An EGFR targeting polypeptide and its use in preparing a targeted drug delivery system for treating diseases with abnormal EGFR expression

Abstract

The present invention provides an EGFR targeting polypeptide and its use in preparing a targeted drug delivery system for treating diseases with abnormal EGFR expression, belonging to the pharmaceutical field. The amino acid sequence of the EGFR targeting polypeptide (named NPTE-1) is shown in SEQ ID NO:1. The NPTE-1 polypeptide has a strong affinity specificity with the EGFR on the cell surface, and can be used to prepare a probe for detecting diseases with abnormal EGFR expression, and can also be used as an EGFR targeting ligand to achieve a targeted effect on tumor cells with abnormal EGFR expression. The present invention further provides a multifunctional targeting polypeptide (named EMC) having an amino acid sequence as shown in SEQ ID NO:2. The drug-loaded liposome modified with the EMC polypeptide of the present invention can effectively improve the killing effect of anticancer drugs on tumor cells, reverse the drug resistance of doxorubicin-resistant tumors, significantly inhibit tumor growth, and prolong the survival of tumor-bearing mice, and has broad application prospects in the preparation of a targeted drug delivery system for treating diseases with abnormal EGFR expression.

Description

Technical Field

[0001] The present invention belongs to the pharmaceutical field, and specifically relates to an EGFR targeting polypeptide and use thereof in preparing a targeted drug delivery system for treating diseases with abnormal EGFR expression. Background Art

[0002] At present, tumors have become one of the most important factors threatening human health. Among them, breast cancer is the most common malignant tumor in women. Its morbidity and mortality rate rank first among various tumors, which poses a huge risk to women's health. Triple negative breast cancer (TNBC) is a subtype of breast cancer, which has the characteristics of high malignancy, rapid pathological progression, poor prognosis, strong invasiveness and easy recurrence and metastasis. Due to its lack of expression of human epidermal growth factor receptor type 2 (HER2), estrogen receptor (ER) and progesterone receptor (PR), it lacks sensitivity to targeted drugs such as trastuzumab, tamoxifen and Herceptin. Therefore, broad-spectrum chemotherapy drugs are currently important clinical drugs for the treatment of TNBC. However, the development of tumor cell resistance to chemotherapy drugs is often an important factor in the failure of TNBC treatment, which increases the risk of death in patients. Therefore, it is necessary to explore new treatment options to improve the therapeutic effect of anti-tumor drugs and reverse the drug resistance of cancer cells.

[0003] In order to improve the therapeutic effect of tumors, prolong the survival of tumor patients, and improve the quality of life of patients, people have established a variety of treatment methods including surgery, radiotherapy, chemotherapy, immunotherapy, photothermal and photodynamic therapy. Among them, chemotherapy, as an economical and effective treatment method, is still an important means of systemic treatment and postoperative adjuvant treatment of malignant tumors. However, the chemotherapy drugs currently used in clinical practice have more or less disadvantages such as poor water solubility, short circulation time in the body, poor targeting, drug resistance and systemic toxicity, which has caused the dilemma of limited clinical application scope of chemotherapy drugs. In recent years, changing the clinical dosage form of chemical drugs or introducing nanodrug carriers to overcome the shortcomings of chemotherapy drugs has pointed out the direction for the expansion of their clinical application. For example, paclitaxel injection ( Paclitaxel injection has become the first-line and second-line treatment for ovarian cancer, breast cancer and non-small cell lung cancer, but this type of dosage form still has typical problems such as poor stability, injection pain, acute allergic reaction (Allergy Asthma Immunol Res 2016, 8: 174-177), neurotoxicity (Clin Breast Cancer 2011, 11: 73-81) and hyperlipidemia (J Clin Oncol 2014, 32: 573-573). Compared with fat emulsion dosage forms, nano drug carriers have shown great advantages and application potential. For example, the FDA-approved albumin paclitaxel preparation Abraxane has the advantages of less toxic side effects, short administration time and low adverse reactions (Drugs 2017, 74: 1757-1768). However, this dosage form is limited in clinical application due to limited albumin sources, microbial contamination risks and high costs. Among the numerous nano dosage forms, liposomes have received extensive attention and application in the fields of biomedicine and tumor treatment. By 2000, the sales of liposome products worldwide reached 1.2 billion U.S. dollars, and increased to 3.3 billion U.S. dollars by 2005. So far, liposomes such as amphotericin, doxorubicin, and daunorubicin have entered clinical trials, and more than 30 liposome anticancer drugs are in the clinical trial stage or waiting for approval for listing, further indicating the application prospects of liposome nano drugs in the field of anticancer. However, liposome drugs themselves also have some problems, such as lack of targeting, poor permeability to solid tumors, etc., which limit the therapeutic effect of liposome drugs. Therefore, developing new liposome drugs and improving their targeting and permeability to tumors have broad application prospects.

