CN114702564A - Exosome peptide and preparation method and application thereof - Google Patents

Abstract

The invention discloses an exosome peptide, which takes an exosome as a carrier and comprises a target FKBPL polypeptide, wherein the target FKBPL polypeptide consists of 24 amino acids, and the amino acid sequence of the target FKBPL polypeptide is QIRQQPRDPPTETLELEVSPDPAS. Preparing a sequence containing an FKBPL gene or a gene fragment encoding a target FKBPL polypeptide and cloning the sequence into a plasmid vector, transfecting a processing cell with the plasmid vector, and collecting the processing cell expressing the target FKBPL polypeptide after culturing; the exosome is obtained by naturally secreting the processing cell, or the exosome of the processing cell is prepared by adopting an extrusion method to obtain the exosome peptide. The exosome peptide is an exosome loading polypeptide, and is a novel drug loading system for treating triple negative breast cancer.

Description

Exosome peptide and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, relates to a drug loading system for treating triple negative breast cancer, and particularly relates to an exosome peptide and a preparation method and application thereof.

Background

In recent years, breast cancer has become the highest incidence and second-most mortality cancer in women worldwide over lung cancer. The triple negative breast cancer is a special subtype high invasive breast cancer, and has the characteristics of poor curative effect of conventional treatment, poor prognosis of patients, high relapse drug resistance rate and low overall survival rate. Currently, surgery, chemotherapy, radiation therapy, endocrine therapy, molecular targeted therapy and immunotherapy remain the most common treatment regimens for breast cancer. Among them, neoadjuvant chemotherapy has become one of the standard treatment regimens for triple negative breast cancer. The breast cancer chemotherapy drugs commonly used in clinic comprise micromolecules and proteins, and the action mechanism of the breast cancer chemotherapy drugs is to interfere the biosynthesis and the function of nucleic acid and protein, so that the proliferation of tumor cells is inhibited, and the apoptosis of the cells is induced. Wherein, the common small molecule drugs for treating the breast cancer, such as tamoxifen, exemestane, paclitaxel and the like, can specifically block signal transduction pathways necessary in the growth and proliferation processes of the tumor. The protein drugs mainly refer to antibody drugs, such as trastuzumab and lapatinib, which enter tumor cells by combining with target cell antigens and release cytotoxic drugs to kill the cells after being cracked. However, adverse reactions and breast cancer resistance rates continue to increase due to widespread drug combinations and dysregulation of the breast cancer cell cycle. Hormone receptors of triple negative breast cancer and human epidermal growth factor receptor-2 (HER 2) genes are negative, and conventional endocrine and targeted therapy is still indiscriminate, so that a new safe and effective therapeutic drug needs to be discovered.

Compared with small molecular drugs, the polypeptide drug has higher affinity and specificity to target tumors, has lower toxic and side effects, and can increase the sensitivity of tumors to other treatment methods. Compared with antibody drugs, the polypeptide drugs have small volume, are easier to permeate tissues and are easier to modify. The polypeptide medicine mainly plays a role by inducing apoptosis and necrosis of tumor cells, inhibiting tumor angiogenesis, activating anti-tumor immune response and other mechanisms. In 2018, the first drug 177 Lu-dotate, a chelate of somatostatin analogues and radionuclides, was approved by the U.S. Food and Drug Administration (FDA) for the treatment of gastroenteropancreatic neuroendocrine tumor patients, via polypeptide receptor-mediated radionuclide-targeted therapy (PRRT). Melflufen (Melphalan Flufenamide, also known as Pepaxto) is the first anti-cancer peptide conjugate approved by the U.S. Food and Drug Administration (FDA) and used to treat adult relapsed/refractory multiple myeloma patients. FK506-binding protein-like protein (FKBPL), a member of the immunoaffinity protein family, is a potent secreted antiangiogenic protein. The highly potent antiangiogenic effect of FKBPL is due to the inhibition of angiogenesis by a CD 44-dependent mechanism by a unique sequence within the N-terminal region, namely the 24 peptide of FKBPL amino acids 34-58 (McClements L, Yakkundi A, Papaspyropoulos A, Harrison H, Ablett MP, Juthsh PV, et al.targeting therapeutic-resistant bacterial cells with FKBPL and peptides derivative, AD-01, via the CD44 pathway. Clin cancer.2013; 19: 3881-20193.). Meanwhile, FKBPL is also a prognostic biomarker of breast cancer, and can effectively predict the sensitivity of endocrine treatment of the breast cancer.

