A polypeptide specifically binding to MUC1 protein, a targeted drug co-delivery system, and preparation method and application thereof

Abstract

The present invention provides a polypeptide specifically binding to MUC1 protein, a targeted drug-carrying co-delivery system, and a preparation method and application thereof. The targeted drug-carrying co-delivery system comprises a targeted drug-carrying long-circulating liposome and a targeted drug-carrying micelle. The polypeptide specifically binding to MUC1 protein is prepared by solid-phase peptide synthesis, and the general formula of its amino acid sequence is X1-DR-X2-VRTQFNQYDTRAASD-X3-G-X4-X5, wherein X1 is the amino terminus and X5 is the carboxyl terminus. The targeted drug-carrying long-circulating liposome comprises phospholipids, cholesterol, an amphiphilic polymer, a polypeptide-amphiphilic polymer and a pharmaceutically active component. The targeted drug-carrying micelle comprises an amphiphilic polymer, a polypeptide-amphiphilic polymer and a pharmaceutically active component. The polypeptide specifically binding to MUC1 protein, the targeted drug-carrying long-circulating liposome and the targeted drug-carrying micelle can be used for the preparation of anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a polypeptide specifically binding to a MUC1 protein, a targeted drug-carrying co-delivery system, and a preparation method and application thereof. Background Art

[0002] Leukemia is a malignant tumor formed by the malignant clonal proliferation of hematopoietic stem cells in the blood system bone marrow. Among them, acute myeloid leukemia (AML) is the most common type of leukemia in adult patients with the highest mortality rate, accounting for about 70% of adult acute leukemia. At present, the treatment methods for leukemia mainly include standard combined chemotherapy, radiotherapy, bone marrow transplantation, small molecule targeted drug therapy and immunotherapy. Among them, bone marrow transplantation treatment is limited by factors such as patient age, physical fitness and high cost, while targeted drug therapy is aimed at special types of hematological malignancies such as specific gene mutations, and is not widely applicable. Therefore, chemotherapy is still the most commonly used treatment for leukemia. The traditional treatment method is cytarabine combined with anthracycline drugs to induce remission, followed by consolidation chemotherapy and (or) hematopoietic stem cell transplantation, but many AML patients have a poor prognosis and are prone to relapse. The long-term disease-free survival rate of AML patients under 60 years old remains at around 40%, especially high-risk refractory patients and elderly patients, who have worse tolerance to chemotherapy. Therefore, AML treatment has always faced major challenges, and there is an urgent need to develop new technologies and treatments that are more efficient and less toxic.

[0003] MUC1 is a transmembrane protein of the mucin family. Studies have shown that MUC1 protein is abnormally expressed in hematological tumors such as multiple myeloma, B-cell non-Hodgkin's lymphoma and acute myeloid leukemia. On the one hand, the expression quantity increases, which is more than 10 times that of normal cells. On the other hand, the glycosylation level of MUC1 changes, exposing new protein or sugar chain epitopes in the core protein of MUC1. These epitopes become ideal target molecules for attacking tumor cells. Therefore, MUC1 protein has attracted extensive attention from scholars.

[0004] CN111201240A discloses an antibody or an antigenic fragment thereof that specifically binds to MUC1, wherein the antibody or the antigenic fragment thereof can recognize a polypeptide of at least five consecutive amino acids contained in the C-terminal extracellular domain of MUC1, and the antibody or the antigenic fragment thereof can be conjugated with a drug to obtain an antibody-drug conjugate, wherein the drug is a chemotherapeutic agent or a toxin, etc. The antibody or the antigenic fragment thereof has significant affinity and binding force to MUC1, and the antibody-drug conjugate can specifically bind to cells expressing MUC1 and efficiently transport the drug, and can be effectively used in the treatment of MUC1-related diseases such as cancer.

[0005] CN112574283A discloses a human MUC1 specific binding polypeptide, whose amino acid sequence is HKFIPHRDTSIA. The polypeptide is obtained by bio-panning and positive phage amplification, and is verified by methods such as DNA sequence determination of the polypeptide presented by the phage and ELISA determination of the screened positive phage. The polypeptide has a small molecular weight, is easy to synthesize, has low immunogenicity, can specifically bind to human MUC1 protein, and has good application value in the field of tumor diagnosis and treatment targeting human MUC1 protein.

[0006] Based on the above research, we can see that MUC1 protein can be used as a tumor-associated antigen and is an ideal broad-spectrum tumor target molecule. However, at present, the research on MUC1 protein in the field of tumor diagnosis and treatment still has certain limitations. The polypeptide sequence that can specifically bind to MUC1 protein needs further development and research. At the same time, the preparation of corresponding anti-tumor drugs has great practical significance for clinical treatment. Summary of the invention

[0007] The purpose of the present invention is to provide a polypeptide specifically binding to MUC1 protein, a targeted drug-carrying co-delivery system, and a preparation method and application thereof. The targeted drug-carrying co-delivery system comprises targeted drug-carrying long-circulating liposomes and targeted drug-carrying micelles. The polypeptide specifically binding to MUC1 protein, targeted drug-carrying long-circulating liposomes and targeted drug-carrying micelles can be used for the preparation and development of anti-tumor drugs, which is of great significance for the clinical treatment of tumors.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a polypeptide that specifically binds to the MUC1 protein, wherein the amino acid sequence of the polypeptide that specifically binds to the MUC1 protein is represented by the general formula X 1 -DR-X 2 -VRTQFNQYDTRAASD-X 3 -GX 4 -X 5 .

[0010] Among them, X 1 Deleted or a peptide fragment of the amino acid sequence GG, X 2 Delete or be the peptide fragment of amino acid sequence RTINVDR, X 3 A peptide fragment or amino acid D, X 4 is any one of the peptide fragments of the amino acid sequence GC, GDGV, GDDD or DDD, X 5 The deletion or peptide fragment of the amino acid sequence TSAPRTD.

[0011] Preferably, the X1 is the amino terminus, the X 5 The carboxyl terminus.

[0012] Preferably, the amino acid sequence of the polypeptide that specifically binds to the MUC1 protein includes any one of the sequences shown in SEQ ID NOs: 1-5, and the specific sequence is as follows:

[0013] SEQ ID NO: 1: DRVRTQFNQYDTRAASDDGGDGVTSAPRTD;

[0014] SEQ ID NO:2:DRRTINVDRVRTQFNQYDTRAASDDGGDGVTSAPRTD;

[0015] SEQ ID NO:3: GGDRRTINVDRVRTQFNQYDTRAASDDGGC;

[0016] SEQ ID NO:4: DRVRTQFNQYDTRAASDDGGDDDTSAPRTD;

[0017] SEQ ID NO:5: DRVRTQFNQYDTRAASDYRGDDDTSAPRTD.