[0004] At present, clinical liposomes are mostly prepared by phospholipids, cholesterol and PEGylated phospholipids, and liposomes lack targeting to tumors or other lesions, so that liposome drugs are inevitably enriched in normal tissues and organs, causing unnecessary toxic side effects. To improve the targeted enrichment effect of lipids on tumors, people have further modified liposomes, and the targeting ligands of tumor-related markers are connected to the liposome surface, such as antibodies, folic acid and hyaluronic acid molecules and polypeptides, etc., to improve the targeting of liposomes in tumor lesions. However, in view of the high mesenchymal pressure (Nat Rev Cancer 2006, 6: 583-592) inside solid tumors, anticancer drugs are significantly inhibited from arriving inside tumors, reducing the therapeutic effect of solid tumors, for this reason, researchers have modified tissue penetration molecules to liposome surfaces, such as TAT ​​polypeptide molecules, but these molecules lack the targeting specificity to target tissues or organs, aggravating their toxicity to normal tissues. Simultaneously modifying the liposome surface with targeting ligands and molecules with permeability can obtain liposome drugs with tissue targeting and permeability. However, the increase in surface modifications increases the steric hindrance of the liposome surface. The effect of the linking ratio of the two molecules on the therapeutic effect of liposome drugs is also an important issue. At the same time, multi-molecular modification of the liposome surface also increases the difficulty of quality control of liposome drugs and increases their production costs.

[0005] Therefore, it is of great clinical significance to construct a targeted drug delivery system that has both targeted penetration capability and the ability to reverse tumor resistance using a simple, easy and low-cost method. Summary of the invention

[0006] The first object of the present invention is to provide a new EGFR targeting polypeptide: NPTE-1; the second object of the present invention is to provide a new multifunctional targeting peptide based on NPTE-1: EMC; the third object of the present invention is to provide a multifunctional targeted drug delivery system modified by a targeting polypeptide.

[0007] The present invention provides a polypeptide, whose amino acid sequence is shown in SEQ ID NO:1.

[0008] The present invention also provides another polypeptide, whose amino acid sequence is shown in SEQ ID NO:2.

[0009] The present invention also provides the use of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 in preparing a detection probe for a disease with abnormal EGFR expression or a targeted drug delivery system for treating a disease with abnormal EGFR expression.

[0010] Furthermore, the disease with abnormal EGFR expression is head and neck tumor, renal cancer, breast cancer, prostate cancer, non-small cell lung cancer, colon cancer, ovarian cancer, bladder cancer, glioma, pancreatic cancer, esophageal cancer, gastric cancer, uterine body cancer or cervical cancer.

[0011] Furthermore, the breast cancer is triple-negative breast cancer.

[0012] Furthermore, the triple-negative breast cancer is drug-resistant triple-negative breast cancer, preferably doxorubicin-resistant triple-negative breast cancer.

[0013] The present invention also provides a targeted drug delivery system, which is prepared using a polypeptide having an amino acid sequence such as SEQ ID NO: 1 or SEQ ID NO: 2, a drug carrier and an active drug ingredient as raw materials.

[0014] Furthermore, it is prepared with lipid materials and active pharmaceutical ingredients as raw materials, wherein the lipid materials include phospholipids and / or phospholipid derivatives modified with a polypeptide having an amino acid sequence such as SEQ ID NO: 1 or SEQ ID NO: 2.

[0015] Further, the active pharmaceutical ingredients are chemotherapeutic drugs and P-gp inhibitors;

[0016] The lipid material is composed of phospholipids and / or phospholipid derivatives, phospholipids and / or phospholipid derivatives modified by a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0017] Further, the chemotherapy drug is one or more of the following drugs or their salts: nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, glyphosate mustard, doxiflurane, docefloxacin, 5-fluorouracil, mercaptopurine, thioguanine, fluguanidine, tegafur, gemcitabine, carmofur, hydroxyurea, methotrexate, ancitabine, actinomycin D, doxorubicin, daunorubicin, epirubicin, mitomycin, pelocybin, flat The invention relates to phenytoin, pirarubicin, irinotecan, harringtonine, hydroxycamptothecin, vinorelbine, paclitaxel, taxotere, topotecan, docetaxel, vincristine, camptothecin, hydroxycamptothecin, vindesine, vincamide, vinblastine, teniposide, etoposide, elemene, atamestane, anastrozole, tamoxifen, carboplatin, cisplatin, oxaliplatin, oxaliplatin, oxaliplatin, mitoxantrone; preferably, the chemotherapy drug is doxorubicin or a salt thereof;

[0018] The P-gp inhibitor is 2-aldehyde-4-methoxyphenylboronic acid;

[0019] The phospholipid derivative is one or more of maleimide-modified phospholipids, polyethylene glycol phospholipids, and maleimide-modified polyethylene glycol phospholipids;

[0020] The phospholipids are one or more of dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylglycerol (DMPG), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), soybean lecithin, hydrogenated soybean lecithin, distearoylphosphatidylethanolamine (DSPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylglycerol (DOPG), and dieucoylphosphatidylcholine (DEPC), preferably dioleoylphosphatidylcholine and distearoylphosphatidylethanolamine.

[0021] Furthermore, the mass ratio of the chemotherapeutic drug to the P-gp inhibitor is (1-20):(1-20), preferably 1:1;

[0022] In the lipid material, the molar number of phospholipids and / or phospholipid derivatives modified with the polypeptide having the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 accounts for 1%-20% of the total molar number of the lipid material, preferably 5%.