Although the polypeptide medicament has remarkable curative effect in the anti-tumor process, the polypeptide medicament cannot stably play a role due to the defects of easy protease hydrolysis, poor physicochemical stability, short in-vivo half-life, low oral bioavailability and the like. Therefore, polypeptide drugs need to be delivered to triple negative breast cancer cells by means of drug carriers, so as to kill tumor cells stably, quasi and strongly.

The exosome is an extracellular vesicle which is secreted by cells and has the diameter of 100-1000 nm, and originates from the outward budding and fission of a cell membrane to wrap rich contents such as RNA, protein, lipid and the like. The exosome not only can be used as a marker for tumor diagnosis, but also can be used as an important carrier of tumor treatment drugs. The microvesicles can be loaded with modified RNA, chemotherapeutic drugs and the like, are delivered to breast cancer cells in a targeted manner, improve the killing efficiency on tumor cells, reduce the damage on other normal tissue cells, and show high-efficiency and specific anti-tumor characteristics. Genetic modification of exosomes results in multivalent antibodies that redirect exosomes, recognizing CD36 and HER2, and thus activating cytotoxic T cells, specifically attacking HER2 positive Breast Cancer cells (Shi X, Cheng Q, Hou T, Han M, Smbatan G, Lang JE, et al. genetic engineering Cell-Derived Nanoparticles for Targeted Breast Cancer immunology. molecular therapy: the journal of the American Society of Gene therapy.2020; 28: 536-47.). Multiple studies have shown that exosome-loaded polypeptides have a good therapeutic effect in animal models of various diseases, such as the mouse model of muscular dystrophy (Gao X, Ran N, Dong X, Zuo B, Yang R, Zhou Q, et al. Anchor peptide peptides, targets, and adhesives assays for diagnosis and therapy.2018; 10.), the rat model of intracranial glioblastoma (Kim G, Kim M, Lee Y, Byun JW, Hwang DW, Lee M.M. delivery System of microRNA-21antisense oligonucleotides to the repair T38-peptide modified oligonucleotides, J. reaction.2020; 317: 273-81) and the mouse model (shake X, osteoarthritis, shake X. repair. K, tissue K. of osteoarthritis, et al. repair. 10. animal model, and 10. animal model of cartilage peptides, tissue reaction.J. Has no obvious toxic or side effect while obtaining good curative effect. Compared with liposome, the exosome can avoid the difficulties of immune recognition, inflammatory toxicity, rapid elimination and the like of other biological materials due to the endogenesis and high biocompatibility of the exosome.

Disclosure of Invention

The invention aims to provide a novel drug-loading system for treating triple negative breast cancer so as to improve the bioavailability and targeting property of the drug, and the drug-loading system takes an anti-angiogenesis polypeptide FKBPL as an anti-cancer drug and takes an exosome as a carrier to carry an anti-tumor active polypeptide naturally secreted by cells, so that the drug-loading system named as an exosome peptide for treating triple negative breast cancer is constructed.

Another object of the present invention is to provide a method for preparing the exosome peptide.

The invention also aims to provide application of the exosome peptide in preparation of a medicine for treating triple negative breast cancer.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

an exosome peptide, characterized in that an exosome is a vector, which comprises a desired FKBPL polypeptide consisting of 24 amino acids whose amino acid sequence is QIRQQPRDPPTETLELEVSPDPAS.

The preparation method of the exosome peptide comprises the following steps:

1) preparing a sequence of an FKBPL gene or gene fragment comprising a sequence encoding an FKBPL polypeptide of interest and cloning into a plasmid vector;

2) transfecting a processing cell by using the plasmid vector, and collecting the processing cell expressed by the target FKBPL polypeptide after culturing;

3) the exosome is obtained by naturally secreting the processing cell, or the exosome of the processing cell is prepared by adopting an extrusion method to obtain the exosome peptide.

