A FAPα hydrolysis-induced charge flipping liposome and composition as well as preparation method and application thereof

Abstract

The present invention belongs to the technical field of biomedical polymer materials, and specifically relates to a FAPα hydrolysis-induced charge reversal liposome and composition, and a preparation method and application thereof. The FAPα hydrolysis-induced charge reversal liposome has a structure shown in formula (1). The FAPα hydrolysis-induced charge reversal liposome provided by the present invention has the characteristics of deep tumor penetration, charge reversal by utilizing the specific enzymatic cleavage of FAPα overexpressed in tumor tissue, and enhancing the drug uptake and delivery efficiency of CAFs. In the hypoxic tumor microenvironment that affects the individualized therapeutic effect of most conventional tumors, the FAPα-responsive liposome can effectively deliver drugs to the interior of the tumor, deeply kill the tumor, and achieve significant tumor inhibition.

Description

Technical Field

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

[0002] In recent years, great progress has been made in the application of nanomedicine in tumor treatment, and a variety of anti-tumor nanomedicines have been launched or entered clinical trials. A large number of studies have shown that the existence of a complex tumor microenvironment (TME) plays an important role in tumor cell resistance and drug intratumoral penetration, and is a key factor limiting the efficacy of existing nanomedicines. Therefore, how to reshape the TME to improve tumor treatment resistance and enhance the in vivo delivery efficiency of anti-tumor nanomedicines is of great significance for overcoming the 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 such as 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 diffuse transport of nanomedicines (extravasation and diffusion), and competitive 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 response ability, which, on the one hand, meets the application requirements of clear structure and long-term plasma stability, and on the other hand, 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, thereby 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 the 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 reversal 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-flipping liposomes.

[0006] The third object of the present invention is to provide a lipid composition induced by hydrolysis of FAPα and resulting in 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-flipping liposome provided by the present invention has a structure shown in formula (1):

[0009]

[0010] R 1 is a lipid group or a lipid-like group;

[0011] R 2 is absent or is connected to R by various chemical bonds formed by carbon-carbon bonds, phosphoester bonds, ester bonds, amide bonds, urethane bonds, ether bonds, thioether bonds or bioorthogonal chemical reactions. 1 Bonded C 1 -C 10 Alkyl, C 6 -C 20 An aromatic or hydrophilic group;

[0012] R 3 is absent or is bonded to proline through an amino group and is bonded to R through a carbon-carbon bond, an ester bond, an amide bond, an ether bond, a thioether bond, or various chemical bonds formed by bioorthogonal chemical reactions. 2 Bonded C 1 -C 10 Alkyl, C 6 -C 20 aromatic group, amino acid group, polypeptide group, polyether group, polyester group, polyamide group, polythioether group or polypolypeptide group;

[0013] R 4 It is a zwitterionic group;

[0014] n is 1 to 8, and specifically can be 1, 2, 3, 4, 5, 6, 7 or 8.

[0015] In a preferred embodiment, R 1 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.

[0016] In a preferred embodiment, R 1 Has at least one of the following structures:

[0017] m = 1 to 10, n = 1 to 10;

[0018] m = 10 to 20, n = 10 to 20;

[0019] m = 1 to 10, n = 1 to 10;

[0020]

[0021] m = 10 to 20, n = 10 to 20;

[0022] m = 5 to 15;

[0023] m=1~10, n=5~15, p=1~10;

[0024] m=1~10, n=1~10, p=1~10, q=1~10;

[0025] m=10~20, n=10~20, p=1~10;

[0026] m = 10-15, n = 10-20;

[0027] n = 10 to 20;

[0028] n = 10 to 20;

[0029] m=5~20,n=4~15,R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;

[0030] m=5~30,n=4~20,R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;

[0031] m=3~15,n=3~20,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=2~20,n=2~15,R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;

[0034] m=2~15,n=2~20,R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;

[0035] m=2~10,n=2~20,R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;

[0036] m=2~10,n=2~20,R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-;

[0037]

[0038]

[0039] m = 8-15, n = 10-20;

[0040] m = 10 to 20, n = 10 to 20;

[0041] m=1~15, n=10~20, p=1~5;

[0042] m=1~15, n=10~20, p=1~5.

[0043] In a preferred embodiment, R 2 wherein the hydrophilic group is derived from at least one of polyethylene glycol, poly(N-(2-hydroxypropyl)methacrylamide), polyvinyl pyrrolidone, poly(2-methacryloyloxyethyl phosphorylcholine) and poly(methacryloylethyl sulfobetaine). 2 The various chemical bonds formed by carbon-carbon bonds, phosphoester bonds, ester bonds, amide bonds, urethane bonds, ether bonds, thioether bonds or bioorthogonal chemical reactions with R 1 When the hydrophilic groups are bonded, the stability of the liposomes can be enhanced, and the duration of drug action can be increased, so that the drug can act for a long time in the blood and have a better anti-tumor effect.