[0023] The present invention also provides a method for preparing the above-mentioned targeted drug delivery system, the method comprising the following steps:

[0024] (1) using a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, a phospholipid and / or a phospholipid derivative as a raw material to obtain a phospholipid and / or a phospholipid derivative modified with a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2;

[0025] (2) dissolving the phospholipid and / or phospholipid derivative modified with the polypeptide having the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 and the remaining lipid material in an organic solvent and mixing them to obtain a mixed solution;

[0026] (3) adding an organic solvent solution of a P-gp inhibitor to the mixed solution, mixing the mixture evenly, and evaporating the mixture under vacuum to obtain a thin film;

[0027] (4) adding an aqueous solution of a chemotherapeutic drug into the film to hydrate the film, and extruding the film through an extruder to obtain a targeted drug delivery system; the chemotherapeutic drug is a water-soluble drug;

[0028] Or, the method comprises the following steps:

[0029] (1') using a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, a phospholipid and / or a phospholipid derivative as a raw material to obtain a phospholipid and / or a phospholipid derivative modified with a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2;

[0030] (2′) dissolving a phospholipid and / or a phospholipid derivative modified with a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 and the remaining lipid material in an organic solvent and mixing them to obtain a mixed solution;

[0031] (3') adding an organic solvent solution of a P-gp inhibitor and a chemotherapeutic drug to the mixed solution, mixing them evenly, and vacuum evaporating them to form a film to obtain a thin film; the chemotherapeutic drug is a water-insoluble drug;

[0032] (4') Deionized water is added into the film to hydrate the film, and the film is extruded through an extruder to obtain a targeted drug delivery system.

[0033] Furthermore, in step (1) or step (1'), the phospholipid and / or phospholipid derivative is distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide; the molar ratio of the polypeptide having an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2 to the phospholipid and / or phospholipid derivative is (0.5-3): 1, preferably 1.5:1;

[0034] In step (2) or (2'), the remaining lipid material is dioleoylphosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol 2000; the molar ratio of dioleoylphosphatidylcholine to distearoylphosphatidylethanolamine-polyethylene glycol 2000 is (0.5-20):(0.5-20), preferably 18:1; the organic solvent is a mixture of dichloromethane and methanol;

[0035] In step (3) or (3'), the organic solvent is dimethyl sulfoxide.

[0036] In the present invention, polyethylene glycol phospholipid refers to a phospholipid modified with polyethylene glycol.

[0037] In the polyethylene glycol phospholipids and maleimide-modified polyethylene glycol phospholipids, the molecular weight of polyethylene glycol is 500-10000, preferably 1000-3400, and more preferably 2000.

[0038] The structural formula of doxorubicin hydrochloride is shown in formula (1):

[0039] The structural formula of Tariquidar is shown in formula (2):

[0040] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0041] 1. The present invention provides a novel EGFR targeting polypeptide: NPTE-1, which has strong affinity and specificity with EGFR on the cell surface. The NPTE-1 polypeptide can be used to prepare detection probes for tumors with abnormal EGFR expression (such as head and neck tumors, kidney cancer, breast cancer, prostate cancer, non-small cell lung cancer, colon cancer, ovarian cancer, bladder cancer, glioma, pancreatic cancer, esophageal cancer, gastric cancer, uterine body cancer, cervical cancer), and can also be used as an EGFR targeting ligand to achieve targeting effects on tumor cells with abnormal EGFR expression.

[0042] 2. Based on the NPTE-1 polypeptide, the present invention further synthesizes an EMC polypeptide that has EGFR targeting and can respond to changes in the tumor microenvironment. The EMC polypeptide consists of three parts: an NPTE-1 polypeptide fragment, a PLGVRG fragment, and a polyarginine fragment with tissue penetration and cell membrane penetration. The EMC polypeptide has both the NPTE-1 functional sequence and the polyarginine functional sequence, which are connected by the MMP-2 substrate PLGVRG fragment, so that the EMC polypeptide-modified nanoliposome drug can respond to MMP-2 in the tumor microenvironment to unload the NPTE-1 fragment, exposing the polyarginine fragment therein, thereby improving the cell / tissue penetration ability of the nanoliposome drug and promoting the penetration of the nanoliposome drug into tumor tissues and cells.

[0043] 3. The present invention uses EMC polypeptide as a targeting polypeptide to modify the surface of liposomes, realizing the composite function of drug targeted delivery and tissue / cell penetration. In the EMC polypeptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes provided by the present invention, the EMC polypeptide can increase the accumulation of liposomes in tumor lesions through targeting and the permeation effect of responding to MMP-2. The liposomes taken up by tumor cells quickly release drugs under the action of weakly acidic conditions in endosomes or lysosomes. At the same time, tariquidar in the liposomes inhibits the efflux of P-gp on drugs, effectively improving the killing effect of anticancer drugs on tumor cells, reversing the drug resistance of doxorubicin-resistant TNBC, significantly inhibiting tumor growth, and prolonging the survival of tumor-bearing mice. The preparation method of the EMC polypeptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes of the present invention is simple, low-cost, and has broad application prospects.

[0044] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0045] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 .The affinity of NPTE-1 peptide to EGFR on the surface of MDA-MB-231, HBMECs, and MCF-7 cells was measured by flow cytometry.

[0047] Figure 2 .The results of the affinity test between NPTE-1 peptide and EGFR by SPRi, K D =9.84×10 -9 M.

[0048] Figure 3 .The results of the affinity and specificity of NPTE-1 peptide to EGFR detected by SPRi. Among them, **** means P < 0.0001.

[0049] Figure 4 .Mass spectrometry results of EMC peptide.

[0050] Figure 5 .HPLC results of EMC peptide and MMP-2 and mass spectrometry results of corresponding components.