[0018] The present invention designs a series of targeted polypeptides for MUC1 protein, which is abnormally expressed in tumors related to the hematopoietic system, such as multiple myeloma or acute myeloid leukemia, and the expression amount increases. The polypeptides involved in the present invention can specifically bind to MUC1 protein, thereby playing a targeting role, providing ideas for the clinical treatment of related tumors.

[0019] In a second aspect, the present invention provides a method for preparing a polypeptide that specifically binds to the MUC1 protein as described in the first aspect, wherein the preparation method comprises a solid phase polypeptide synthesis method.

[0020] In a third aspect, the present invention provides a targeted drug-loaded long-circulating liposome that specifically binds to MUC1 protein, wherein the raw materials for preparing the targeted drug-loaded long-circulating liposome that specifically binds to MUC1 protein include phospholipids, cholesterol, amphiphilic polymers, polypeptide-amphiphilic polymers and active pharmaceutical ingredients.

[0021] The amphiphilic polymer includes DSPE-PEG and / or DSPE-mPEG, the polypeptide-amphiphilic polymer includes DSPE-PEG-polypeptide and / or DSPE-mPEG-polypeptide, and the DSPE-PEG-polypeptide and / or DSPE-mPEG-polypeptide are the products of the polypeptide specifically binding to MUC1 protein as described in the first aspect connected with PEG.

[0022] The present invention constructs a targeted drug-loaded long-circulating liposome that specifically binds to the MUC1 protein. The surface of the targeted drug-loaded long-circulating liposome is modified with a polypeptide that can specifically bind to the MUC1 protein, thereby targeting the drug-loaded long-circulating liposome to tumor cells, killing tumor cells more effectively, while reducing the damage of chemotherapy drugs to normal cells, thereby reducing toxic side effects. In addition, the targeted drug-loaded long-circulating liposome contains an amphiphilic polymer component, which increases the flexibility and hydrophilicity of the targeted drug-loaded long-circulating liposome, avoids being engulfed by the reticuloendothelial system, and prolongs its circulation time in the body.

[0023] Preferably, the molar ratio of the phospholipid, cholesterol, amphiphilic polymer, polypeptide-amphiphilic polymer and pharmaceutically active ingredient is 1:(0.01-5):(0.001-5):(0.001-1):(0.001-1), preferably 1:(0.01-1):(0.01-1):(0.001-0.5):(0.01-0.5).

[0024] The specific point values ​​of the (0.01-5) can be 0.01, 0.1, 0.5, 0.8, 1, 2, 3, 4 or 5, etc.

[0025] The specific point values ​​of the (0.001-5) can be 0.001, 0.005, 0.01, 0.05, 0.08, 1, 2, 3, 4 or 5, etc.

[0026] The specific point value of the (0.001-1) can be 0.001, 0.003, 0.005, 0.008, 0.01, 0.05, 0.08, 0.1, 0.5, 0.8 or 1, etc.

[0027] Other point values ​​within the above range can be selected and will not be described in detail here.

[0028] Preferably, the phospholipid includes any one of soybean lecithin, hydrogenated soybean lecithin or egg yolk lecithin, or a combination of at least two of them. The combination can be, for example, a combination of soybean lecithin and hydrogenated soybean lecithin or a combination of hydrogenated soybean lecithin and egg yolk lecithin, and any other combinations can be selected and will not be described one by one here. Soybean lecithin is preferred.

[0029] The phospholipid in the present invention is preferably soybean lecithin, because soybean lecithin has good solubility in anhydrous ethanol.

[0030] Preferably, the active pharmaceutical ingredient includes any one of homoharringtonine, vincristine, doxorubicin hydrochloride, paclitaxel or cyclophosphamide, or a combination of at least two of them. For example, the combination may be a combination of homoharringtonine and vincristine or a combination of vincristine and doxorubicin hydrochloride, and any other combinations may be selected and will not be described in detail here.

[0031] In a fourth aspect, the present invention provides a method for preparing the targeted drug-loaded long-circulating liposomes that specifically bind to the MUC1 protein as described in the third aspect, wherein the preparation method adopts a thin film-ultrasound method and specifically comprises the following steps:

[0032] (1) mixing phospholipids, cholesterol, an amphiphilic polymer, a polypeptide-amphiphilic polymer, a pharmaceutically active component, and an organic solvent to obtain a first mixed solution;

[0033] (2) evaporating and removing the organic solvent in the first mixed solution to obtain a thin film;

[0034] (3) Mixing the film obtained in step (2) with water to obtain a second mixed solution, and subjecting the solution to ultrasound to obtain the targeted drug-loaded long-circulating liposomes.

[0035] Preferably, the organic solvent includes any one of anhydrous ethanol, chloroform, ether or methanol, or a combination of at least two thereof. The combination may be, for example, a combination of anhydrous ethanol and chloroform or a combination of chloroform and ether, and any other combinations may be selected and will not be described in detail here. Anhydrous ethanol is preferred.

[0036] In the present invention, anhydrous ethanol is preferably used as the organic solvent because of its low toxicity and safer operation.

[0037] Preferably, the frequency of the ultrasound is 100-500W, for example, it can be 100W, 150W, 200W, 250W, 300W, 350W, 400W, 450W or 500W, etc., preferably 100-200W.

[0038] The number of ultrasound cycles is 5-30 times, for example, 5 times, 15 times, 18 times, 20 times, 22 times, 25 times, 28 times or 30 times, etc., preferably 5-10 times.

[0039] The ultrasound time is 3-10 s, for example, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s or 10 s.

[0040] The ultrasound interval time is 2-20 s, for example, it can be 2s, 4s, 6s, 8s, 10s, 12s, 14s, 16s, 18s or 20s, etc., preferably 2-10s.

[0041] Other point values ​​within the above range can be selected and will not be described in detail here.

[0042] In a fifth aspect, the present invention provides a targeted drug-loaded micelle that specifically binds to the MUC1 protein, wherein the raw materials for preparing the targeted drug-loaded micelle include an amphiphilic polymer, a polypeptide-amphiphilic polymer and a pharmaceutically active component.

[0043] The amphiphilic polymer includes DSPE-PEG and / or DSPE-mPEG, the polypeptide-amphiphilic polymer includes DSPE-PEG-polypeptide and / or DSPE-mPEG-polypeptide, and the DSPE-PEG-polypeptide and / or DSPE-mPEG-polypeptide are the products of the polypeptide specifically binding to MUC1 protein as described in the first aspect connected with PEG.

[0044] The present invention constructs a targeted drug-loaded micelle that specifically binds to the MUC1 protein. The surface of the targeted drug-loaded micelle is modified with a polypeptide, and the polypeptide can specifically bind to the MUC1 protein, thereby targeting the drug-loaded micelle to tumor cells, killing tumor cells more effectively, while reducing the damage of chemotherapy drugs to normal cells, thereby reducing toxic side effects. In addition, the targeted drug-loaded micelle contains an amphiphilic polymer component, which increases the flexibility and hydrophilicity of the targeted drug-loaded micelle, avoids being engulfed by the reticuloendothelial system, and prolongs its circulation time in the body.