[0044] In a preferred embodiment, R 2 Has at least one of the following structures:

[0045] n = 1 to 6;

[0046] m=0~6,n=0~6;

[0047] m=0~6,n=0~6;

[0048] n=1~115;

[0049] n=1~115;

[0050] n=1~115;

[0051] n=1~6;

[0052] m=0~6,n=0~6;

[0053] m=0~6,n=0~6;

[0054] n=1~115;

[0055] n=1~115;

[0056] n=1~115;

[0057] n=1~115;

[0058] n=1~115;

[0059] n=1~115;

[0060] n=1~115;

[0061] n=1~115;

[0062] n=1~6;

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

[0064] m=0~6,n=0~6;

[0065] n=1~6;

[0066] m=0~6,n=0~6;

[0067] m = 0 to 6, n = 0 to 6;

[0068] n = 1 to 115;

[0069] n = 1 to 115;

[0070] n = 1 to 115;

[0071] n = 1 to 115;

[0072] n = 1 to 115;

[0073] n = 20 to 400;

[0074] n = 20 to 400;

[0075] n = 20 to 400;

[0076] n=20~400.

[0077] In a preferred embodiment, R 3 Has at least one of the following structures:

[0078] n = 1 to 3;

[0079] n = 1 to 12;

[0080]

[0081]

[0082] m and n are each independently 1 to 5;

[0083]

[0084] n = 1 to 6;

[0085] n = 1 to 6;

[0086] n = 1 to 12;

[0087] n = 1 to 6;

[0088]

[0089]

[0090] In the above formulas, R 31 For nothing, n = 1 to 15;

[0091] In the above formulas, R 31 ` and R 32 ` are each independently O or -NH-; R 31 `` is H or C 1 -C 5 The alkyl group;

[0092] In the above formulas, R 32 For nothing,

[0093] In a preferred embodiment, R 4 Has at least one of the following structures:

[0094] m = 2 to 5;

[0095] m = 1 to 5;

[0096] m = 2 to 5, n = 1 to 4;

[0097] n = 1 to 5;

[0098] n = 1 to 5;

[0099]

[0100] In a preferred embodiment, the FAPα hydrolysis-induced charge-flipping liposome has at least one of the following structures:

[0101] 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;

[0102]

[0103] m is 10 to 20, n is 1 to 10, p is 1 to 5, and q is 1 to 5;

[0104] 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;

[0105]

[0106] m is 5 to 15, n is 5 to 15, p is 1 to 5, q is 1 to 50, o is 1 to 50, r is 1 to 5, s is 1 to 5, t is 1 to 5;

[0107] m is 40 to 50, n is 1 to 10;

[0108] m is 1 to 5, n is 1 to 50;

[0109] n is 1 to 10;

[0110] m is 1 to 5, n is 1 to 10, and p is 1 to 10;

[0111] m is 5 to 15, n is 5 to 15, p is 15 to 25, q is 5 to 10;

[0112] 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;

[0113] 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 11 to 5;

[0114] 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.

[0115] The preparation method of FAPα hydrolysis-induced charge-flipping liposomes provided by the present invention is carried out according to the following steps:

[0116] Method (I):

[0117] S11, R 2 The source compound is bonded to R 1 On the source compound, the R 2 The source compound is capable of increasing R 1 Substances that improve the stability of source compounds to obtain stable lipid / lipid compounds;

[0118] S12, R 3The source compound is bonded to the stable lipid / lipid compound to obtain a branched lipid / lipid compound;

[0119] S13, conjugating glycine-proline dipeptide to branched lipid / lipid compound; when the glycine-proline dipeptide contains zwitterionic groups, the resulting conjugation product is FAPα hydrolysis-induced charge flip liposome; when the glycine-proline dipeptide does not contain zwitterionic groups, a zwitterionic group is conjugated to the end of the glycine-proline dipeptide of the resulting conjugation product to obtain FAPα hydrolysis-induced charge flip liposome; or,

[0120] Method (II):

[0121] S21, R 3 The source compound is bonded to R 1 On the source compound, a branched lipid / lipid compound is obtained;

[0122] S22, conjugating glycine-proline dipeptide to branched lipid / lipid compound; when the glycine-proline dipeptide contains zwitterionic groups, the resulting conjugation product is FAPα hydrolysis-induced charge flip liposome; when the glycine-proline dipeptide does not contain zwitterionic groups, a zwitterionic group is conjugated to the end of the glycine-proline dipeptide of the resulting conjugation product to obtain FAPα hydrolysis-induced charge flip liposome; or,

[0123] Method (III):

[0124] S31, R 2 The source compound is bonded to R 1 On the source compound, the R 2 The source compound is capable of increasing R 1 Substances that improve the stability of source compounds to obtain stable lipid / lipid compounds;

[0125] S32. Bind the glycine-proline dipeptide to a stable lipid / lipid compound; when the glycine-proline dipeptide contains a zwitterionic group, the resulting binding product is the FAPα hydrolysis-induced charge-flipped liposome; when the glycine-proline dipeptide does not contain a zwitterionic group, a zwitterionic group is attached to the end of the glycine-proline dipeptide of the resulting binding product to obtain the FAPα hydrolysis-induced charge-flipped liposome.

[0126] In a preferred embodiment, the R 1 The 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.

[0127] In a preferred embodiment, after the reaction is completed, the R 1 The remaining residue of the source compound has the structure shown in formula (2) to formula (29).

[0128] In a preferred embodiment, after the reaction is completed, the R 2 The remaining residues of the source compounds have structures shown in formula (30) to formula (61).

[0129] In a preferred embodiment, after the reaction is completed, the R 3 The remaining residues of the source compound have structures shown in formula (62) to formula (75).