[0051] Figure 6 .DSPE-PEG 2000 -Mal and DSPE-PEG 2000 -EMC mass spectrometry results.

[0052] Figure 7 .TEM and DLS results of EMC peptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes.

[0053] Figure 8 .Zeta potential results of EMC peptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes before and after MMP-2 treatment.

[0054] Fig. 9 .Drug release results of EMC peptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes under different pH conditions.

[0055] Fig.10 .The results of the cytotoxicity of doxorubicin to MDA-MB-231 and MDA-MB-231 / ADR.

[0056] Fig.11.The cytotoxicity results of free doxorubicin, doxorubicin + tariquidar, doxorubicin liposomes, co-loaded doxorubicin / tariquidar liposomes, EMC peptide-modified doxorubicin liposomes, and EMC peptide-modified co-loaded doxorubicin / tariquidar liposomes on MDA-MB-231 / ADR.

[0057] Fig.12 .Results of apoptosis of MDA-MB-231 / ADR cells induced by free doxorubicin, doxorubicin + Tariquidar, co-loaded doxorubicin / tariquidar liposomes, and EMC peptide-modified co-loaded doxorubicin / tariquidar liposomes.

[0058] Fig.13 .MDA-MB-231 / ADR uptake results of free doxorubicin, doxorubicin + Tariquidar, doxorubicin / tariquidar co-loaded liposomes and EMC peptide-modified doxorubicin / tariquidar co-loaded liposomes. * indicates P < 0.05, ** indicates P < 0.01.

[0059] Fig.14 .The results of the changes in tumor volume during the treatment of TNBC mice with normal saline, free doxorubicin, doxorubicin + Tariquidar, doxorubicin / tariquidar co-loaded liposomes, and EMC peptide-modified doxorubicin / tariquidar co-loaded liposomes. *** indicates P < 0.001.

[0060] Fig.15 .Graph showing the changes in survival of TNBC mice after treatment with normal saline, free doxorubicin, doxorubicin + Tariquidar, doxorubicin / tariquidar co-loaded liposomes, and EMC peptide-modified doxorubicin / tariquidar co-loaded liposomes. DETAILED DESCRIPTION

[0061] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0062] Example 1: Synthesis of targeting peptide NPTE-1

[0063] The amino acid sequence of NPTE-1 is shown in SEQ ID NO:1.

[0064] According to the amino acid sequence, NPTE-1 is directly synthesized using conventional methods in the art.

[0065] Example 2: Synthesis of targeting peptide EMC

[0066] The amino acid sequence of EMC is shown in SEQ ID NO:2.

[0067] According to the amino acid sequence, MC is directly synthesized using conventional methods in the art.

[0068] Table 1 Peptide names and sequences

[0069] Peptide name Sequence number Amino acid sequence NPTE-1 SEQ ID NO:1 FDFRYYWRY EMC SEQ ID NO:2 FDFRYYWRYPLGVRGRRRRRRRRC

[0070] Example 3: Construction of EMC polypeptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes

[0071] 1. Preparation of EMC polypeptide functionalized polyethylene glycol phospholipids: DSPE-PEG 2000 -EMC

[0072] Distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG 2000 -Mal) and EMC polypeptide, respectively, DSPE-PEG 2000 -Mal and EMC peptide were dissolved in DMSO to prepare a solution with a concentration of 2 mg / mL. The two dissolved solutions were mixed evenly and reacted at room temperature in the dark for 48 h. Then, DSPE-PEG was dialyzed with a dialysis bag with a molecular cutoff of 3400 Da to obtain 2000 -EMC, freeze-dried for later use.

[0073] 2. Preparation of EMC peptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes

[0074] 10 mg of dioleoylphosphatidylcholine (DOPC) was added to a 50 mL round-bottom flask, and then DOPC, distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000 ), DSPE-PEG 2000 Weigh DSPE-PEG at a molar ratio of 9:0.5:0.5 2000 and DSPE-PEG 2000 -EMC was added to the flask, and 20 mL of dichloromethane-methanol mixture (V 二氯甲烷 : V 甲醇 =1:1) to dissolve it completely.

[0075] 2-Formyl-4-methoxyphenylboronic acid (tariquidar) was dissolved in DMSO to prepare a 20 mg / mL solution. 75 μL of tariquidar solution (containing 1.5 mg tariquidar) was added to the above solution and mixed evenly. The solution was evaporated under reduced pressure at 37° C. for 6 h using a rotary evaporator to form a thin film.

[0076] Doxorubicin hydrochloride was dissolved in ultrapure water to prepare a solution with a concentration of 1 mg / mL, 1.5 mL of doxorubicin hydrochloride solution (containing 1.5 mg of doxorubicin hydrochloride) was added to the above film, and the film was hydrated by water bath ultrasound for 30 min, and then repeatedly extruded 11 times through a 100 nm micro-liposome extruder, dialyzed overnight to remove unloaded doxorubicin, and concentrated by 100 KDa ultrafiltration to obtain EMC polypeptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes.