[0045] Preferably, the molar ratio of the amphiphilic polymer, polypeptide-amphiphilic polymer and pharmaceutically active ingredient is 1:(0.001-10):(0.01-30), preferably 1:(0.1-1):(0.1-10).

[0046] The specific point values ​​of the (0.001-10) can be 0.001, 0.005, 0.01, 0.05, 0.1, 1, 3, 5, 7, 9 or 10, etc.

[0047] The specific point values ​​of the (0.01-30) can be 0.01, 0.1, 0.5, 1, 5, 8, 10, 15, 18, 20, 25 or 30, etc.

[0048] Other point values ​​within the above range can be selected and will not be described in detail here.

[0049] Preferably, the active pharmaceutical ingredient includes any one of homoharringtonine, vincristine, doxorubicin hydrochloride, paclitaxel or cyclophosphamide, or a combination of at least two of them. For example, the combination may be a combination of homoharringtonine and vincristine or a combination of vincristine and doxorubicin hydrochloride, and any other combinations may be selected and will not be described in detail here.

[0050] In a sixth aspect, the present invention provides a method for preparing a targeted drug-loaded micelle that specifically binds to the MUC1 protein as described in the fifth aspect, wherein the preparation method adopts a thin film-hydration method and specifically comprises the following steps:

[0051] (1) mixing an amphiphilic polymer, a polypeptide-amphiphilic polymer, a pharmaceutically active component, and an organic solvent to obtain a first mixed solution;

[0052] (2) evaporating and removing the organic solvent in the first mixed solution to obtain a thin film;

[0053] (3) Mixing the film obtained in step (2) with water to obtain a second mixed solution, ultrasonicating, water bathing, and standing to obtain the targeted drug-loaded micelles.

[0054] Preferably, the organic solvent includes any one of anhydrous ethanol, chloroform, ether or methanol, or a combination of at least two thereof. The combination may be, for example, a combination of anhydrous ethanol and chloroform or a combination of anhydrous ethanol and methanol, and any other combinations may be selected and will not be described in detail here. Anhydrous ethanol is preferred.

[0055] In the present invention, anhydrous ethanol is preferably used as the organic solvent because of its low toxicity and safer operation.

[0056] Preferably, the frequency of the ultrasound is 80-120W, for example, it can be 80W, 82W, 85W, 88W, 90W, 95W, 100W, 105W, 110W, 115W or 120W, etc.

[0057] The ultrasound time is 15-25 min, for example, it can be 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min or 25 min, etc.

[0058] Preferably, the temperature of the water bath is 20-65°C, for example, it can be 20°C, 30°C, 40°C, 50°C, 52°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 62°C, 64°C or 65°C, etc., preferably 50°C.

[0059] The water bath time is 25-40 min, for example, it can be 25 min, 26 min, 27 min, 28 min, 30 min, 32 min, 34 min, 35 min, 36 min, 38 min or 40 min, etc.

[0060] Preferably, the standing time is 1-3 h, for example, it can be 1 h, 1.4 h, 1.5 h, 1.8 h, 2 h, 2.2 h, 2.4 h, 2.6 h, 2.8 h or 3 h.

[0061] Other point values ​​within the above range can be selected and will not be described in detail here.

[0062] In the seventh aspect, the present invention provides a use of a polypeptide specifically binding to MUC1 protein as described in the first aspect, a targeted drug-loaded long-circulating liposome specifically binding to MUC1 protein as described in the third aspect, or a targeted drug-loaded micelle specifically binding to MUC1 protein as described in the fifth aspect in the preparation of anti-tumor drugs.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] (1) The present invention provides a polypeptide that specifically binds to the MUC1 protein, wherein the amino acid sequence of the polypeptide that specifically binds to the MUC1 protein includes any one of the sequences shown in SEQ ID NOs: 1-5;

[0065] (2) The present invention provides a targeted drug-loaded long-circulating liposome that specifically binds to the MUC1 protein and a preparation method thereof. The targeted drug-loaded long-circulating liposome contains an amphiphilic polymer component and is surface-modified with a polypeptide. The polypeptide can specifically bind to the MUC1 protein to play a targeting role. The amphiphilic polymer can increase the flexibility and hydrophilicity of the targeted drug-loaded long-circulating liposome;

[0066] (3) The present invention provides a targeted drug-loaded micelle that specifically binds to the MUC1 protein and a preparation method thereof. The targeted drug-loaded micelle contains an amphiphilic polymer component and is surface-modified with a polypeptide. The polypeptide can specifically bind to the MUC1 protein to play a targeting role. The amphiphilic polymer can increase the flexibility and hydrophilicity of the targeted drug-loaded micelle.

[0067] (4) The polypeptide specifically binding to MUC1 protein, the targeted drug-loaded long-circulating liposomes and the targeted drug-loaded micelles described in the present invention can be used to prepare anti-tumor drugs, which are of great significance for the clinical treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 This is the result of the test of the binding rate between MUC1 antibody and KG1a;

[0069] Figure 2 is a graph showing the binding rate detection result of the polypeptide obtained in Preparation Example 1 of the present invention and KG1a;

[0070] Figure 3 is a graph showing the binding rate detection result of the polypeptide obtained in Preparation Example 2 of the present invention and KG1a;

[0071] Figure 4 is a graph showing the results of the binding rate detection between the polypeptide obtained in Preparation Example 3 of the present invention and KG1a;

[0072] Figure 5 is a graph showing the binding rate detection result of the polypeptide obtained in Preparation Example 4 of the present invention and KG1a;

[0073] Figure 6 is a graph showing the results of the binding rate test between the polypeptide obtained in Preparation Example 5 of the present invention and KG1a;

[0074] Figure 7 This is the result of the test of the binding rate between MUC1 antibody and KG1;

[0075] Figure 8 This is a graph showing the results of the binding rate test between the polypeptide obtained in Preparation Example 1 of the present invention and KG1;

[0076] Fig. 9 is a graph showing the results of the binding rate detection between the polypeptide obtained in Preparation Example 2 of the present invention and KG1;

[0077] Fig.10 is a graph showing the results of the binding rate test between the polypeptide obtained in Preparation Example 3 of the present invention and KG1;

[0078] Fig.11 is a graph showing the results of the binding rate test between the polypeptide obtained in Preparation Example 4 of the present invention and KG1;

[0079] Fig.12 is a graph showing the results of the binding rate detection between the polypeptide obtained in Preparation Example 5 of the present invention and KG1;

[0080] Fig.13 This is a graph showing the results of the binding rate test between the polypeptide obtained in Preparation Example 1 of the present invention and NRK;