[0130] In the preparation process of the FAPα hydrolysis-induced charge flip liposome provided by the present invention, the R 1 The source compound needs to contain 2 Source compound or R 3 The reactive group of the source compound, the R 2 The source compound needs to contain 1 The R 3 The source compound needs to contain a lipid / lipid compound or R 1 The active group that reacts with the source compound and the glycine-proline dipeptide, the glycine-proline dipeptide needs to contain an active group that can react with the branched lipid / lipid compound and optionally contains an active group that reacts with the zwitterionic compound. The type of the active group is not particularly limited, and can be at least one of a carboxyl group, an amino group, a hydroxyl group, etc.

[0131] In the preparation process of FAPα hydrolysis-induced charge flipping liposomes provided by the present invention, the bonding, linking, combining, and joining are only used to distinguish the chemical reactions in different steps for the convenience of description, and have no other special meanings. The conditions of the bonding, linking, combining, and joining can be selected according to the various reaction raw materials and reaction types, which are known to those skilled in the art and will not be described in detail here.

[0132] The FAPα hydrolysis-induced charge-flipping lipid composition provided by the present invention contains the FAPα hydrolysis-induced charge-flipping liposome.

[0133] 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 radionuclides.

[0134] 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.

[0135] In a preferred embodiment, the molecular targeted drug is selected from at least one of imatinib, gefitinib, icotinib and sunitinib.

[0136] In a preferred embodiment, the protein drug is selected from at least one of nimotuzumab, cetuximab, trastuzumab, bevacizumab, catalase, glucose oxidase and pegaspargase.

[0137] In a preferred embodiment, the gene drug is selected from at least one of therapeutic DNA, siRNA, microRNA and mRNA.

[0138] In a preferred embodiment, the ultrasound contrast agent is sulfur hexafluoride and / or propane octafluoride.

[0139] In a preferred embodiment, the fluorescent dye is selected from at least one of rhodamine, indocyanine green, fluorescein and BODIPY.

[0140] In a preferred embodiment, the nuclear magnetic resonance contrast agent is selected from at least one of gadolinium ions, manganese ions and iron ions.

[0141] In a preferred embodiment, the CT contrast agent is selected from at least one of iohexol, diatrizoate and gold nanoparticles.

[0142] In a preferred embodiment, the photosensitizer is at least one selected from porphyrin, porphin and indocyanine green.

[0143] 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.

[0144] In a preferred embodiment, the FAPα hydrolysis-induced charge flipping lipid composition further contains auxiliary phospholipids and optional cholesterol, wherein the use of auxiliary phospholipids and cholesterol can enhance the stability of liposomes and prolong blood circulation time.

[0145] In a preferred embodiment, the molar ratio of the FAPα hydrolysis-induced charge-flipping liposome, the auxiliary phospholipid and cholesterol is 1:(0.1-5):(0-2). Specifically, based on the amount of the FAPα hydrolysis-induced charge-flipping liposome as 1 mol, 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.

[0146] 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).

[0147] The present invention also provides the use of the above-mentioned FAPα hydrolysis-induced charge-flipping liposome and / or FAPα hydrolysis-induced charge-flipping lipid composition in tumor diagnosis, treatment and surgical navigation.

[0148] The FAPα hydrolysis-induced charge-flipped 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 it has 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

[0149] Figure 1 This is a Zeta potential charge reversal curve of the FAPα hydrolysis-induced charge-flipping lipid composition obtained in Example 2 under FAPα enzyme catalysis;

[0150] Figure 2 This is a diagram of the cellular endocytosis experiment of the charge-flipping lipid composition induced by FAPα hydrolysis obtained in Example 2;

[0151] Figure 3 This is a diagram showing an in vitro tumor penetration experiment of the FAPα hydrolysis-induced charge-flipping lipid composition obtained in Example 2;

[0152] Figure 4 This is a graph showing the tumor inhibition effect of the FAPα hydrolysis-induced charge-flipping lipid composition obtained in Example 2 in subcutaneous 4T1 breast cancer tumor model mice;

[0153] Figure 5 This is a graph showing the tumor inhibition effect of the FAPα hydrolysis-induced charge-flipping lipid composition obtained in Example 2 in subcutaneous Hepg-2 liver cancer model mice. DETAILED DESCRIPTION

[0154] The present invention will be described in detail below through examples.

[0155] Preparation Example 1

[0156]

[0157] (1a) Synthesis of intermediate c

[0158] Compound b (1 mmol), DCC (1.1 mmol) and DMAP (1.1 mmol) were added to DCM (10 mL), mixed and stirred at room temperature for 4 h, and then compound a (5 mmol) was added and stirred for 48 h. After the reaction was completed, the by-products were filtered out, and the DCM was concentrated and precipitated in ether three times to obtain product c.

[0159] (1b) Synthesis of intermediate e

[0160] Compound d (1 mmol), EDC (2 mmol) and NHS (2 mol) were mixed evenly in DCM (10 mL), and stirred at room temperature for 4 h. Then, product c (0.2 mmol) was added and stirred for 48 h. After the reaction was completed, 10 mL of TFA was added and stirred for 1 h. The product e was obtained by concentration, dialyzation and drying.

[0161] (1c) Synthesis of intermediate g

[0162] Boc-Gly-pro-OH (Boc-glycine-L-proline, 1 mmol), EDC (2 mmol) and NHS (2 mol) were mixed evenly in DCM (10 mL) and stirred at room temperature for 4 h. Then, product e (0.2 mmol) was added and stirred for 48 h. Subsequently, 10 mL of TFA was added and stirred for 1 h, followed by concentration, dialyzation and drying to obtain product g.

