentrated under reduced pressure on a rotary evaporator to obtain a crude product. The crude product was further purified by column chromatography (using a silica gel column and a petroleum ether / ethyl acetate mixed solvent as the eluent) to collect the target component and concentrate to obtain pure compound k.

[0293] (13f) Synthetic product 1

[0294] Compound k and compound 1 were added to DMF (10 mL) and refluxed at 40° C. for 2 d. The reaction mixture was dialyzed to remove small molecule by-products.

[0295] The chemical shifts of product 1 are as follows: 1H NMR (400 MHz, CDCL3, δ): 8.68 (s, 4H), 8.62 (s, 2H), 8.57 (s, 2H), 7.86 (s, 1H), 7.68 (s, 1H), 7.61 (s, 1H), 7.51 (d, 6H), 7.42 (s, 4H), 4.34 (dd, 1H), 4.28 (dd, J = 7.0, 0.7 Hz, 2H), 4.23 (dd, 1H), 4.20–4.16 (m, 5H), 4.16–4.07 (m, 4H), 4.06–3.98 (m, 5 H),3.65–3.51(m,11H),3.50–3.30(m,8H),3.24–3.04(m,25H),3.03–2.83(m,15H),2.80–2.70(m,16H),2.69–2.49(m,16 H),2.49–2.28(m,11H),2.28–2.16(m,3H),1.92–1.63(m,22H),1.56–1.40(m,9H),1.37–1.22(m,6H),0.92–0.84(m,3H).

[0296] Example 1

[0297] The ammonium sulfate gradient method was used to prepare DOX-loaded FAPα hydrolysis-induced charge-flipped liposomes. The preparation process is as follows:

[0298] (1) In a 250 mL flask, HSPC (hydrogenated soybean lecithin) (23.6 mg, 0.03 mmol), CHOL (cholesterol) (7.7 mg, 0.02 mmol), the product of Preparation Example 1 (43.0 mg, 0.03 mmol), and 28.06 mg of DSPE-PEG 2000 at a molar ratio of 3:2:3:1 were dissolved in dichloromethane (10 mL) and rotary evaporated under reduced pressure to form a lipid film.

[0299] (2) Add ammonium sulfate solution (0.3 M, 20 mL) to the lipid film, ultrasonically hydrate, and extrude the solution through a polycarbonate membrane (100 nm) to obtain a blank liposome solution. Centrifuge the solution at 5000 rpm for 60 min in an ultrafiltration tube (molecular weight cutoff of 100 kDa) to remove excess ammonium sulfate solution and obtain concentrated liposomes.

[0300] (3) 52.5 mg of concentrated liposomes and 10 mg of DOX (doxorubicin) were mixed in 5.0 mL of histidine buffer (10 mM, pH 6.5) containing 10% (w / w) sucrose, and the mixture was shaken in an oscillator (100 rpm, 50°C) for 4 h to obtain a FAPα hydrolysis-induced charge reversal lipid composition, which was designated as FAPα-1@DOX.

[0301] Example 2

[0302] A FAPα hydrolysis-induced charge-flipped lipid composition was prepared according to the method of Example 1, except that the product of Preparation Example 1 was replaced by the product of Preparation Example 2 in the same molar amount. The remaining conditions were the same as in Example 1 to obtain a FAPα hydrolysis-induced charge-flipped lipid composition, which was recorded as FAPα-2@DOX.

[0303] Example 3

[0304] A FAPα hydrolysis-induced charge-flipped lipid composition was prepared according to the method of Example 1, except that the product of Preparation Example 1 was replaced by the product of Preparation Example 3 in the same molar amount. The other conditions were the same as in Example 1 to obtain a FAPα hydrolysis-induced charge-flipped lipid composition, which was recorded as FAPα-3@DOX.

[0305] Example 4

[0306] A FAPα hydrolysis-induced charge-flipped lipid composition was prepared according to the method of Example 1, except that the product of Preparation Example 1 was replaced by the product of Preparation Example 4 in the same molar amount. The remaining conditions were the same as in Example 1 to obtain a FAPα hydrolysis-induced charge-flipped lipid composition, which was recorded as FAPα-4@DOX.

[0307] Example 5

[0308] A FAPα hydrolysis-induced charge-flipped lipid composition was prepared according to the method of Example 1, except that the product of Preparation Example 1 was replaced by the product of Preparation Example 5 in the same molar amount. The other conditions were the same as in Example 1 to obtain a FAPα hydrolysis-induced charge-flipped lipid composition, which was recorded as FAPα-5@DOX.