[0081] Fig.14 This is a graph showing the results of the binding rate test between the polypeptide obtained in Preparation Example 2 of the present invention and NRK;

[0082] Fig.15 This is a graph showing the results of the binding rate test between the polypeptide obtained in Preparation Example 3 of the present invention and NRK;

[0083] Fig.16 This is a graph showing the results of the binding rate test between the polypeptide obtained in Preparation Example 4 of the present invention and NRK;

[0084] Fig.17 This is a graph showing the results of the binding rate test between the polypeptide obtained in Preparation Example 5 of the present invention and NRK;

[0085] Fig.18 The invention relates to a targeted drug-loaded long-circulating liposome and a targeted drug-loaded micelle for the regulation of GFP in the peripheral blood of mice. + Effect of scale results graph;

[0086] Fig.19The invention relates to a targeted drug-loaded long-circulating liposome and a targeted drug-loaded micelle for the regulation of GFP in the spleen of mice. + Effect of scale results graph;

[0087] Fig. 20 The invention relates to a targeted drug-loaded long-circulating liposome and a targeted drug-loaded micelle for the regulation of GFP in the bone marrow of mice. + Effect of scale results graph;

[0088] Fig.21 The invention relates to a targeted drug-loaded long-circulating liposome and a targeted drug-loaded micelle for the regulation of GFP in the liver of mice. + Effect of scale results graph;

[0089] Fig. 22 The invention relates to a targeted drug-loaded long-circulating liposome and a targeted drug-loaded micelle for the regulation of GFP in the lung of mice. + Effect of scale results graph;

[0090] Fig.23 This is a result diagram of the effects of the targeted drug-loaded long-circulating liposomes and targeted drug-loaded micelles on the number of leukocytes in the peripheral blood of mice;

[0091] Fig.24 This is a result diagram of the effects of the targeted drug-loaded long-circulating liposomes and targeted drug-loaded micelles on the number of red blood cells in the peripheral blood of mice;

[0092] Fig.25 This is a graph showing the effects of the targeted drug-loaded long-circulating liposomes and targeted drug-loaded micelles on the number of platelets in the peripheral blood of mice. DETAILED DESCRIPTION

[0093] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0094] In the following preparation examples and embodiments, the abbreviation G is glycine, the abbreviation R is arginine, the abbreviation T is threonine, the abbreviation I is isoleucine, the abbreviation N is asparagine, the abbreviation D is aspartic acid, the abbreviation V is valine, the abbreviation Q is glutamine, the abbreviation Y is tyrosine, the abbreviation A is alanine, the abbreviation F is phenylalanine, the abbreviation S is serine, and the abbreviation C is cysteine.

[0095] The sources of the corresponding materials and raw materials involved in the following embodiments and test examples are as follows:

[0096] The soybean lecithin used was purchased from Shanghai Huyu Biochemical Reagent Co., Ltd., with the product number LE030A; the egg yolk lecithin used was purchased from Shanghai Huyu Biochemical Reagent Co., Ltd., with the product number P0051; AML1-ETO (AE) & C-KIT D816V The cells were constructed and provided by Professor Wang Jianxiang's team from the Hematology Hospital of the Chinese Academy of Medical Sciences.

[0097] Preparation Example 1

[0098] This preparation example provides a polypeptide that specifically binds to the MUC1 protein. The amino acid sequence of the polypeptide that specifically binds to the MUC1 protein is the sequence shown in SEQ ID NO:1: DRVRTQFNQYDTRAASDDGGDGVTSAPRTD, wherein the left side of the sequence is the amino terminus and the right side of the sequence is the carboxyl terminus. The polypeptide that specifically binds to the MUC1 protein is prepared by solid phase peptide synthesis.

[0099] Preparation Example 2

[0100] This preparation example provides a polypeptide that specifically binds to the MUC1 protein. The amino acid sequence of the polypeptide that specifically binds to the MUC1 protein is the sequence shown in SEQ ID NO:2: DRRTINVDRVRTQFNQYDTRAASDDGGDGVTSAPRTD, wherein the left side of the sequence is the amino terminus and the right side of the sequence is the carboxyl terminus. The polypeptide that specifically binds to the MUC1 protein is prepared by solid phase peptide synthesis.

[0101] Preparation Example 3

[0102] This preparation example provides a polypeptide that specifically binds to the MUC1 protein. The amino acid sequence of the polypeptide that specifically binds to the MUC1 protein is the sequence shown in SEQ ID NO:3: GGDRRTINVDRVRTQFNQYDTRAASDDGGC, wherein the left side of the sequence is the amino terminus and the right side of the sequence is the carboxyl terminus. The polypeptide that specifically binds to the MUC1 protein is prepared by solid phase peptide synthesis.

[0103] Preparation Example 4

[0104] This preparation example provides a polypeptide that specifically binds to the MUC1 protein. The amino acid sequence of the polypeptide that specifically binds to the MUC1 protein is the sequence shown in SEQ ID NO:4: DRVRTQFNQYDTRAASDDGGDDDTSAPRTD, wherein the left side of the sequence is the amino terminus and the right side of the sequence is the carboxyl terminus. The polypeptide that specifically binds to the MUC1 protein is prepared by solid phase peptide synthesis.

[0105] Preparation Example 5

[0106] This preparation example provides a polypeptide that specifically binds to the MUC1 protein. The amino acid sequence of the polypeptide that specifically binds to the MUC1 protein is the sequence shown in SEQ ID NO:5: DRVRTQFNQYDTRAASDYRGDDDTSAPRTD, wherein the left side of the sequence is the amino terminus and the right side of the sequence is the carboxyl terminus. The polypeptide that specifically binds to the MUC1 protein is prepared by solid phase peptide synthesis.

[0107] Example 1

[0108] This embodiment provides a targeted drug-loaded long-circulating liposome that specifically binds to the MUC1 protein and a preparation method thereof, the preparation method comprising the following steps:

[0109] (1) 4 μmol soybean lecithin, 2 μmol cholesterol, 0.12 μmol DSPE-PEG2000, 0.2 μmol homoharringtonine, 0.03 μmol DSPE-PEG2000-peptide and 150 mL of anhydrous ethanol were mixed to obtain a first mixed solution;

[0110] The DSPE-PEG2000-polypeptide is the product obtained by connecting the polypeptide obtained in Preparation Example 1 with PEG2000;

[0111] (2) evaporating and removing anhydrous ethanol in the first mixed solution to form a thin film;

[0112] (3) The film was mixed with 2 mL of normal saline, and ultrasonicated 10 times at 100 W, each ultrasonication for 3 seconds, with an ultrasonication interval of 2 seconds, to obtain the targeted drug-loaded long-circulating liposomes.