Step 1), the FKBPL gene (NCBI no: 63943) Is derived from human F-box protein 31.

In step 1), the plasmid vector contains full-length FKBPL gene or FKBPL gene fragment, and the full-length FKBPL gene or FKBPL gene fragment can be expressed in a processing cell to obtain the target FKBPL polypeptide with 24 amino acids. The amino acid sequence of the full-length FKBPL gene code is shown in Table 1, the length of the polypeptide is 1-349 th amino acid, and the cloned plasmid vector is marked as FK 1. The amino acid sequence of the FKBPL gene segment code is shown in Table 2, the length of the polypeptide is 1-85 amino acids, and the cloned plasmid vector is marked as FK 2; or the amino acid sequence coded by FKBPL gene segment is shown in Table 3, the length of the polypeptide is 1-150 amino acids, and the cloned plasmid vector is marked as FK 3. The plasmid vector is preferably FK2, wherein the FKBPL gene fragment encodes amino acids 1-85 of FKBPL polypeptide.

In step 2), 293T cells or immune cells commonly used for obtaining exosomes can be used as the processing cells, and the cells include NK cells, T cells, macrophages, DC cells and the like. Preferably, the processing cell is 293T cell, and the production of the FKBPL polypeptide of interest is higher after transfection with FK1, FK2 or FK3 plasmid vector.

In the step 3), the natural secretion of the processing cell refers to that the exosome is obtained by collecting a culture solution of the processing cell after the plasmid vector is transfected to the processing cell and performing centrifugal purification. Specifically, FK1, FK2 or FK3 plasmid vectors were transformed into cells, exosome-free serum was replaced after 12 hours, and then cell culture fluid was collected and fresh exosome-free culture fluid was added at 24, 48 and 72 hours, respectively; and (3) centrifuging the culture solution at a high speed, and collecting exosomes naturally secreted by the processed cells.

In step 3), extrusion is preferably used. The extrusion method is that after the plasmid vector is transfected to the processing cell, the processing cell is collected and the cell suspension is extruded by a micro extruder, and the cell filtrate is centrifugally purified to obtain the exosome which is several times of the naturally secreted exosome. Specifically, FK1, FK2 or FK3 plasmid vectors were transformed into processed cells, exosome-free sera were replaced after 12 hours and processed cells were collected after 48 hours. The cells are then continuously extruded through polycarbonate filters (e.g., 10 μm, 5 μm and 1 μm), and the resulting cell filtrate is centrifuged at high speed and the exosomes are collected.

The invention also relates to application of the exosome peptide in preparation of a medicine for treating triple negative breast cancer.

The exosome peptide can be taken by triple-negative breast cancer cells in a large quantity, reduces the expression levels of DLL4 and CD44 in the triple-negative breast cancer cells, interferes with the dryness maintenance of tumor cells, and destroys the tube forming capability of endothelial cells so as to resist the generation of tumor neovascularization.

Has the advantages that: although the polypeptide medicament has obvious curative effect in the process of resisting tumors, the polypeptide medicament can not stably play a role due to the defects of easy hydrolysis by protease, poor physicochemical stability, short in-vivo half-life, low oral bioavailability and the like. The exosome peptide takes the anti-angiogenesis polypeptide FKBPL as an anti-cancer drug and an exosome as a carrier, constructs a drug-loading system which is naturally secreted by cells and carries the anti-tumor polypeptide drug, and improves the bioavailability and targeting property of the drug. The FKBPL polypeptide with the amino acid length suitable for cell autocrine is selected and cloned into a vector, so that the cell can stably produce the target anti-tumor active polypeptide without being degraded by intracellular enzymes. The FKBPL exosome peptide shows better cell entering capability and has good anti-angiogenesis and anti-cancer effects. The exosome peptide provided by the invention can be used as a drug loading system, can be used for treating triple negative breast cancer, has a good application prospect, and is expected to promote the development and clinical application transformation of polypeptide drugs.