[0309] Example 6

[0310] A FAPα hydrolysis-induced charge-flipped lipid composition was prepared according to the method of Example 1, except that the product of Preparation Example 1 was replaced by the product of Preparation Example 6 in the same molar amount. The remaining conditions were the same as in Example 1 to obtain a FAPα hydrolysis-induced charge-flipped lipid composition, which was recorded as FAPα-6@DOX.

[0311] Example 7

[0312] A FAPα hydrolysis-induced charge-flipped lipid composition was prepared according to the method of Example 1, except that the product of Preparation Example 1 was replaced by the product of Preparation Example 7 in the same molar amount. The other conditions were the same as in Example 1 to obtain a FAPα hydrolysis-induced charge-flipped lipid composition, which was recorded as FAPα-7@DOX.

[0313] Example 8

[0314] A FAPα hydrolysis-induced charge-flipped lipid composition was prepared according to the method of Example 1, except that the product of Preparation Example 1 was replaced by the product of Preparation Example 8 in the same molar amount. The other conditions were the same as in Example 1 to obtain a FAPα hydrolysis-induced charge-flipped lipid composition, which was recorded as FAPα-4@DOX.

[0315] Example 9

[0316] A FAPα hydrolysis-induced charge-flipped lipid composition was prepared according to the method of Example 1, except that the product of Preparation Example 1 was replaced by the product of Preparation Example 9 in the same molar amount. The remaining conditions were the same as in Example 1 to obtain a FAPα hydrolysis-induced charge-flipped lipid composition, which was recorded as FAPα-9@DOX.

[0317] Example 10

[0318] A FAPα hydrolysis-induced charge-flipped lipid composition was prepared according to the method of Example 1, except that the product of Preparation Example 1 was replaced by the product of Preparation Example 10 in the same molar amount. The other conditions were the same as in Example 1 to obtain a FAPα hydrolysis-induced charge-flipped lipid composition, which was recorded as FAPα-10@DOX.

[0319] Example 11

[0320] A FAPα hydrolysis-induced charge-flipped lipid composition was prepared according to the method of Example 1, except that the product of Preparation Example 1 was replaced by the product of Preparation Example 11 in the same molar amount. The other conditions were the same as in Example 1 to obtain a FAPα hydrolysis-induced charge-flipped lipid composition, which was recorded as FAPα-11@DOX.

[0321] Example 12

[0322] A FAPα hydrolysis-induced charge-flipped lipid composition was prepared according to the method of Example 1, except that the product of Preparation Example 1 was replaced by the product of Preparation Example 12 in the same molar amount. The other conditions were the same as in Example 1 to obtain a FAPα hydrolysis-induced charge-flipped lipid composition, which was recorded as FAPα-12@DOX.

[0323] Example 13

[0324] A FAPα hydrolysis-induced charge-flipped lipid composition was prepared according to the method of Example 1, except that the product of Preparation Example 1 was replaced by the product of Preparation Example 13 in the same molar amount. The other conditions were the same as in Example 1 to obtain a FAPα hydrolysis-induced charge-flipped lipid composition, which was recorded as FAPα-13@DOX.

[0325] Comparative Preparation Example 1

[0326] A FAPα hydrolysis-induced charge-flipping lipid composition was prepared according to the method of Example 2, except that the product of Preparation Example 1 was replaced with the same molar amount of DSPE-PEG 2000. The other conditions were the same as in Example 1 to obtain a reference FAPα hydrolysis-induced charge-flipping lipid composition, which was recorded as PEG@DOX.

[0327] Test Case

[0328] (1) Characterization of liposome particle size and potential

[0329] The FAPα hydrolysis-induced charge-flipped lipid compositions obtained in Examples 1 to 5 were diluted to a concentration of 0.5 mg / mL using PBS buffer (10 mM, pH 7.4). The particle size and potential of the liposomes were then tested using a dynamic light scattering analyzer. The results are shown in Table 1.