[0113] Example 2

[0114] This embodiment provides a targeted drug-loaded micelle that specifically binds to the MUC1 protein and a preparation method thereof, the preparation method comprising the following steps:

[0115] (1) 0.03 μmol DSPE-PEG2000, 0.016 μmol DSPE-PEG2000-peptide, 0.2 μmol homoharringtonine and 60 mL anhydrous ethanol were mixed to obtain a first mixed solution;

[0116] The DSPE-PEG2000-polypeptide is the product obtained by connecting the polypeptide obtained in Preparation Example 1 with PEG2000;

[0117] (2) evaporating and removing anhydrous ethanol in the first mixed solution to form a thin film;

[0118] (3) The film was mixed with 2 mL of physiological saline to obtain a second mixed solution, which was ultrasonicated at 100 W for 20 min, then placed in a water bath at 55° C. for 30 min, and allowed to stand at 25° C. for 2 h to obtain the targeted drug-loaded micelles.

[0119] Comparative Example 1

[0120] This comparative example provides a drug-loaded long-circulating liposome and a preparation method thereof. Compared with Example 1, the raw materials for preparing the drug-loaded long-circulating liposome do not include DSPE-PEG2000-polypeptide, the 0.12 μmol DSPE-PEG2000 is replaced by 0.15 μmol DSPE-PEG2000, and the other parameters and steps are consistent with Example 1.

[0121] Comparative Example 2

[0122] This comparative example provides a long-circulating liposome and a preparation method thereof. Compared with Example 1, the raw materials for preparing the long-circulating liposome do not include DSPE-PEG2000-peptide and homoharringtonine, the 0.12 μmol DSPE-PEG2000 is replaced by 0.15 μmol DSPE-PEG2000, and the other parameters and steps are consistent with Example 1.

[0123] Comparative Example 3

[0124] This comparative example provides a drug-loaded micelle and a preparation method thereof. Compared with Example 8, the raw material for preparing the drug-loaded micelle does not include DSPE-PEG2000-polypeptide, the 0.03 μmol DSPE-PEG2000 is replaced by 0.046 μmol DSPE-PEG2000, and the other parameters and steps are consistent with Example 8.

[0125] Comparative Example 4

[0126] This comparative example provides a micelle and a preparation method thereof. Compared with Example 8, the raw materials for preparing the micelle do not include DSPE-PEG2000-polypeptide and homoharringtonine, the 0.03 μmol DSPE-PEG2000 is replaced by 0.046 μmol DSPE-PEG2000, and the other parameters and steps are consistent with Example 8.

[0127] Test Example 1

[0128] This test example detects the binding of the MUC1 antibody and the polypeptide obtained in Preparation Example 1-5 to the acute myeloid leukemia cell line KG1a.

[0129] The detection method of MUC1 antibody binding to KG1a is as follows:

[0130] (1) KG1a was inoculated in a 24-well plate at 2×10 5 1 well, 200 μL culture medium / well, incubate at 37°C with 5% CO 2 Incubate in an incubator for 12 hours. There are 3 wells in each of the blank control group, negative control group, and experimental group;

[0131] (2) Collect the cells into 9 1.5 mL Eppendorf tubes, add 1 mL PBS to each tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 100 μL 1% BSA / PBS to resuspend the cells, add 1 μL anti-MUC1 antibody to each tube of the experimental group, and add 1 μL 1% BSA / PBS to each tube of the blank control group and the negative control group, and incubate at 4°C for 1 h;

[0132] (3) After incubation, add 1 mL of 1% BSA / PBS to each of the nine Eppendorf tubes, centrifuge at 1000 rpm for 5 min, and discard the supernatant;

[0133] (4) Add 100 μL 1% BSA / PBS to each of the 9 Eppendorf tubes to resuspend the cells. Add 2 μL goat anti-mouse secondary antibody (1:100 dilution) to each tube of the negative control group and experimental group, and add 2 μL 1% BSA / PBS to each tube of the blank control group. Incubate at 4°C in the dark for 30 min.

[0134] (5) After incubation, 1 mL of 1% BSA / PBS was added to each of the nine Eppendorf tubes and centrifuged at 1000 rpm for 5 min;

[0135] (6) After centrifugation, discard the supernatant and add 200 μL PBS to each of the nine Eppendorf tubes for detection by flow cytometry.

[0136] The method for detecting the binding of KG1a to the peptide is as follows:

[0137] (1) KG1a was inoculated into a 24-well plate, 2×10 5 The blank control group included 3 wells, and the experimental groups were divided into 5 groups, with experimental group 1 including 6 wells and experimental groups 2-5 including 3 wells respectively;

[0138] (2) 300 μL of culture medium was added to each well of the blank control group, 10 μM FITC-labeled polypeptide obtained in Preparation Example 1 was added to 3 wells of the experimental group 1, 20 μM FITC-labeled polypeptide obtained in Preparation Example 1 was added to the remaining 3 wells, and 20 μM FITC-labeled polypeptide obtained in Preparation Examples 2-4 was added to 3 wells of the experimental groups 2-4, respectively;

[0139] (3) 37°C, 5% CO 2After incubation in the incubator for 2 h, the cells were collected into Eppendorf tubes, washed three times with PBS, centrifuged at 1000 rpm for 5 min, and 200 μL PBS was added to each tube for uniform mixing, and then detected by flow cytometry.

[0140] Test results such as Figure 1-6 As shown by Figure 1 The data show that the binding rate of MUC1 antibody to KG1a is 97.9%, the result of the negative control group is 0.4%, and the result of the blank control group is 0.4%. Figure 2 The data show that the binding rate of the polypeptide obtained in Preparation Example 1 to KG1a is 98.9% at 20 μM and 77.3% at 10 μM, while the result of the blank control group is 0.2%. Figure 3 The data show that the binding rate of the polypeptide obtained in Preparation Example 2 to KG1a at 20 μM is 97%, while the result of the blank control group is 0.2%. Figure 4 The data show that the binding rate of the polypeptide obtained in Preparation Example 3 to KG1a at 20 μM is 97.2%, while the result of the blank control group is 0.2%. Figure 5 From the data, it can be seen that the binding rate of the polypeptide obtained in Preparation Example 4 to KG1a at 20 μM is 98.2%, while the result of the blank control group is 0.2%. Figure 6 The data show that the binding rate of the polypeptide obtained in Preparation Example 5 to KG1a is 98% at 10 μM, while the result of the blank control group is 0.2%.

[0141] Test Example 2

[0142] This test example detects the binding of the MUC1 antibody and the polypeptide obtained in Preparation Examples 1-5 to the acute myeloid leukemia cell line KG1.