[0077] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0078] Experimental Example 1: Specific affinity of peptide NPTE-1 to cell surface EGFR

[0079] 1. Experimental methods

[0080] Synthesize NPTE-1 polypeptide, extend an arginine in the C segment, and then couple FITC to the side chain of the arginine to obtain FITC-labeled NPTE-1 polypeptide (FITC-NPTE-1) as shown in formula (3);

[0081]

[0082] The above-mentioned FITC-NPTE-1 polypeptide was prepared into solutions with concentrations of 0, 0.3125, 0.625, 1.25, 2.5, 5, 10, and 20 μM using 1×PBS, and then incubated with MDA-MB-231 (EGFR overexpression), HBMEC (EGFR medium expression) and MCF-7 (EGFR negative) cells for 1 h, and the affinity of the polypeptide to the cell surface EGFR was detected by flow cytometry.

[0083] 2. Experimental results

[0084] Depend on Figure 1 It can be seen that the NPTE-1 polypeptide has a higher affinity with the MDA-MB-231 cells that overexpress EGFR, but has no specific binding effect with the EGFR-negative MCF-7 cells, indicating that the NPTE-1 polypeptide has a specific affinity with the EGFR on the cell surface.

[0085] Experimental Example 2: Affinity and specificity of peptide NPTE-1 and cell surface EGFR

[0086] 1. Experimental methods

[0087] Synthesize NPTE-1 polypeptide, and extend a cysteine ​​in the C segment to obtain NPTE-1-Cys polypeptide shown in formula (4);

[0088]

[0089] The above-mentioned NPTE-1-Cys polypeptide was prepared into a 1 mg / mL solution with ultrapure water, and then the NPTE-1-Cys solution or the same volume of ultrapure water was spotted on the chip, incubated overnight under humid conditions at 4°C, and then washed twice with 10×PBS, 1×PBS and deionized water in sequence, each time for 10 min. After washing, the chip was immersed in 5% milk and blocked overnight, and further washed twice with 10×PBS, 1×PBS and deionized water, each time for 10 min. After drying with nitrogen gas, the chip was loaded into a biomacromolecule interaction analyzer (model: Biacore X100, GE, USA);

[0090] 1×PBS was used to prepare EGFR protein solutions with concentrations of 5.68nM, 11.4nM, 22.8nM, 45.6nM and 91.2nM. Then 1×PBS and the EGFR protein of the above series of concentrations were flowed through the microchannel of the SPRi chip, and the SPRi signal was recorded and analyzed.

[0091] EGFR, HER2, HER3, HSA, IgG and IgM were further prepared into a 45.6 nM solution using 1×PBS, and then passed through the SPRi chip microchannel in sequence to record and analyze the SPRi signal.

[0092] 2. Experimental results

[0093] Depend on Figure 2 It can be seen that the SPRi signal of NPTE-1 polypeptide gradually increases with the increase of protein concentration, indicating that the NPTE-1 polypeptide of the present invention has a strong binding effect with EGFR, and its affinity dissociation constant (K D ) reaches 9.84×10 -8 M, close to the affinity of the antibody. Figure 3 It can be seen that the binding signals of NPTE-1 polypeptide with HER2, HER3, HSA, IgG and IgM are very weak, which further indicates that NPTE-1 polypeptide has strong affinity specificity with EGFR.

[0094] The above results indicate that the NPTE-1 polypeptide of the present invention can be used as a probe molecule targeting EGFR, and can be used for the detection and targeted treatment of diseases with abnormal EGFR expression.

[0095] Experimental Example 3: Cleavage of EMC by MMP-2

[0096] 1. Experimental methods

[0097] The EMC peptide was synthesized and its molecular weight was characterized by MALDI-TOF-MS technique.

[0098] The EMC polypeptide was prepared into a solution with a concentration of 1 mg / mL using 1×PBS, and then matrix metalloproteinase 2 (MMP-2) was added at a final concentration of 1 μM. After incubation at 37°C for 6 hours, the solution was passed through an ultrafiltration tube with a molecular cutoff of 10KD, the filtrate was collected, and the shearing effect of MMP-2 on the polypeptide was analyzed by HPLC and mass spectrometry.

[0099] 2. Experimental results

[0100] Depend on Figure 4 It can be seen that the molecular weight of EMC polypeptide is 3347.2Da; Figure 5 It can be seen that the EMC polypeptide can be cleaved by the MMP-2 polypeptide into two fragments with molecular weights of 1682.8 and 1683.8 Da, indicating that the EMC polypeptide can be effectively cleaved from PLGVRG by MMP-2.

[0101] Experimental Example 4: Structural characterization of EMC peptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes

[0102] 1. Experimental methods

[0103] The DSPE-PEG synthesized in step 1 of Example 3 was analyzed by MALDI-TOF-MS. 2000 -EMC characterization.

[0104] The EMC polypeptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes synthesized in step 1 of Example 3 were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM).

[0105] 2. Experimental results

[0106] Depend on Figure 6 It can be seen that DSPE-PEG 2000 The molecular weight of -Mal is about 2877Da, and DSPE-PEG after linking EMC 2000 -EMC molecular weight increased to 6238Da, indicating that the present invention successfully coupled the EMC polypeptide to DSPE-PEG 2000 -Mal.

[0107] Depend on Figure 7 It can be seen that the EMC polypeptide modified multifunctional doxorubicin-tariquidar co-loaded liposomes present a monodisperse spherical structure with a particle size of 136.18 nm and a PDI of 0.121.

[0108] Experimental Example 5: Response of EMC peptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes to MMP-2

[0109] 1. Experimental methods

[0110] The EMC polypeptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes synthesized in Example 3 were dispersed in 1×PBS at pH 7.0, and MMP-2 protein was added to make the final concentration of 1 μM. After incubation at 37°C for 2 h, the liposomes were collected by an ultrafiltration tube with a molecular cutoff of 100 KD. After dispersion, the surface potential of the liposomes before and after the action of MMP-2 was analyzed by Zeta potential.