[0330] Table 1

[0331] (2) FAPα protease enzyme-responsive charge reversal assay

[0332] 2 mg of each of the FAPα hydrolysis-induced charge-flipped liposomes (FAPα-1@DOX to FAPα-5@DOX) obtained in Examples 1 to 5 and the reference liposome PEG@DOX obtained in Comparative Example 1 were accurately weighed into 1 mL of Hepes buffer (pH 7.4, 10 mM). 10 U of FAPα protease was added and the mixture was incubated at 37°C, 100 rpm, on a constant-temperature shaker for 24 hours. Samples were taken at 0, 2, 4, 8, 16, and 24 hours, and the zeta potential of the nanoparticles under the catalytic conditions of FAPα protease was characterized using a particle size analyzer. Three replicates were run for each experiment, and the average results were calculated. See Table 2 for details. The results for the FAPα hydrolysis-induced charge-flipped liposomes (FAPα-2@DOX) from Example 2 are shown in Figure 1. As can be seen from the results in Table 2 and Figure 1, the FAPα hydrolysis-induced charge-flipped liposomes provided by the present invention exhibit charge-flipping properties.

[0333] Table 2

[0334] (3) Cell endocytosis experiment

[0335] Mouse breast cancer cells (4T1) were cultured at 10 5 Cells were evenly plated on a confocal microscopy dish and cultured in DMEM for 24 hours. DOX, FAPα@DOX, and PEG@DOX control groups were set up. The drug concentration was calculated based on the DOX concentration at 0.49 μg / mL. After 1 hour, 4 hours, and 8 hours of culture, the drug-containing medium was removed, and the cells were fixed with paraformaldehyde solution for 30 minutes. After removing the paraformaldehyde solution, the cells were washed three times with PBS, and the nuclei were stained with DAPI for 15 minutes. After washing with PBS, the cells were observed under a confocal microscope. The results showed that the FAPα-responsive liposomes provided by the present invention all had a higher endocytosis rate. The results corresponding to the FAPα-2@DOX liposomes obtained in Example 2, which were induced by FAPα hydrolysis and charge reversal, are shown in Figure 2.

[0336] (4) MTT cytotoxicity assay

[0337] Mouse breast cancer cells (4T1) in good growth condition and in the logarithmic phase were added to a 96-well plate at a density of 5000 cells / well. The outer wells of the well plate were discarded and filled with PBS buffer (blank peripheral PBS). After the cells adhered to the wall, the old culture medium was aspirated and 100 μL of diluted FAPα hydrolysis-induced charge-flipped DOX liposomes were added. The concentration gradient was 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, and 10.0 μg / mL DOX. After incubation in the incubator for 24 hours, 10 μL of MTT solution and 90 μL of DMEM culture medium were added to each well and incubated for another 1 hour. Finally, the absorbance was measured at 545 nm to calculate the cell survival rate. The relative cell survival rate was calculated using formula (1):

[0338] Relative cell survival rate (%) = (A x -A0) / (A c -A0)×100………………(1)

[0339] Among them A x , A0 and A c They are the absorbance values ​​of the experimental group, the zero-adjustment group and the negative control group, respectively. The zero-adjustment group is the blank peripheral PBS group (also known as the background group), and the negative control group is not added with liposomes and DOX.

[0340] IC 50 The IC value is the drug concentration that causes 50% cell death and can be used to measure the cytotoxicity of the drug. That is, the stronger the cytotoxicity of the drug, the higher the IC value. 50 The lower the value. 50 The cell survival rate at different concentrations was used to draw a curve and after fitting, the drug concentration corresponding to 50% cell survival rate was calculated. 50 The values ​​are shown in Table 3. From the experimental results in Table 3, it can be seen that the FAPα hydrolysis-induced charge-flipped liposomes provided by the present invention have a high cell growth inhibitory effect.

[0341] Table 3

[0342] (5) In vitro tumor penetration experiment

[0343] 3D tumor sphere experiments were used to verify the tumor penetration effect. 3D tumor spheres were obtained in a 96-well hanging drop plate (3D Biomatrix, Michigan, USA). The specific operation steps were as follows: 40 μL of well-growing human pancreatic cancer cells (BxPC-3) in the logarithmic phase were added to each well at 3×10 5Cells were seeded in plates and incubated at 37°C with 5% CO2 for 14 days, with fresh RPMI-1640 medium replaced daily. When the tumor spheres reached the desired size, 0.49 μg / mL DOX equivalent of free DOX, FAP@DOX, and PEG@DOX were added to each well and incubated for 4 hours. Images were captured using a laser confocal microscope in z-axis scanning mode. In the inhibition experiments, the 3D spheres were incubated with 1.0×10 -5 After pretreatment with a FAPα protease inhibitor, the cells were incubated with FAP@DOX liposomes for another 4 hours. 3D tumor spheres were then observed under a laser confocal microscope using the same conditions. The results demonstrated that the FAP@DOX liposomes provided by the present invention possessed deep tumor penetration. The results corresponding to the charge-flipped lipid composition induced by FAPα hydrolysis obtained in Example 2 are shown in Figure 3.