[0143] The detection method of MUC1 antibody binding to KG1 is as follows:

[0144] (1) KG1 was inoculated in a 24-well plate at 2×10 5 1 well, 200 μL culture medium / well, incubate at 37°C with 5% CO 2 Incubate in an incubator for 12 hours. There are 3 wells in each of the blank control group, negative control group, and experimental group;

[0145] (2) Collect the cells into 9 1.5 mL Eppendorf tubes, add 1 mL PBS to each tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 100 μL 1% BSA / PBS to resuspend the cells, add 1 μL anti-MUC1 antibody to each tube of the experimental group, and add 1 μL 1% BSA / PBS to each tube of the blank control group and the negative control group, and incubate at 4°C for 1 h;

[0146] (3) After incubation, add 1 mL of 1% BSA / PBS to each of the nine Eppendorf tubes, centrifuge at 1000 rpm for 5 min, and discard the supernatant;

[0147] (4) Add 100 μL 1% BSA / PBS to each of the 9 Eppendorf tubes to resuspend the cells. Add 2 μL goat anti-mouse secondary antibody (1:100 dilution) to each tube of the negative control group and experimental group, and add 2 μL 1% BSA / PBS to each tube of the blank control group. Incubate at 4°C in the dark for 30 min.

[0148] (5) After incubation, 1 mL of 1% BSA / PBS was added to each of the nine Eppendorf tubes and centrifuged at 1000 rpm for 5 min;

[0149] (6) After centrifugation, discard the supernatant and add 200 μL PBS to each of the nine Eppendorf tubes for detection by flow cytometry.

[0150] The method for detecting the binding of KG1 to peptide is as follows:

[0151] (1) KG1 was inoculated into a 24-well plate at 2×10 5 The blank control group included 3 wells, and the experimental groups were divided into 5 groups, with experimental group 1 including 6 wells and experimental groups 2-5 including 3 wells respectively;

[0152] (2) 300 μL of culture medium was added to each well of the blank control group, 10 μM FITC-labeled polypeptide obtained in Preparation Example 1 was added to 3 wells of the experimental group 1, 20 μM FITC-labeled polypeptide obtained in Preparation Example 1 was added to the remaining 3 wells, and 20 μM FITC-labeled polypeptide obtained in Preparation Examples 2-4 was added to 3 wells of the experimental groups 2-4, respectively;

[0153] (3) 37°C, 5% CO 2 After incubation in the incubator for 2 h, the cells were collected into Eppendorf tubes, washed three times with PBS, centrifuged at 1000 rpm for 5 min, and 200 μL PBS was added to each tube for uniform mixing, and then detected by flow cytometry.

[0154] Test results such as Figure 7-12 As shown by Figure 7 The data show that the binding rate of MUC1 antibody to KG1 is 61.8%, the result of the negative control group is 0.7%, and the result of the blank control group is 0.6%. Figure 8 The data show that the binding rate of the polypeptide obtained in Preparation Example 1 to KG1 is 99.9% at 20 μM and 89.3% at 10 μM, while the result of the blank control group is 0.3%. Fig. 9 From the data, it can be seen that the binding rate of the polypeptide obtained in Preparation Example 2 to KG1 at 20 μM is 94.1%, while the result of the blank control group is 1.4%. Fig.10The data show that the binding rate of the polypeptide obtained in Preparation Example 3 to KG1 at 20 μM is 90.4%, while the result of the blank control group is 0.3%. Fig.11 The data show that the binding rate of the polypeptide obtained in Preparation Example 4 to KG1 at 20 μM is 93%, while the result of the blank control group is 0.1%. Fig.12 The data show that the binding rate of the polypeptide obtained in Preparation Example 5 to KG1 at 20 μM is 92.7%, while the result of the blank control group is 0.1%.

[0155] Test Example 3

[0156] This test example detects the binding of the polypeptide obtained in Preparation Example 1-5 to the normal rat kidney cell line NRK. The detection method is as follows:

[0157] (1) NRK was inoculated in a 24-well plate, 2×10 5 / well, the white control group includes 3 wells, the experimental groups are divided into 5 groups, experimental group 1 includes 6 wells, and experimental groups 2-5 include 3 wells respectively;

[0158] (2) 300 μL of culture medium was added to each well of the blank control group, 10 μM FITC-labeled polypeptide obtained in Preparation Example 1 was added to 3 wells of the experimental group 1, 20 μM FITC-labeled polypeptide obtained in Preparation Example 1 was added to the remaining 3 wells, and 20 μM FITC-labeled polypeptide obtained in Preparation Examples 2-4 was added to 3 wells of the experimental groups 2-4, respectively;

[0159] (3) 37°C, 5% CO 2 After incubation in the incubator for 2 h, the cells were collected into Eppendorf tubes, washed three times with PBS, centrifuged at 1000 rpm for 5 min, and 200 μL PBS was added to each tube for uniform mixing, and then detected by flow cytometry.

[0160] Test results such as Figure 13-17 As shown by Fig.13 From the data, it can be seen that the binding rate of the polypeptide obtained in Preparation Example 1 to NRK is 21.4% at 20 μM and 9.7% at 10 μM, and the result of the blank control group is 1.5%. Fig.14 The data show that the binding rate of the polypeptide obtained in Preparation Example 2 to NRK at 20 μM is 11.3%, while the result of the blank control group is 6.1%. Fig.15 The data show that the binding rate of the polypeptide obtained in Preparation Example 3 to NRK at 20 μM is 11.4%, while the result of the blank control group is 6.1%. Fig.16 The data show that the binding rate of the polypeptide obtained in Preparation Example 4 to NRK at 20 μM is 27.2%, while the result of the blank control group is 0.2%. Fig.17The data show that the binding rate of the polypeptide obtained in Preparation Example 5 to NRK is 33% at 20 μM, while the result of the blank control group is 0.3%.

[0161] Test Example 4

[0162] This test example detects the inhibitory activity of long-circulating liposomes and micelles on acute myeloid leukemia cells at the animal level. The detection method is as follows:

[0163] Using AML1-ETO-GFP and C-KIT D816V GFP retrovirus was used to infect hematopoietic stem / progenitor cells sorted from C57 mouse bone marrow cells to obtain AML1-ETO(AE)&C-KIT D816V Cells. The cells were transplanted into recipient mice irradiated with a lethal dose of X-rays, and the primary diseased mice in a moribund state were killed. The spleen was separated in a sterile operating environment, ground and filtered with a 300-mesh sieve to obtain a spleen single cell suspension, which was frozen at -80°C for later use;

[0164] The frozen spleen cells were revived and 1×10 6 Cells. Waiting for AML1-ETO(AE)&C-KIT D816V The mouse was in a moribund state. The spleen single cell suspension of the mouse was isolated in a sterile operating environment and inoculated into C57 female mice 4 hours after semi-lethal X-ray dose irradiation. The number of inoculated cells and irradiation conditions were the same as above. After cell transplantation, the cell ratio in peripheral blood can be detected by BDC6 flow cytometer. Mice with appropriate disease status can be selected for animal experiments according to needs. Blood was collected and grouped on the seventh day of modeling. The drug was injected into the tail vein on the eighth day. The drug was administered three times a week, and the mice were killed 48 hours after drug withdrawal. The animals were divided into 6 groups for drug administration, including: (1) blank liposome group (injection of the long-circulating liposomes prepared in Comparative Example 2); (2) homoharringtonine liposome group (injection of the drug-loaded long-circulating liposomes prepared in Comparative Example 1); (3) targeted drug-loaded long-circulating liposome group (injection of the targeted drug-loaded long-circulating liposomes prepared in Example 1); (4) blank micelle group (injection of the micelles prepared in Comparative Example 4); (5) homoharringtonine micelle group (injection of the drug-loaded micelles prepared in Comparative Example 3); (6) targeted drug-loaded micelle group (injection of the targeted drug-loaded micelles prepared in Example 2).