[0163] (1d) Synthesis product i

[0164] Compound h (1 mmol), EDC (2 mmol) and NHS (2 mol) were mixed evenly in DCM (10 mL), and stirred at room temperature for 4 h, then product g (0.2 mmol) was added and stirred for 48 h. After the reaction was completed, 10 mL of TFA was added and stirred for 1 h, and then concentrated, dialyzed and dried to obtain product i.

[0165] The chemical shift of product i is as follows: 1 H NMR (400 MHz, CDCl 3 , δ): 4.89 (p, 1H), 4.53-4.45 (m, 4H), 4.40 (ddd, 1H), 4.32 (dt, 2H), 4.25 (s, 1H), 4.23-4.21 (m, 5H), 4.19-4.09 (m, 11H), 4.05 (t, 2H), 3.80 (t, 2H), 3.63-3.51 (m, 7H), 3.44 (td, 2H), 3.2 7(s, 8H), 2.95-2.87(m, 2H), 2.85-2.77(m, 2H), 2.71-2.52(m, 21H), 2.42(td, 2H), 2.30(t, 2 H), 1.98-1.87 (m, 5H), 1.82 (dddt, 2H), 1.71-1.59 (m, 30H), 1.06 (s, 3H), 0.90-0.84 (m, 3H).

[0166] Preparation Example 2

[0167]

[0168] (2a) Synthesis of intermediate DSPE-Lys

[0169] First, Boc-Lys(Boc)-OH (1 mmol), EDC (2 mmol) and NHS (2 mmol) were added to DCM (10 mL) and stirred at room temperature for 4 h, and then DSPE (1 mmol) was added and stirred for 12 h. After the reaction was completed, 10 mL of trifluoroacetic acid (TFA) was added and stirred for 1 h, and the mixture solution was concentrated by rotary evaporation, and then the mixture solution was dialyzed in methanol solution for 24 h. After the dialysis was completed, it was dried to obtain Lys-modified DSPE (DSPE-Lys, about 0.65 g, yield 74.5%).

[0170] (2b) Synthesis of intermediate DSPE-Lys-diGP

[0171] 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, DSPE-Lys (0.5 mmol) was added and stirred for 24 h, then 10 mL TFA was added, the mixture was stirred for 1 h, and DSPE-Lys-diGP (about 0.51 g, yield 68.9%) was obtained after concentration, dialyzation and drying.

[0172] (2c) Synthetic product DSPE-Lys-diGPS

[0173] 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.

[0174] The chemical shifts of DSPE-Lys-diGPS are as follows: 1 H NMR (400 MHz, CDCl 3 , 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).

[0175] Preparation Example 3

[0176]

[0177] (3a) Synthesis of intermediate c

[0178] Compound a (1 mmol), EDC (2 mmol) and NHS (2 mmol) were mixed evenly in DCM (10 mL) and stirred at room temperature for 4 h, then compound b (0.2 mmol) was added and stirred for 24 h. After the reaction was completed, 10 mL of TFA was added and stirred for 1 h, followed by concentration, dialyzation and drying to obtain product c.

[0179] (3b) Synthesis of intermediate e

[0180] Boc-Lys(Boc)-OH (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 (0.2 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 mixture was then concentrated, dialyzed and dried to obtain product e.

[0181] (3c) Synthetic product g

[0182] 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, then compound e (0.2 mmol) was added and stirred for 48 h. After the reaction was completed, 10 mL of TFA was added and stirred for 1 h, followed by concentration, dialyzation and drying to obtain product g.

[0183] The chemical shift of product g is as follows: 1 H NMR (400 MHz, CDCl 3 , δ): 7.06-6.96 (m, 3H), 6.63-6.57 (m, 2H), 5.52 (ddq, 1H), 5.45 (d, 1H ), 4.53-4.47(m, 1H), 4.22(d, 2H), 4.05(t, 1H), 3.89(dt, 1H), 3.66-3. 60 (m, 1H), 3.57 (t, 14H), 3.21 (t, 12H), 2.89 (s, 6H), 2.85-2.73 (m, 2H) , 2.72-2.47(m, 5H), 1.98-1.87(m, 5H), 1.82(dddt, 1H), 1.14(dd, 3H).

[0184] Preparation Example 4

[0185]

[0186] (4a) Synthesis of intermediate c

[0187] Compound a (1 mmol), EDC (2 mmol) and NHS (2 mol) were mixed evenly in DCM (10 mL), and stirred at room temperature for 4 h. Compound b (0.2 mmol) was then added and stirred for 48 h. 10 mL of TFA was added and stirred for 1 h, followed by concentration, dialyzation and drying to obtain product c.

[0188] (4b) Synthesis of intermediate e

[0189] Compound d (1 mmol), EDC (2 mmol) and NHS (2 mol) were mixed evenly in DCM (10 mL) and stirred at room temperature for 4 h, then product c (0.2 mmol) was added and stirred for 48 h. After the reaction was completed, 10 mL of TFA was added and stirred for 12 h, and then concentrated, dialyzed and dried to obtain product e.

[0190] (4c) Synthesis of intermediate g

[0191] Boc-Gly-pro-OH (Boc-glycine-L-proline, 1 mmol), EDC (2 mmol) and NHS (2 mol) were mixed evenly in DCM (10 mL) and stirred at room temperature for 4 h. Then, product e (0.2 mmol) was added and stirred for 48 h. After that, 10 mL TFA was added and stirred for 1 h. After that, the mixture was concentrated, dialyzed and dried to obtain product g.