[0111] 2. Experimental results

[0112] Figure 8 The results showed that after 2 hours of MMP-2 action, the surface potential of the liposomes increased from -7.8±1.4mV to 2.47±1.36mV. It is speculated that this may be related to the exposure of polyarginine, indicating that the EMC polypeptide on the surface of the liposomes can unload the EGFR targeting fragment under the action of MMP-2.

[0113] Experimental Example 6: Drug release study of EMC peptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes

[0114] 1. Experimental methods

[0115] The EMC polypeptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes synthesized in Example 3 were dispersed in 1×PBS containing 0.1% Tween-20, the final concentration of doxorubicin was 200 μM, and the pH of PBS was 5.5 and 7.4, respectively. The above liposomes were transferred to a dialysis bag with a molecular cutoff of 3400 Da, and then immersed in 50 mL of the corresponding PBS buffer, and incubated in a constant temperature shaker at 37° C. and 100 rpm. 1 mL of the solution outside the dialysis bag was taken at 0, 1, 2, 4, 6, 8, 10, 12, 24, 36, and 48 h, and the same volume of the corresponding buffer was added. The released doxorubicin and Tariquidar were analyzed by a fluorescence spectrophotometer and a high performance liquid chromatograph, respectively.

[0116] 2. Experimental results

[0117] Depend on Fig. 9 It can be seen that under the condition of pH 5.5, doxorubicin and tariquidar can be quickly released from the liposomes, indicating that the EMC polypeptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes of the present invention have the function of releasing drugs in response to the weakly acidic microenvironment of the tumor.

[0118] Experimental Example 7: In vitro study on reversal of tumor drug resistance by EMC peptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes

[0119] 1. Experimental methods

[0120] The doxorubicin-resistant TNBC cells MDA-MB-231 (MDA-MB-231 / ADR for short) used in this experimental example were induced with a culture medium containing doxorubicin. The concentration of doxorubicin was gradually increased from 0.2 μg / mL to 4 μg / mL. Each concentration gradient induced the cells for 3-4 weeks until the cells were able to grow stably.

[0121] MDA-MB-231 / ADR cells were seeded into 96-well plates. After 24 hours, the drugs to be tested were added. The concentrations of doxorubicin were 0, 0.01, 0.05, 0.25, 1.25, 6.25, 31.25, and 156.25 μM, respectively. After incubation at 37°C for 48 hours, the cell activity was detected using a CCK-8 kit. According to the grouping, the drugs to be tested were: (1) free doxorubicin; (2) doxorubicin + tariquidar; (3) doxorubicin liposomes; (4) unmodified EMC peptide-loaded doxorubicin / tariquidar liposomes; (5) EMC peptide-modified doxorubicin liposomes; (6) EMC peptide-modified doxorubicin / tariquidar liposomes.

[0122] MDA-MB-231 / ADR cells were inoculated in 6-well plates and cultured at 37°C for 24 hours. The drugs to be tested were added, and the concentration of doxorubicin was 5 μM. After incubation at 37°C overnight, the cells were collected and the apoptosis of MDA-MB-231 / ADR cells was evaluated using the V-FITC / 7-AAD kit. According to the grouping, the drugs to be tested were: (1) free doxorubicin; (2) doxorubicin + Tariquidar, (3) unmodified EMC peptide co-loaded doxorubicin / Tariquidar liposomes; (4) EMC peptide modified co-loaded doxorubicin / Tariquidar liposomes.

[0123] The above-mentioned EMC polypeptide-modified doxorubicin / Tariquidar co-loaded liposomes are the EMC polypeptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes prepared in Example 3.

[0124] 2. Experimental results

[0125] Depend on Fig.10 It can be seen that the method of the present invention for inducing drug resistance of MDA-MB-231 cells by using a culture medium containing doxorubicin successfully obtains doxorubicin-resistant MDA-MB-231 cells.

[0126] Depend on Fig.11It can be seen that compared with the free drug group, the liposome drug group without EMC peptide modification and the doxorubicin liposome group modified with EMC peptide, the killing effect of the multifunctional doxorubicin-tariquidar co-loaded liposome modified with EMC peptide on MDA-MB-231 / ADR was significantly improved; Fig.12 It can be seen that compared with the free drug group and the liposome drug group without EMC peptide modification, the EMC peptide-modified liposomes significantly enhanced the doxorubicin-mediated apoptosis of MDA-MB-231 / ADR cells.

[0127] Experimental Example 8: In vitro study of the targeted permeability of EMC peptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes

[0128] 1. Experimental methods

[0129] MDA-MB-231 / ADR cells were seeded into 6-well plates and cultured at 37°C for 24 hours. The following drugs were added to the cells: (1) free doxorubicin; (2) doxorubicin + tariquidar; (3) unmodified EMC peptide co-loaded doxorubicin / tariquidar liposomes; (4) EMC peptide modified multifunctional doxorubicin-tariquidar co-loaded liposomes; (5) EMC peptide modified multifunctional doxorubicin-tariquidar co-loaded liposomes + MMP-2 protein. The final concentration of doxorubicin in each control group was 10 μM, and the final concentration of MMP-2 protein in group (5) was 100 nM. After incubation at 37°C for another 4 hours, the cells were digested and collected, and the uptake of doxorubicin by MDA-MB-231 / ADR was detected by flow cytometry.