[0165] The results are as follows Figure 18-22 As shown, Fig.18 GFP in peripheral blood + The proportional result diagram, Fig.19 GFP in spleen + The proportional result diagram, Fig. 20 GFP in bone marrow + The proportional result diagram, Fig.21GFP in liver + The proportional result diagram, Fig. 22 GFP in lung + The data in the figure show that compared with the blank liposome group, the targeted drug-loaded long-circulating liposome group reduced the GFP expression in peripheral blood, bone marrow, liver and lung. + cells, among which, significantly reduced GFP in liver and lung + cells, 24.81% and 42.61% respectively; compared with the homoharringtonine liposome group, the targeted drug-loaded long-circulating liposome group reduced GFP to varying degrees + The number of cells in the lungs was significantly reduced by 34.31%. The results showed that the drug-loaded long-circulating liposomes modified with targeted peptides on the surface can kill leukemia cells more effectively.

[0166] It can be seen from the data in the figure that compared with the blank micelle group, the targeted drug-loaded micelle group reduced the GFP expression in peripheral blood, bone marrow, liver and lung to varying degrees. + cells, among which, significantly reduced myeloid GFP + 13.59% of cells showed significant decrease in lung GFP + Compared with the homoharringtonine micelle group, the targeted drug-loaded micelle group reduced the GFP expression in peripheral blood, bone marrow, liver and lung to varying degrees. + cells, among which, significantly reduced myeloid GFP + The results showed that micelles modified with targeting peptides on the surface can kill leukemia cells more effectively.

[0167] Figure 23-25 This is a graph showing the number of white blood cells, red blood cells, and platelets in peripheral blood. Fig.23 The result graph of the number of white blood cells is shown in Figure 2. Fig.24 The result graph of the number of red blood cells is shown in Figure 2. Fig.25 The result graph of platelet count. The data in the graph show that compared with the blank liposome group, the homoharringtonine liposome group reduced the number of red blood cells and platelets to varying degrees, but the red blood cells in the targeted drug-loaded long-circulation liposome group increased by 37.54%, which was 102.79% higher than the homoharringtonine liposome group; and the platelets increased by 57.22% compared with the blank group.

[0168] From the data in the figure, it can be seen that compared with the blank micelle group, the homoharringtonine micelle group and the targeted drug-loaded micelle group had little effect on the number of red blood cells in peripheral blood. Compared with the blank micelle group, the targeted drug-loaded micelle group increased the number of platelets in peripheral blood by 19.57%.

[0169] The results show that the liposome drug delivery system or micelle drug delivery system with a surface modified with a targeting peptide can carry drugs that specifically kill leukemia cells while reducing damage to normal red blood cells and platelets and reducing toxic side effects.

[0170] In summary, the present invention provides a polypeptide that specifically binds to the MUC1 protein, a targeted drug-loaded long-circulating liposome, and a targeted drug-loaded micelle, which can accurately kill tumor cells that highly express MUC1 without damaging normal cells, can be used for the preparation of anti-tumor drugs, and has great practical significance for the clinical treatment of tumors. The anti-tumor drugs contained in the targeted drug-loaded long-circulating liposome and the targeted drug-loaded micelle are replaceable and have a wide range of uses.

[0171] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention. SEQUENCE LISTING <110> National Center for Nanoscience and Technology Institute of Basic Medicine, Chinese Academy of Medical Sciences <120> A polypeptide specifically binding to MUC1 protein, a targeted drug co-delivery system, and preparation method and application thereof <130> 2021-11-23 <160> 5 <170> PatentIn version 3.3 <210> 1 <211> 30 <212> PRT <213> Synthesis <400> 1 Asp Arg Val Arg Thr Gln Phe Asn Gln Tyr Asp Thr Arg Ala Ala Ser 1 5 10 15 Asp Asp Gly Gly Asp Gly Val Thr Ser Ala Pro Arg Thr Asp 20 25 30 <210> 2 <211> 37 <212> PRT <213> Synthesis <400> 2 Asp Arg Arg Thr Ile Asn Val Asp Arg Val Arg Thr Gln Phe Asn Gln 1 5 10 15 Tyr Asp Thr Arg Ala Ala Ser Asp Asp Gly Gly Asp Gly Val Thr Ser 20 25 30 Ala Pro Arg Thr Asp 35 <210> 3 <211> 30 <212> PRT <213> Synthetic <400> 3 Gly Gly Asp Arg Arg Thr Ile Asn Val Asp Arg Val Arg Thr Gln Phe 1 5 10 15 Asn Gln Tyr Asp Thr Arg Ala Ala Ser Asp Asp Gly Gly Cys 20 25 30 <210> 4 <211> 30 <212> PRT <213> Synthetic <400> 4 Asp Arg Val Arg Thr Gln Phe Asn Gln Tyr Asp Thr Arg Ala Ala Ser 1 5 10 15 Asp Asp Gly Gly Asp Asp Asp Thr Ser Ala Pro Arg Thr Asp 20 25 30 <210> 5 <211> 30 <212> PRT <213> Synthetic <400> 5 Asp Arg Val Arg Thr Gln Phe Asn Gln Tyr Asp Thr Arg Ala Ala Ser 1 5 10 15 Asp Tyr Arg Gly Asp Asp Asp Thr Ser Ala Pro Arg Thr Asp 20 25 30

Claims

1. A polypeptide that specifically binds to MUC1 protein, characterized in that: The amino acid sequence of the polypeptide that specifically binds to the MUC1 protein is any one of the sequences shown in SEQ ID NOs: 1-5.

2. The method for preparing a polypeptide that specifically binds to MUC1 protein according to claim 1, characterized in that: The preparation method comprises a solid phase polypeptide synthesis method.

3. A targeted drug-loaded long-circulating liposome that specifically binds to MUC1 protein, characterized in that: The raw materials for preparing the targeted drug-loaded long-circulating liposomes that specifically bind to the MUC1 protein include phospholipids, cholesterol, amphiphilic polymers, polypeptide-amphiphilic polymers and active pharmaceutical ingredients; The amphiphilic polymer includes DSPE-PEG and / or DSPE-mPEG, the polypeptide-amphiphilic polymer includes DSPE-PEG-polypeptide and / or DSPE-mPEG-polypeptide; the amino acid sequence of the polypeptide is the sequence shown in SEQ ID NO: 1; and the pharmaceutically active ingredient includes homoharringtonine.