[0344] (6) 4T1 subcutaneous model tumor inhibition experiment

[0345] 4T1 subcutaneous breast cancer model mice were divided into 4 groups (3 mice / group), and the experimental groups were designed as follows: PBS group, DOX group, PEG@DOX group, and FAP@DOX group. The drug was injected into the tail vein: the total concentration of the drug was 5 mg / kg of doxorubicin equivalent, 100 μL / mouse, and the drug was given every 2 days for 4 times. Every 2 days, the length (L) and width (W) of the tumor were measured and recorded, and the weight of the mice was weighed and recorded. The tumor volume of the mice was calculated according to formula (2), and the tumor inhibition rate was calculated according to formula (3): V n =1 / 2LW 2 …….………………(2) Inhibition rate (%) = V n / V0…….……(3)

[0346] Among them, V n is the tumor volume on day n, and V0 is the initial tumor volume of the mice dosed. The results demonstrate that the FAPα hydrolysis-induced charge-flipped lipid composition provided by the present invention can achieve significant tumor inhibition. The results corresponding to the FAPα hydrolysis-induced charge-flipped lipid composition obtained in Example 2 are shown in Figure 4. The tumor inhibition rates corresponding to the FAPα hydrolysis-induced charge-flipped lipid compositions obtained in Examples 1-5 are shown in Table 4.

[0347] Table 4

[0348] (7) Tumor inhibition experiment in subcutaneous Hepg-2 liver cancer model

[0349] The subcutaneous humanized liver cancer model mice were divided into 4 groups (3 mice / group), and the experimental groups were designed as follows: PBS group, DOX group, PEG@DOX group, and FAP@DOX group. The drug was injected into the tail vein: the total concentration of the drug was 5 mg / kg of doxorubicin equivalent, 100 μL / mouse, and the drug was given every 2 days for 4 times. The length (L) and width (W) of the tumor were measured and recorded every 2 days, and the weight of the mice was weighed and recorded. The tumor volume of the mice was calculated according to formula (2), and the tumor inhibition rate was calculated according to formula (3): V n =1 / 2LW 2 …….………………(2) Inhibition rate (%) = V n / V0…….……(3)

[0350] Among them, V n is the tumor volume on day n, and V0 is the initial tumor volume of the mice dosed. The results demonstrate that the FAPα hydrolysis-induced charge-flipped lipid composition provided by the present invention can achieve significant tumor inhibition. The results corresponding to the FAPα hydrolysis-induced charge-flipped lipid composition obtained in Example 2 are shown in Figure 5. The tumor inhibition rates corresponding to the FAPα hydrolysis-induced charge-flipped lipid compositions obtained in Examples 1-5 are shown in Table 5.

[0351] Table 5

[0352] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. An FAPa hydrolysis-induced charge reversal liposome, wherein the FAPa hydrolysis-induced charge reversal liposome has a structure represented by Formula (1):l                                                                                                                                                                            lo Y R4Ri-r2—rY_  ,9 O;r^99n        :         1 Formula (1);R1 is a lipid group or a lipid-like group;R2 is absent, or C1-C10 alkyl, C6-C20 aryl or a hydrophilic group which is bonded to R1 via a carbon-carbon bond, a phosphodiester bond, an ester bond, an amide bond, a urethane bond, an ether bond, a thioether bond, or via various chemical bonds formed by bioorthogonal chemical reactions;R3 is absent, or C1-C10 alkyl, C6-C20 aryl, an amino acid-based group, polypeptidyl, a polyether group, a polyester group, a polyamide group, a polythioether group or polypolypeptidyl which is bonded to proline via an amino group and bonded to R2 via a carbon-carbon bond, an ester bond, an amide bond, an ether bond, a thioether bond, or via various chemical bonds formed by bioorthogonal chemical reactions;R4 is a zwitterionic group;n is 1-8.

2. The FAPa hydrolysis-induced charge reversal liposome according to claim 1, wherein R1 is derived from at least one of sterols, steroids, enols, fatty acids, fatty alcohols, fatty amines, fatty acid glycerides, fatty alcohol glycidyl ethers, sphingolipids, phospholipids, polyketides, vitamins and fluorides.