[0192] (4d) Synthetic product i

[0193] The 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 obtain the product i.

[0194] The chemical shift of product i is as follows: 1 H NMR (400 MHz, CDCl 3 , 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, 7H), 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).

[0195] Preparation Example 5

[0196]

[0197] (5a) Synthesis of intermediate c

[0198] 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, and then compound b (0.2 mmol) was added and stirred for 48 h. After the reaction was completed, 10 mL of TFA was added and stirred for 1 h. The stirred product was dialyzed in an aqueous solution and dried to obtain product c.

[0199] (5b) Synthesis of intermediate e

[0200] Compound d (1 mmol), EDC (2 mmol) and NHS (2 mol) were mixed evenly in DCM (10 mL) and stirred at room temperature for 4 h, then product c (0.5 mmol) was added and stirred for 48 h. After the reaction was completed, 10 mL of TFA was added and stirred for 1 h, and then concentrated, dialyzed and dried to obtain product e (yield 68.9%).

[0201] (5c) Synthesis of intermediate g

[0202] Boc-Gly-pro-OH (Boc-glycine-L-proline, 1 mmol), EDC (2 mmol) and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h, then product e (0.2 mmol) was added and stirred for 48 h. After the reaction was completed, 10 mL of TFA was added and stirred for 1 h, and then concentrated, dialyzed and dried to obtain product g.

[0203] (5d) Synthetic product i

[0204] Compound h (Boc-glycine-L-proline, 1 mmol), EDC (2 mmol) and NHS (2 mol) were mixed in DCM (10 mL) and stirred at room temperature for 4 h, then product g (0.2 mmol) was added and stirred for 48 h. After the reaction was completed, 10 mL of TFA was added, the mixture was stirred for 1 h, then concentrated, dialyzed and dried to obtain product i.

[0205] The chemical shift of product i is as follows: 1 H NMR (400 MHz, CDCl 3, δ): 4.57-4.46 (m, 6H), 4.43 (t, 2H), 4.40-4.28 (m, 16H), 4.28-4.15 (m, 8 H), 3.93-3.85 (m, 6H), 3.74 (t, 2H), 3.57 (d, 2H), 3.51 (p, 1H), 3.19 (tdd, 6 H), 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).

[0206] Example 1

[0207] The ammonium sulfate gradient method was used to prepare FAPα hydrolysis-induced charge-flipped liposomes loaded with DOX. The preparation process was as follows:

[0208] (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 DSPE-PEG 2000 in 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.

[0209] (2) Add ammonium sulfate solution (0.3 M, 20 mL) to the lipid film, and after ultrasonic hydration, extrude the solution through a polycarbonate membrane (100 nm) to obtain a blank liposome solution, which is centrifuged at 5000 r / min for 60 min in an ultrafiltration tube (molecular weight cutoff of 100 kDa) to remove excess ammonium sulfate solution to obtain concentrated liposomes.

[0210] (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 r / min, 50°C) for 4 h to obtain a FAPα hydrolysis-induced charge reversal lipid composition, which was recorded as FAPα-1@DOX.

[0211] Example 2

[0212] A FAPα hydrolysis-induced charge-flipping 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, and the other conditions were the same as in Example 1 to obtain a FAPα hydrolysis-induced charge-flipping lipid composition, which was recorded as FAPα-2@DOX.

[0213] Example 3

[0214] A FAPα hydrolysis-induced charge-flipping 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, and the other conditions were the same as in Example 1 to obtain a FAPα hydrolysis-induced charge-flipping lipid composition, which was recorded as FAPα-3@DOX.

[0215] Example 4

[0216] A FAPα hydrolysis-induced charge-flipping 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, and the other conditions were the same as in Example 1 to obtain a FAPα hydrolysis-induced charge-flipping lipid composition, which was recorded as FAPα-4@DOX.

[0217] Example 5

[0218] A FAPα hydrolysis-induced charge-flipping 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, and the other conditions were the same as in Example 1 to obtain a FAPα hydrolysis-induced charge-flipping lipid composition, which was recorded as FAPα-5@DOX.

[0219] Comparative Preparation Example 1

[0220] The 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 by the same molar amount of DSPE-PEG 2000, and 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.

[0221] Test Case

[0222] (1) Characterization of liposome particle size and potential

[0223] The FAPα hydrolysis-induced charge-flipping 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), and then the particle size and potential of the liposomes were tested using a dynamic light scattering analyzer. The results are shown in Table 1.

[0224] Table 1

[0225] Liposomes Size(nm) Zeta(mV) FAPα-1@DOX 87.56 -12.36 FAPα-2@DOX 86.89 -14.25 FAPα-3@DOX 88.06 -12.53 FAPα-4@DOX 83.89 -13.24 FAPα-5@DOX 84.56 -14.23 PEG@DOX 80.42 -9.25

[0226] (2) FAPα protease enzyme-responsive charge reversal assay

[0227] Accurately weigh 2 mg of the FAPα hydrolysis-induced charge-flipping liposomes FAPα-1@DOX~FAPα-5@DOX obtained in Examples 1 to 5 and the reference liposome PEG@DOX obtained in Comparative Example 1, respectively, in 1 mL (pH=7.4, 10 mM) Hepes buffer, add 10 U of FAPα protease, and culture in a constant temperature shaker at 37°C and 100 r / min for 24 hours. Samples were taken at 0h, 2h, 4h, 8h, 16h and 24h, and the Zeta potential of the nanoparticles in the FAPα protease catalytic environment was characterized and monitored by a particle size analyzer. Three parallel experiments were set for each group of experiments, and the results were averaged. See Table 2 for details. Among them, the results of the FAPα hydrolysis-induced charge-flipping liposomes FAPα-2@DOX in Example 2 are shown in Table 2. Figure 1 From Table 2 and Figure 1 It can be seen from the results that the FAPα hydrolysis-induced charge-reversal liposomes provided by the present invention have charge reversal characteristics.