[0130] The EMC polypeptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes were prepared in Example 3.

[0131] 2. Experimental results

[0132] Depend on Fig.13 It can be seen that the multifunctional doxorubicin-tariquidar co-loaded liposomes modified with EMC peptide can significantly increase the drug uptake by MDA-MB-231 / ADR cells; in addition, under the action of MMP-2, the drug uptake by MDA-MB-231 / ADR cells is further increased, which is closely related to the combined effect of the polyarginine permeability function exposed after the EGFR fragment is sheared and unloaded by MMP-2 and tariquidar inhibiting the efflux of doxorubicin by P-glycoprotein (P-gp).

[0133] Experimental Example 9: In vivo study on reversal of tumor resistance by EMC peptide-modified multifunctional doxorubicin-tariquidar co-loaded liposomes

[0134] 1. Experimental methods

[0135] MDA-MB-231 / ADR cells were inoculated into the left flank mammary fat pad of BALB / C nude immunodeficient mice at a tumor cell inoculation density of 2 million and an inoculation volume of 50 μL. 3 The drug to be tested was injected into mice through the tail vein. The dose of doxorubicin was 5 mg / kg and the drug was administered every 2 days for 5 times. The tumor volume of the tumor-bearing mice was recorded every other day, and the changes in the survival period of the tumor-bearing mice were recorded.

[0136] According to the grouping, the drugs to be tested are: (1) free doxorubicin; (2) doxorubicin + tariquidar, (3) unmodified EMC polypeptide co-loaded doxorubicin / tariquidar liposomes; (4) EMC polypeptide modified co-loaded doxorubicin / tariquidar liposomes. In addition, physiological saline without any drug was used as a blank control. The above-mentioned EMC polypeptide modified co-loaded doxorubicin / tariquidar liposomes are the EMC polypeptide modified multifunctional doxorubicin-tariquidar co-loaded liposomes prepared in Example 3.

[0137] 2. Experimental results

[0138] Depend on Fig.14 and Fig.15 It can be seen that the EMC polypeptide-modified co-loaded doxorubicin / tariquidar liposomes of the present invention can significantly inhibit tumor growth in tumor-bearing mice and significantly prolong the survival of tumor-bearing mice. This is due to the targeting effect of the EMC polypeptide in the EMC polypeptide-modified co-loaded doxorubicin / tariquidar liposomes and the permeation enhancement effect in response to MMP-2.

[0139] In summary, the present invention provides an EGFR targeting polypeptide: NPTE-1, which has a strong affinity and specificity with the EGFR on the cell surface, can be used to prepare detection probes for diseases with abnormal EGFR expression, and can also be used as an EGFR targeting ligand to achieve targeted effects on tumor cells with abnormal EGFR expression. The present invention further provides a multifunctional targeting polypeptide: EMC, whose amino acid sequence is shown in SEQ ID NO: 2. The co-loaded doxorubicin / tariquidar liposomes modified with the EMC polypeptide of the present invention can effectively improve the killing effect of anticancer drugs on tumor cells, reverse the drug resistance of doxorubicin-resistant tumors, significantly inhibit tumor growth, and prolong the survival of tumor-bearing mice, and have broad application prospects in the preparation of targeted drug delivery systems for the treatment of diseases with abnormal EGFR expression. SEQUENCE LISTING <110> West China Hospital, Sichuan University <120> An EGFR targeting polypeptide and its use in preparing a targeted drug delivery system for treating diseases with abnormal EGFR expression way <130> GYKH2105-2021P0114413CC <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Artificial sequence <400> 1 Phe Asp Phe Arg Tyr Tyr Trp Arg Tyr 1 5 <210> 2 <211> twenty four <212> PRT <213> Artificial sequence <400> 2 Phe Asp Phe Arg Tyr Tyr Trp Arg Tyr Pro Leu Gly Val Arg Gly Arg 1 5 10 15 Arg Arg Arg Arg Arg Arg Arg Cys 20

Claims

1. A polypeptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

2. A polypeptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

2.

3. Use of the polypeptide according to claim 1 or 2 in the preparation of a detection probe for a disease with abnormal EGFR expression or a targeted drug delivery system for treating a disease with abnormal EGFR expression, wherein the disease with abnormal EGFR expression is head and neck tumors, renal cancer, breast cancer, prostate cancer, non-small cell lung cancer, colon cancer, ovarian cancer, bladder cancer, glioma, pancreatic cancer, esophageal cancer, gastric cancer, uterine body cancer or cervical cancer.

4. The use according to claim 3, characterized in that: The breast cancer is triple-negative breast cancer.

5. The use according to claim 4, characterized in that: The triple-negative breast cancer is drug-resistant triple-negative breast cancer.

6. The use according to claim 5, characterized in that: The drug-resistant triple-negative breast cancer is doxorubicin-resistant triple-negative breast cancer.

7. A targeted drug delivery system, characterized in that: The drug is prepared using the polypeptide described in claim 1 or 2, a drug carrier and active drug ingredients as raw materials.

8. The targeted drug delivery system according to claim 7, characterized in that: The invention is prepared by taking lipid material and active pharmaceutical ingredients as raw materials. The lipid material comprises the phospholipid modified by the polypeptide according to claim 1 or 2 and / or the derivative of the phospholipid.