3. The FAPa hydrolysis-induced charge reversal liposome according to claim 1, wherein R1 has at least one of the following structures:* HN9H OHNFormula (3), m=10-20, and n=10-20;Formula (4), m=1-10, and n=1-10;Formula (5);Formula (6), m=10-20, and n=10-20;■7 'N'o                  Formula (7), m=5-15;Formula (8), m=1-10, n=5-15, and p=1-10;Formula (9), m=1-10, n=1-10, p=1-10, and q=1-10;Formula (10), m=10-20, n=10-20, and p=1-10;NH / O, *Formula (11), m=10-15, and n=10-20;Formula (12), n=10-20;o Formula (13), n=10-20;Formula (14), m=5-20, n=4-15, and R11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;Formula (15), m=5-30, n=4-20, and R11is -O-, -NH-, -CO-, -OCO- or -NHCO-;Formula (16), m=3-15, n=3-20, and R11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;Formula (17), m=2-15, n=2-20, and R11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;Formula (18), m=2-20, n=2-15, and R11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;Formula (19), m=2-15, n=2-20, and R11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;Formula (20), m=2-10, n=2-20, and R11 is -O-, -NH-, -CO-, -OCO-or -NHCO-;Formula (21), m=2-10, n=2-20, and R11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;Formula (22);I Formula (23);Formula (24);Formula (25);Formula (26), m=8-15, and n=10-20;Formula (28), m=1-15, n=10-20, and p=1-5;Formula (29), m=1-15, n=10-20, and p=1-5.

4. The FAPa hydrolysis-induced charge reversal liposome according to claim 1, wherein in R2, the hydrophilic group is derived from at least one of polyethylene glycol, poly(N-(2-hydroxypropyl)methacrylamide), polyvinylpyrrolidone, poly(2-methacryloyloxyethyl phosphorylcholine) and poly(methacryloyethyl sulfobetaine).

5. The FAPa hydrolysis-induced charge reversal liposome according to claim 1, wherein R2 has at least one of the following structures:nH Formula (30), n=1-6;Formula (31), m=0-6, and n=0-6;HN " Formula (32), m=0-6, and n=0-6;Formula (33), n=1-115;Formula (34), n=1-115;oFormula (35), n=1-115;•W oFormula (36), n=1-6;Formula (37), m=0-6, and n=0-6;Formula (38), m=0-6, and n=0-6;Formula (40), n=1-115;Formula (41), n=1-115;Formula (42), n=1-115;0          0Formula (43), n=1-115;Formula (44), n=1-115;oFormula (45), n=1-115;Formula (46), n=1-115;*"N\7'N'x*H / n HFormula (47), n=1-6;Formula (48), m=0-6, and n=0-6;Formula (49), m=0-6, and n=0-6;7h Formula (50), n=1-6;o O' x\ / P-0   / —\’“O HrK / H\n'—'  hn-* Formula (51), m=0-6, and n=0-6;os p-*-dhn-* Formula (52), m=0-6, and n=0-6;Formula (53), n=1-115;Formula (55), n=1-115;Formula (56), n=1-115;Formula (57), n=1-115;Formula (58), n=20-400;Formula (59), n=20-400;Formula (60), n=20-400;Formula (61), n=20-400.

6. The FAPa hydrolysis-induced charge reversal liposome according to claim 1, wherein R3has at least one of the following structures:0Formula (62), n=1-3;0Formula (63), n=1-12;0R31     Formula (64);*R310R3l'R3l'' * Formula (65);Formula (66);Formula (67), m and n are each independently 1-5;IFormula (69);Formula (70), n=1-6;k£0Rr“Formula (71), n=1-6;Formula (72), n=1-12;R32 '     'n           Formula (73), n=1-6;Formula (74);I-Iin the above formulas, R31 is absent, * M * ,  * M *orand n=1-15;in the above formulas, R31' and R32' are each independently O -or -NH-; R31'' is H or C1-C5alkyl;in the above formulas, R32 is absent,*    *              0      ,         0                or7. The FAPa hydrolysis-induced charge reversal liposome according to claim 1, wherein R4 has at least one of the following structures:Formula (76), m=2-5;Formula (77), m=1-5;Formula (78), m=2-5, and n=1-4;Formula (79), n=1-5;o          Formula (80), n=1-5;o          o Formula (81).