[0228] Table 2

[0229]

[0230] (3) Cell endocytosis experiment

[0231] Mouse breast cancer cells (4T1) were cultured at 10 5 The number of cells was evenly spread in a confocal dish and cultured in DMEM culture medium for 24 hours. A control group of DOX, FAPα@DOX, and PEG@DOX were set up. The drug concentration was calculated as 0.49 μg / mL according to the DOX concentration. The drug-containing culture medium was removed after culturing for 1 hour, 4 hours, and 8 hours, respectively. The cells were fixed with a paraformaldehyde solution for 30 minutes, and washed with PBS three times after removing the paraformaldehyde solution. The cell nuclei were stained with DAPI for 15 minutes. After washing with PBS, the cells were observed under a confocal microscope. The results show that the FAPα-responsive liposomes provided by the present invention all have a higher endocytosis rate. Among them, the results corresponding to the FAPα-2@DOX charge-flipping liposomes obtained in Example 2 are shown in Table 2. Figure 2 .

[0232] (4) MTT cytotoxicity assay

[0233] Mouse breast cancer cells (4T1) in good growth state and in the logarithmic phase were added to a 96-well plate at a cell 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 hydrolyzed DOX liposomes with charge flipping 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):

[0234] Relative cell survival rate (%) = (A x -A 0 ) / (A c -A 0 )×100……………………(1)

[0235] Among them A x , A 0 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.

[0236] IC 50 The 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 50 The lower the value. IC 50 The above cell survival rates at different concentrations were used to make a curve and after fitting, the drug concentration corresponding to a cell survival rate of 50% 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 inhibition effect.

[0237] Table 3

[0238] Liposomes <![CDATA[IC 50 (μg / mL)]]> FAPα-1@DOX 0.45 FAPα-2@DOX 0.49 FAPα-3@DOX 0.56 FAPα-4@DOX 0.47 FAPα-5@DOX 0.56 PEG@DOX 8.96

[0239] (5) In vitro tumor penetration experiment

[0240] The 3D tumor sphere experiment was used to verify the tumor penetration effect. 3D tumor spheres were obtained in a 96-well hanging drop plate (3DBiomatrix, Michigan, USA). The specific operation steps were as follows: 40 μL of human pancreatic cancer cells (BxPC-3) in good growth state and in the logarithmic phase were added to each well at 3×10 5The cells were plated in a plate and incubated at 37°C with 5% CO 2 Incubate for 14 days, and replace part of the RPMI-1640 medium with fresh medium every day. When the tumor spheres reached the ideal size, 0.49 μg / mL DOX equivalent of free DOX, FAP@DOX, and PEG@DOX were added to each well and incubated for 4 h. Images were taken using a laser confocal microscope in z-axis scanning mode. In the inhibition experiment, the 3D spheres were incubated with 1.0×10 -5 M FAPα protease inhibitor pretreatment, added FAP@DOX liposomes and incubated for another 4h, and then observed 3D tumor spheres under a laser confocal microscope under the same conditions. The results showed that the FAP@DOX liposomes provided by the present invention have deep tumor penetration ability. Among them, the results corresponding to the FAPα hydrolysis-induced charge flip lipid composition obtained in Example 2 are shown in Figure 3 .

[0241] (6) 4T1 subcutaneous model tumor inhibition experiment

[0242] The 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. 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):

[0243] V n =1 / 2LW 2 ……………………(2)

[0244] Inhibition rate (%) = V n / V 0 …………(3)

[0245] Among them, V n is the tumor volume on day n, V 0 is the initial tumor volume of the mice. The results show that the FAPα hydrolysis-induced charge flip lipid composition provided by the present invention can achieve significant tumor inhibition. Among them, the results corresponding to the FAPα hydrolysis-induced charge flip lipid composition obtained in Example 2 are shown in Figure 4 The tumor inhibition rates corresponding to the lipid compositions induced by hydrolysis of FAPα and charge reversal obtained in Examples 1-5 are shown in Table 4.

[0246] Table 4

[0247]

[0248] (7) Tumor inhibition experiment of subcutaneous Hepg-2 liver cancer model

[0249] 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):

[0250] V n =1 / 2LW 2 ……………………(2)

[0251] Inhibition rate (%) = V n / V 0 …………(3)

[0252] Among them, V n is the tumor volume on day n, V 0 is the initial tumor volume of the mice. The results show that the FAPα hydrolysis-induced charge flip lipid composition provided by the present invention can achieve significant tumor inhibition. Among them, the results corresponding to the FAPα hydrolysis-induced charge flip lipid composition obtained in Example 2 are shown in Figure 5 The tumor inhibition rates corresponding to the lipid compositions induced by hydrolysis of FAPα and charge reversal obtained in Examples 1-5 are shown in Table 5.

[0253] Table 5

[0254]

[0255] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary 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 intent of the present invention.