4. The targeted drug-loaded long-circulating liposome specifically binding to MUC1 protein according to claim 3, characterized in that: The molar ratio of the phospholipid, cholesterol, amphiphilic polymer, polypeptide-amphiphilic polymer and pharmaceutically active component is 1:(0.01-5):(0.001-5):(0.001-1):(0.001-1).

5. The targeted drug-loaded long-circulating liposome specifically binding to MUC1 protein according to claim 4, characterized in that: The molar ratio of the phospholipid, cholesterol, amphiphilic polymer, polypeptide-amphiphilic polymer and pharmaceutically active component is 1:(0.01-1):(0.01-1):(0.001-0.5):(0.01-0.5).

6. The targeted drug-loaded long-circulating liposome specifically binding to MUC1 protein according to claim 3, characterized in that: The phospholipids include any one of soybean lecithin, hydrogenated soybean lecithin or egg yolk lecithin, or a combination of at least two of them.

7. The targeted drug-loaded long-circulating liposome specifically binding to MUC1 protein according to claim 6, characterized in that: The phospholipid is soybean lecithin.

8. The method for preparing targeted drug-loaded long-circulating liposomes that specifically bind to MUC1 protein according to any one of claims 3 to 7, characterized in that: The preparation method adopts a thin film-ultrasound method, which specifically comprises the following steps: (1) mixing phospholipids, cholesterol, an amphiphilic polymer, a polypeptide-amphiphilic polymer, a pharmaceutically active component, and an organic solvent to obtain a first mixed solution; (2) evaporating and removing the organic solvent in the first mixed solution to obtain a thin film; (3) Mixing the film obtained in step (2) with water to obtain a second mixed solution, and ultrasonicating to obtain the targeted drug-loaded long-circulating liposomes.

9. The method for preparing the targeted drug-loaded long-circulating liposomes specifically binding to MUC1 protein according to claim 8, characterized in that: The organic solvent includes any one of anhydrous ethanol, chloroform, ether or methanol, or a combination of at least two thereof.

10. The method for preparing the targeted drug-loaded long-circulating liposomes specifically binding to MUC1 protein according to claim 9, characterized in that: The organic solvent is anhydrous ethanol.

11. The method for preparing targeted drug-loaded long-circulating liposomes specifically binding to MUC1 protein according to claim 8, characterized in that: The frequency of the ultrasound is 100-500W.

12. The method for preparing targeted drug-loaded long-circulating liposomes that specifically bind to MUC1 protein according to claim 11, characterized in that: The frequency of the ultrasound is 100-200W.

13. The method for preparing targeted drug-loaded long-circulating liposomes that specifically bind to MUC1 protein according to claim 8, characterized in that: The number of cycles of the ultrasound is 5-30 times.

14. The method for preparing targeted drug-loaded long-circulating liposomes that specifically bind to MUC1 protein according to claim 13, characterized in that: The number of cycles of the ultrasound is 5-10 times.

15. The method for preparing targeted drug-loaded long-circulating liposomes specifically binding to MUC1 protein according to claim 8, characterized in that: The ultrasound duration is 3-10 s, and the ultrasound interval is 2-20 s.

16. The method for preparing targeted drug-loaded long-circulating liposomes that specifically bind to MUC1 protein according to claim 15, characterized in that: The ultrasound interval is 2-10 s.

17. A targeted drug-loaded micelle that specifically binds to MUC1 protein, characterized in that: The raw materials for preparing the targeted drug-loaded micelles that specifically bind to the MUC1 protein include amphiphilic polymers, polypeptide-amphiphilic polymers and active pharmaceutical ingredients; The amphiphilic polymer includes DSPE-PEG and / or DSPE-mPEG, the polypeptide-amphiphilic polymer includes DSPE-PEG-polypeptide and / or DSPE-mPEG-polypeptide; the amino acid sequence of the polypeptide is the sequence shown in SEQ ID NO: 1; and the pharmaceutically active ingredient includes homoharringtonine.

18. The targeted drug-loaded micelle specifically binding to MUC1 protein according to claim 17, characterized in that: The molar ratio of the amphiphilic polymer, the polypeptide-amphiphilic polymer and the pharmaceutically active component is 1:(0.001-10):(0.01-30).

19. The targeted drug-loaded micelle specifically binding to MUC1 protein according to claim 18, characterized in that: The molar ratio of the amphiphilic polymer, the polypeptide-amphiphilic polymer and the pharmaceutically active component is 1:(0.1-1):(0.1-10).

20. The method for preparing the targeted drug-loaded micelles specifically binding to MUC1 protein according to any one of claims 17 to 19, characterized in that: The preparation method adopts a thin film-hydration method, which specifically comprises the following steps: (1) mixing an amphiphilic polymer, a polypeptide-amphiphilic polymer, a pharmaceutically active component, and an organic solvent to obtain a first mixed solution; (2) evaporating and removing the organic solvent in the first mixed solution to obtain a thin film; (3) Mixing the film obtained in step (2) with water to obtain a second mixed solution, ultrasonicating, bathing, and standing to obtain the targeted drug-loaded micelles.

21. The method for preparing targeted drug-loaded micelles that specifically bind to MUC1 protein according to claim 20, characterized in that: The organic solvent includes any one of anhydrous ethanol, chloroform, ether or methanol, or a combination of at least two thereof.

22. The method for preparing targeted drug-loaded micelles that specifically bind to MUC1 protein according to claim 21, characterized in that: The organic solvent is anhydrous ethanol.

23. The method for preparing targeted drug-loaded micelles that specifically bind to MUC1 protein according to claim 20, characterized in that: The frequency of the ultrasound is 80-120 W, and the time of the ultrasound is 15-25 min.

24. The method for preparing targeted drug-loaded micelles specifically binding to MUC1 protein according to claim 20, characterized in that: The temperature of the water bath is 20-65°C.

25. The method for preparing targeted drug-loaded micelles that specifically bind to MUC1 protein according to claim 24, characterized in that: The temperature of the water bath was 50°C.

26. The method for preparing targeted drug-loaded micelles that specifically bind to MUC1 protein according to claim 20, characterized in that: The water bath time is 25-40 min.

27. The method for preparing targeted drug-loaded micelles that specifically bind to MUC1 protein according to claim 20, characterized in that: The standing time is 1-3 h.

28. Use of the polypeptide specifically binding to MUC1 protein according to claim 1, the targeted drug-loaded long-circulating liposome specifically binding to MUC1 protein according to any one of claims 3 to 7, or the targeted drug-loaded micelle specifically binding to MUC1 protein according to any one of claims 17 to 19 in the preparation of anti-acute myeloid leukemia drugs.

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