8. The FAPa hydrolysis-induced charge reversal liposome according to claim 1, wherein the FAPa hydrolysis-induced charge reversal liposome has at least one of the following structures:Formula (82), m is 1-10, n is 1-10, p is1-10, q is 1-10, s is 1-5, and t is 1-5;Formula (83);Formula (84), m is 10-20, n is 1-10, p is 1-5, and q is 1-5;Formula (85), m is 10-15, n is 10-15, p is 1-10, q is 1-10, s is 1-10, t is 1-10, and r is 1-10;Formula (86), m is 5-15, n is5-15, p is 1-5, o is 1-50, r is 1-5, s is 1-5, and t is 1-5;0HFormula (87), mis 40-50, and n is 1-10;Formula (88), m is 1-5, and n is 1-50;nh2oh Formula (91), m is 5-15, n is 5-15,p is 15-25, and q is 5-10;Formula (92), m is 10-20, n is 10-20, pis 1-5, q is 1-5, r is 1-5, s is 1-5, and t is 1-5;0 o 0          Formula (93), m is 5-15, n is 5-10, p is 5-15, q is 100-150, s is 1-5, and r is 1-5;o5, n is 5-15, p is 5-15, q is 1-10, o is 1-5, r is 1-5, s is 1-5, and t is 1-5.Formula (94), m is 1-9. A preparation method for the FAPa hydrolysis-induced charge reversal liposome according to claim 1, wherein the method is performed by the following steps:Method (I):S11, bonding an R2-source compound to an R1-source compound to obtain a stable lipid / lipid-like compound, wherein the R2-source compound is a substance capable of increasing the stability of the R1-source compound;S12, bonding an R3-source compound to the stable lipid / lipid-like compound to obtain a branched lipid / lipid-like compound;S13, conjugating a glycyl-proline dipeptide to the branched lipid / lipid-like compound, wherein when the glycyl-proline dipeptide contains a zwitterionic group, the obtained conjugate is the FAPa hydrolysis-induced charge reversal liposome; and when the glycyl-proline dipeptide contains no zwitterionic group, the zwitterionic group is conjugated to the terminal of the glycyl-proline dipeptide of the obtained conjugate to obtain the FAPa hydrolysis-induced charge reversal liposome; or,Method (II):S21, bonding an R3-source compound to an R1-source compound to obtain a branched lipid / lipid-like compound;S22, conjugating a glycyl-proline dipeptide to the branched lipid / lipid-like compound, wherein when the glycyl-proline dipeptide contains a zwitterionic group, the obtained conjugate is the FAPa hydrolysis-induced charge reversal liposome; and when the glycyl-proline dipeptide contains no zwitterionic group, the zwitterionic group is conjugated to the terminal of the glycyl-proline dipeptide of the obtained conjugate to obtain the FAPa hydrolysis-induced charge reversal liposome; or,Method (III):S31, bonding an R2-source compound to an R1-source compound to obtain a stable lipid / lipid-like compound, wherein the R2-source compound is a substance capable of increasing the stability of the R1-source compound; andS32, conjugating a glycyl-proline dipeptide to the stable lipid / lipid-like compound, wherein when the glycyl-proline dipeptide contains a zwitterionic group, the obtained conjugate is the FAPa hydrolysis-induced charge reversal liposome; and when the glycyl-proline dipeptide contains no zwitterionic group, the zwitterionic group is conjugated to the terminal of the glycyl-proline dipeptide of the obtained conjugate to obtain the FAPa hydrolysis-induced charge reversal liposome.

10. The preparation method for the FAPa hydrolysis-induced charge reversal liposome according to claim 9, wherein the R1-source compound is selected from at least one of sterols, steroids, enols, fatty acids, fatty alcohols, fatty amines, fatty acid glycerides, fatty alcohol glycidyl ethers, sphingolipids, phospholipids, polyketides, vitamins and fluorides, each of which contains a reactive group.

11. The preparation method for the FAPa hydrolysis-induced charge reversal liposome according to claim 9, wherein upon completion of the reaction, the residual groups in the R1-source compound have structures represented by Formula (2)-Formula (29); and / or,upon completion of the reaction, the residual groups in the R2-source compound have structures represented by Formula (30)-Formula (61); and / or,upon completion of the reaction, the residual groups in the R3-source compound have structures represented by Formula (62)-Formula (75).