Claims

1. A FAPα hydrolysis-induced charge flip liposome, characterized in that: The FAPα hydrolysis-induced charge-flipping liposome has a structure shown in formula (1): n` is 1 to 8; R1 is a lipid group or a lipid group; R2 is C1-C2+ or C1-C2+ bonded to R1 through a carbon-carbon bond, a phosphoester bond, an ester bond, an amide bond, an urethane bond, an ether bond, a thioether bond, or various chemical bonds formed by bioorthogonal chemical reactions. 10 Alkyl, C6-C 20 An aromatic or hydrophilic group; R3 is C1-C2-hydroxy-2-nitropropene or is bonded to proline via an amino group and is bonded to R2 via a carbon-carbon bond, an ester bond, an amide bond, an ether bond, a thioether bond, or various chemical bonds formed by bioorthogonal chemical reactions. 10 Alkyl, C6-C 20 aromatic group, amino acid group, polypeptide group, polyether group, polyester group, polyamide group, polythioether group or polypolypeptide group; R3 has at least one of the following structures: n＝1～3； n＝1～12； m and n are each independently 1 to 5; n＝1～6； n＝1～6； n＝1～12； n＝1～6； In the above formulas, R 31 For nothing, n＝1～15； In the above formulas, R 31 ` and R 32 ` are each independently O or -NH-; R 31 `` is H or C1-C5 alkyl; In the above formulas, R 32 For nothing, n＝1～15； RR4 is a zwitterionic group; R4 has at least one of the following structures: m＝2～5； m＝1～5； m=2~5,n=1~4; n＝1～5； n＝1～5； 2. The FAPα hydrolysis-induced charge-flipping liposome according to claim 1, characterized in that: 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.

3. The FAPα hydrolysis-induced charge-flipping liposome according to claim 1, characterized in that: R1 has at least one of the following structures: m=1~10,n=1~10; m=10~20,n=10~20; m=1~10,n=1~10; m=10~20,n=10~20; m＝5～15； m=1~10,n=5~15,p=1~10; m=1~10,n=1~10,p=1~10,q=1~10; m=10~20,n=10~20,p=1~10; m=10~15,n=10~20; n＝10～20； n＝10～20； m=5~20,n=4~15,R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-; m=5~30,n=4~20,R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-; m=3~15,n=3~20,R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-; m = 2 to 15, n = 2 to 20, R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-; m = 2 to 20, n = 2 to 15, R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-; m = 2 to 15, n = 2 to 20, R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-; m = 2 to 10, n = 2 to 20, R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-; m = 2 to 10, n = 2 to 20, R 11 is -O-, -NH-, -CO-, -OCO- or -NHCO-; m=8~15,n=10~20; m=10~20,n=10~20; m=1~15,n=10~20,p=1~5; m=1~15,n=10~20,p=1~5。 4. The FAPα hydrolysis-induced charge-flipped liposome according to claim 1, characterized in that: In R2, the hydrophilic group is derived from at least one of polyethylene glycol, poly(N-(2-hydroxypropyl)methacrylamide), polyvinyl pyrrolidone, poly(2-methacryloyloxyethyl phosphorylcholine) and poly(methacryloylethyl sulfobetaine).

5. The FAPα hydrolysis-induced charge-flipping liposome according to claim 1, characterized in that: R2 has at least one of the following structures: n＝1～6； m=0~6,n=0~6; m=0~6,n=0~6; n＝1～115； n＝1～115； n＝1～115； n＝1～6； m=0~6,n=0~6; m=0~6,n=0~6; n＝1～115； n＝1～115； n＝1～115； n＝1～115； n＝1～115； n＝1～115； n＝1～115； n＝1～115； n＝1～6； m=0~6,n=0~6; m=0~6,n=0~6; n＝1～6； m=0~6,n=0~6; m=0~6,n=0~6; n＝1～115； n＝1～115； n＝1～115； n＝1～115； n＝1～115； n＝20～400； n＝20～400； n＝20～400； n＝20～400。 6. The FAPα hydrolysis-induced charge-flipped liposome according to any one of claims 1 to 5, characterized in that: The FAPα hydrolysis-induced charge flipping liposome has at least one of the following structures: 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; m is 10 to 20, n is 1 to 10, p is 1 to 5, and q is 1 to 5; 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; m is 5 to 15, n is 5 to 15, p is 1 to 5, q is 1 to 50, o is 1 to 50, r is 1 to 5, s is 1 to 5, t is 1 to 5; m is 40 to 50, n is 1 to 10; m is 1 to 5, n is 1 to 50; n is 1 to 10; m is 1 to 5, n is 1 to 10, and p is 1 to 10; m is 5 to 15, n is 5 to 15, p is 15 to 25, q is 5 to 10; 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; 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; 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.

7. The method for preparing the charge-flipped liposomes induced by hydrolysis of FAPα according to any one of claims 1 to 6, characterized in that: The method proceeds as follows: Method (I): 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; S12, bonding the R3 source compound to the stable lipid / lipid compound to obtain a branched lipid / lipid compound; S13, conjugating glycine-proline dipeptide to branched lipid / lipid compound; when the glycine-proline dipeptide contains zwitterionic groups, the resulting conjugation product is FAPα hydrolysis-induced charge flip liposome; when the glycine-proline dipeptide does not contain zwitterionic groups, a zwitterionic group is conjugated to the end of the glycine-proline dipeptide of the resulting conjugation product to obtain FAPα hydrolysis-induced charge flip liposome; or, Method (II): S21, bonding the R3 source compound to the R1 source compound to obtain a branched lipid / lipid compound; S22, conjugating the glycine-proline dipeptide to a branched lipid / lipid compound; when the glycine-proline dipeptide contains a zwitterionic group, the resulting conjugation 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 end of the glycine-proline dipeptide of the resulting conjugation product to obtain a FAPα hydrolysis-induced charge-flipped liposome; or, Method (III): 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; S32. Bind the glycine-proline dipeptide to a stable lipid / lipid compound; when the glycine-proline dipeptide contains a zwitterionic group, the resulting binding product is the FAPα hydrolysis-induced charge-flipped liposome; when the glycine-proline dipeptide does not contain a zwitterionic group, a zwitterionic group is attached to the end of the glycine-proline dipeptide of the resulting binding product to obtain the FAPα hydrolysis-induced charge-flipped liposome.