9. The targeted drug delivery system according to claim 8, characterized in that: The active pharmaceutical ingredients are chemotherapeutic drugs and P-gp inhibitors; The lipid material consists of phospholipids and / or phospholipid derivatives modified by the polypeptide according to claim 1 or 2, phospholipids and / or phospholipid derivatives.

10. The targeted drug delivery system according to claim 9, characterized in that: The chemotherapy drug is one or more of the following drugs or their salts: nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, glyphosate mustard, doxorubicin, docefloxacin, 5-fluorouracil, mercaptopurine, thioguanine, fluguanidine, tegafur, gemcitabine, carmofur, hydroxyurea, methotrexate, ancitabine, actinomycin D, doxorubicin, daunorubicin, epirubicin, mitomycin, peplomycin, bleomycin, pirarubicin, irinotecan, harringtonine, hydroxycamptothecin, vinorelbine, paclitaxel, taxotere, topotecan, vincristine, camptothecin, hydroxycamptothecin, vindesine, vinamide, vinblastine, teniposide, etoposide, elemene, atamestane, anastrozole, tamoxifen, carboplatin, cisplatin, oxaliplatin, mitoxantrone; The P-gp inhibitor is 2-aldehyde-4-methoxyphenylboronic acid; The phospholipid derivative is one or more of maleimide-modified phospholipids, polyethylene glycol phospholipids, and maleimide-modified polyethylene glycol phospholipids; The phospholipid is one or more of dipalmitoylphosphatidylcholine, dimyristoylphosphatidylglycerol, dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine, soybean lecithin, hydrogenated soybean lecithin, distearoylphosphatidylethanolamine, distearoylphosphatidylcholine, dioleoylphosphatidylglycerol, and dierucoylphosphatidylcholine.

11. The targeted drug delivery system according to claim 10, characterized in that: The chemotherapeutic drug is doxorubicin or its salt; The phospholipids are dioleoylphosphatidylcholine and distearoylphosphatidylethanolamine.

12. The targeted drug delivery system according to claim 10, characterized in that: The mass ratio of the chemotherapeutic drug to the P-gp inhibitor is (1-20): (1-20); In the lipid material, the molar number of the phospholipid and / or phospholipid derivative modified by the polypeptide according to claim 1 or 2 accounts for 1%-20% of the total molar number of the lipid material.

13. The targeted drug delivery system according to claim 12, characterized in that: The mass ratio of the chemotherapy drug to the P-gp inhibitor is 1:1; In the lipid material, the molar number of the phospholipid and / or phospholipid derivative modified by the polypeptide according to claim 1 or 2 accounts for 5% of the total molar number of the lipid material.

14. A method for preparing the targeted drug delivery system according to any one of claims 7 to 13, characterized in that: The method comprises the following steps: (1) Using the polypeptide, phospholipid and / or phospholipid derivative of claim 1 or 2 as raw materials to obtain the phospholipid and / or phospholipid derivative modified with the polypeptide of claim 1 or 2; (2) dissolving the polypeptide-modified phospholipid and / or phospholipid derivative according to claim 1 or 2 and the remaining lipid material in an organic solvent and mixing them to obtain a mixed solution; (3) adding an organic solvent solution of a P-gp inhibitor to the mixed solution, mixing the mixture evenly, and evaporating the mixture under vacuum to obtain a thin film; (4) adding an aqueous solution of a chemotherapeutic drug into the film to hydrate the film, and extruding the film through an extruder to obtain a targeted drug delivery system; the chemotherapeutic drug is a water-soluble drug; Or, the method comprises the following steps: (1') using the polypeptide, phospholipid and / or phospholipid derivative according to claim 1 or 2 as raw materials to obtain the phospholipid and / or phospholipid derivative modified with the polypeptide according to claim 1 or 2; (2') dissolving the polypeptide-modified phospholipid and / or phospholipid derivative according to claim 1 or 2 and the remaining lipid material in an organic solvent and mixing them to obtain a mixed solution; (3') adding an organic solvent solution of a P-gp inhibitor and a chemotherapeutic drug to the mixed solution, mixing them evenly, and vacuum evaporating them to form a film to obtain a thin film; the chemotherapeutic drug is a water-insoluble drug; (4') Deionized water is added into the film to hydrate the film, and the film is extruded through an extruder to obtain a targeted drug delivery system.

15. The method according to claim 14, characterized in that: In step (1) or step (1'), the phospholipid and / or phospholipid derivative is distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide; The molar ratio of the polypeptide according to claim 1 or 2 to the phospholipid and / or the phospholipid derivative is (0.5-3):1; In step (2) or (2'), the remaining lipid material is dioleoylphosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol 2000; the molar ratio of dioleoylphosphatidylcholine to distearoylphosphatidylethanolamine-polyethylene glycol 2000 is (0.5-20):(0.5-20); the organic solvent is a mixture of dichloromethane and methanol; In step (3) or (3'), the organic solvent is dimethyl sulfoxide.

16. The method according to claim 15, characterized in that: In step (1) or step (1'), the molar ratio of the polypeptide according to claim 1 or 2 to the phospholipid and / or the phospholipid derivative is 1.5:1; In step (2) or (2'), the molar ratio of dioleoylphosphatidylcholine to distearoylphosphatidylethanolamine-polyethylene glycol 2000 is 18:1.

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