12. An FAPa hydrolysis-induced charge reversal lipid composition, wherein the FAPa hydrolysis-induced charge reversal lipid composition contains the FAPa hydrolysis-induced charge reversal liposome according to claim 1.

13. The FAPa hydrolysis-induced charge reversal lipid composition according to claim 12, wherein the FAPa hydrolysis-induced charge reversal liposome is loaded with at least one of a chemotherapeutic agent, a molecular targeted agent, a protein drug, a gene drug, an ultrasound contrast agent, a fluorescent dye, a magnetic resonance imaging (MRI) contrast agent, a computed tomography (CT) contrast agent, a photosensitizer and a radionuclide.

14. The FAPa hydrolysis-induced charge reversal lipid composition according to claim 13, wherein the chemotherapeutic agent is selected from at least one of paclitaxel, docetaxel, doxorubicin, epirubicin, amphotericin, camptothecin, 7-ethyl-10-hydroxycamptothecin, irinotecan, gemcitabine, cisplatin, gambogic acid, dansylic acid, methotrexate, maytansine, a-amanitin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), DM4, calicheamicin, exatecan, mitomycin C and arsenic trioxide; and / or,the molecular targeted agent is selected from at least one of imatinib, gefitinib, icotinib and sunitinib; and / or,the protein drug is selected from at least one of nimotuzumab, cetuximab, trastuzumab, bevacizumab, catalase, glucose oxidase and pegaspargase; and / or,the gene drug is selected from at least one of therapeutic DNA, siRNA, microRNA and mRNA; and / or,the ultrasound contrast agent is selected from at least one of sulfur hexafluoride and / or perfluoropropane; and / or,the fluorescent dye is selected from at least one of rhodamine, indocyanine green, fluorescein and BODIPY; and / or,the MRI contrast agent is selected from at least one of gadolinium ions, manganese ions and iron ions; and / or,the CT contrast agent is selected from at least one of iohexol, diatrizoic acid and gold nanoparticles; and / or,the photosensitizer is selected from at least one of porphyrins, porphine and indocyanine green; and / or,the radionuclide is selected from at least one of fluorine-18, technetium-99m, iodine-123, iodine-125, iodine-124, iodine-131, cobalt-60, cesium-137, iridium-192, gold-198, tantalum-182, strontium-90, yttrium-90, gallium-68 and lutetium-177.

15. The FAPa hydrolysis-induced charge reversal lipid composition according to claim 12, wherein the FAPa hydrolysis-induced charge reversal lipid composition further contains an auxiliary phospholipid and optional cholesterol.

16. The FAPa hydrolysis-induced charge reversal lipid composition according to claim 15, wherein a molar ratio of the FAPa hydrolysis-induced charge reversal liposome to the auxiliary phospholipid to the cholesterol is 1:(0.1-5):(0-2).

17. The FAPa hydrolysis-induced charge reversal lipid composition according to claim 15, wherein the auxiliary phospholipid is selected from at least one of lysophosphatidylethanolamine, N-palmitoyl-D-erythro-sphingosylphosphocholine, 1,2-dilauroyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphoric acid (sodium salt), N-(carbonyl-methoxypolyethylene glycol)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine           (sodium           salt),1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoric acid (sodium      salt),      1,2-dimyristoyl-sn-glycero-3-phosphoglycerol      (sodium      salt),distearoylphosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphoglycerol (sodium salt), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine,     1-stearoyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine,            dipalmitoylphosphatidylglycerol,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol               (sodium               salt),1,2-dioleoyl-sn-glycero-3-phosphocholine,          1,2-dierucoyl-sn-glycero-3-phosphocholine,L-a-dioleoylphosphatidyl-DL-glycerol                     (sodium                     salt),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1-myristoyl-2-lyso-sn-glycero-3-phosphocholine,1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium                salt),                 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine,distearoyl-rac-glycerol-polyethylene glycol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol,                                                  N-(carbonyl-methoxypolyethyleneglycol)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine      (sodium      salt)      andN-(carbonyl-methoxypolyethylene    glycol)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine(sodium salt).

18. Use of the FAPa hydrolysis-induced charge reversal liposome according to claim 1 in tumor diagnosis, treatment and surgical navigation.

19. Use of the FAPa hydrolysis-induced charge reversal lipid composition according to claim 12 in tumor diagnosis, treatment and surgical navigation.

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