8. The method for preparing the charge-flipped liposomes induced by FAPα hydrolysis according to claim 7, characterized in that: 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.

9. The method for preparing the charge-flipped liposomes induced by FAPα hydrolysis according to claim 7, characterized in that: After the reaction is completed, the remaining residue of the R1 source compound has a structure shown in Formula (2) to Formula (29).

10. The method for preparing the charge-flipped liposomes induced by FAPα hydrolysis according to claim 7, characterized in that: When the reaction is completed, the remaining residue of the R2 source compound has a structure shown in Formula (30) to Formula (61).

11. The method for preparing FAPα hydrolysis-induced charge-flipping liposomes according to claim 7, characterized in that: When the reaction is completed, the remaining residue of the R3 source compound has a structure shown in Formula (62) to Formula (75).

12. A lipid composition induced by hydrolysis of FAPα and resulting in charge flipping, characterized in that: The FAPα hydrolysis-induced charge-flipping lipid composition contains the FAPα hydrolysis-induced charge-flipping liposome according to any one of claims 1 to 6.

13. The FAPα hydrolysis-induced charge-flipping lipid composition according to claim 12, characterized in that: The FAPα hydrolysis-induced charge flip liposome is loaded with at least one of chemotherapeutic drugs, molecular targeted drugs, protein drugs, gene drugs, ultrasound contrast agents, fluorescent dyes, nuclear magnetic contrast agents, CT contrast agents, photosensitizers and radioactive nuclides.

14. The FAPα hydrolysis-induced charge-flipping lipid composition according to claim 13, characterized in that: 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, macrosulfonic acid, methotrexate, maytansine, alpha-amanitin, MMAE, MMAF, DM4, calicheamicin, exitecan, mitomycin C and arsenic trioxide.

15. The FAPα hydrolysis-induced charge-flipping lipid composition according to claim 13, characterized in that: The molecular targeted drug is selected from at least one of imatinib, gefitinib, icotinib and sunitinib.

16. The FAPα hydrolysis-induced charge-flipping lipid composition according to claim 13, characterized in that: The protein drug is selected from at least one of nimotuzumab, cetuximab, trastuzumab, bevacizumab, catalase, glucose oxidase and pegaspargase.

17. The FAPα hydrolysis-induced charge-flipping lipid composition according to claim 13, characterized in that: The gene drug is selected from at least one of therapeutic DNA, siRNA, microRNA and mRNA.

18. The FAPα hydrolysis-induced charge-flipping lipid composition according to claim 13, characterized in that: The ultrasound contrast agent is sulfur hexafluoride and / or propane octafluoride.

19. The FAPα hydrolysis-induced charge-flipping lipid composition according to claim 13, characterized in that: The fluorescent dye is selected from at least one of rhodamine, indocyanine green, fluorescein and BODIPY.

20. The FAPα hydrolysis-induced charge-flipping lipid composition according to claim 13, characterized in that: The nuclear magnetic resonance contrast agent is selected from at least one of gadolinium ions, manganese ions and iron ions.

21. The FAPα hydrolysis-induced charge-flipping lipid composition according to claim 13, characterized in that: The CT contrast agent is selected from at least one of iohexol, diatrizoate and gold nanoparticles.

22. The FAPα hydrolysis-induced charge-flipping lipid composition according to claim 13, characterized in that: The photosensitizer is selected from at least one of porphyrin, porphin and indocyanine green.

23. The FAPα hydrolysis-induced charge-flipping lipid composition according to claim 13, characterized in that: 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.

24. The FAPα hydrolysis-induced charge-flipping lipid composition according to any one of claims 12 to 23, characterized in that: The FAPα hydrolysis-induced charge-flipping lipid composition further contains auxiliary phospholipids and optionally cholesterol.

25. The FAPα hydrolysis-induced charge-flipping lipid composition according to claim 24, characterized in that: The molar ratio of the FAPα hydrolyzed charge-reversed liposome, auxiliary phospholipid and cholesterol is 1:(0.1-5):(0-2).

26. The FAPα hydrolysis-induced charge-flipping lipid composition according to claim 24, characterized in that: 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), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphate (sodium salt), 1,2-di Myristoyl-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, dipalmitoylphosphatidylcholine acylglycerol, 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-dipalmitoyl- 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), N-(carbonyl-methoxypolyethylene glycol)-1,2-distearoyl-SN-glycero-3-phosphoethanolamine (sodium salt).

27. Use of the FAPα hydrolysis-induced charge-flipping liposomes according to any one of claims 1 to 6 and / or the FAPα hydrolysis-induced charge-flipping lipid composition according to any one of claims 12 to 26 in tumor diagnosis and treatment.

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