Drawings

FIG. 1 is a schematic flow diagram of the preparation of an exosome peptide of the present invention;

FIG. 2 construction and cellular expression of FKBPL polypeptide plasmid vectors, (A) FKBPL plasmid structure maps; (B) sequencing peak diagram of FKBPL plasmid; (C) enzymatic cleavage of FKBPL plasmid; (D) western blot analysis of FKBPL;

FIG. 3 preparation of exosome peptides, (A) representative TEM image of exosome peptides, with 500nm scale; (B) particle size distribution of exosome peptides; (C) western blot verification of exosome marker proteins CD9 and CD 63;

fig. 4 identification of target FKBPL polypeptide in exosome peptide, (a) liquid phase high resolution high precision mass spectrometry (LC-HRMS) analysis profile; (B) FKBPL polypeptide content of exosome peptides, wherein the number of exosome peptides of a-f is 1000, 2000, 4000, 8000, 16000, 32000 respectively;

figure 5 anticancer effect of exosome peptides, (a) representative confocal picture of the uptake of exosome peptides by HCC1937 cells; (B) HCC1937 cells DLL4 and CD44 expression; (C) representative photographs of endothelial cell looping experiments, wherein a is a control group, b and c are exosome peptide-treated groups containing 100nM and 500nM FKBPL short peptides, respectively;

FIG. 6 shows the near-infrared fluorescence imaging photograph of mouse tumor with the multimode nano-contrast agent, and the excitation wavelength is 780 nm.

Detailed Description

The technical solutions of the present invention are further described in detail with reference to the following embodiments, but it should be noted that the following embodiments are only used for describing the content of the present invention and do not limit the scope of the present invention.

The following examples describe the exosome peptides of the present invention and their preparation method, and their results of qualitative and quantitative studies as drug-loading systems for triple negative breast cancer treatment.

The main reagents and materials for implementing the invention comprise human kidney epithelial cells 293T cells, purchased from China Shanghai Chinese academy of sciences cell banks, cultured in DMEM medium containing 10% fetal calf serum and 1% penicillin-streptomycin. Human umbilical vein endothelial cell HUVEC cell and human triple negative breast cancer cell HCC1937 cell, purchased from Shanghai Zhongji cell bank, China, were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 1% penicillin-streptomycin. DMEM medium, RPMI-1640 medium, trypsin, Phosphate Buffered Saline (PBS), and paraformaldehyde were purchased from Kyoho Kay Biotechnology Ltd. Fetal Bovine Serum (FBS) was purchased from Gibco, usa.

Example 1 preparation of exosomes

The exosome peptide of the invention takes exosome of processing cells as a carrier, and comprises FKBPL gene target short peptide generated by the transfection of the processing cells, wherein the target short peptide comprises 24 amino acids, namely amino acids 34-58 of FKBPL protein, and the amino acid sequence of the target short peptide is QIRQQPRDPPTETLELEVSPDPAS.

The preparation process of the exosome peptide of the present invention is shown in figure 1, and the exosome peptide is prepared by firstly carrying out FKBPL transfection on tool cells, collecting the cells after 48 hours, and carrying out a mechanical extrusion method. The method specifically comprises the following steps:

1) construction of vectors expressing FKBPL Polypeptides

The human FKBPL gene is cloned into a plasmid vector PCDNA3.1 through Kpn1/BamH1 double enzyme digestion reaction to construct an FKBPL gene target short peptide expression vector.

Designing enzyme cutting sites at the whole length of the target gene or two ends of the target gene according to the enzyme cutting sites of the plasmid vector PCDNA3.1, and carrying out whole gene synthesis. Subsequently, the plasmid vector PCDNA3.1 and the synthesized target gene were reacted at 37 ℃ for 1 hour in an enzyme digestion reaction system containing 16. mu.L (1. mu.g) of the vector plasmid/target gene, 2. mu.L of 10 Xbuffer Cut S, 1. mu.L (15U) of EcoR I and 1. mu.L (15U) of Not I, respectively, and the vector and the target gene fragment were recovered by cutting the gel. After the vector DNA fragment after the enzyme digestion is subjected to gel cutting and purification, a T4 ligase reaction solution system containing 1 mu L of 10 XT 4 DNA ligase Buffer, 3 mu L of the vector fragment after the enzyme digestion, 5.5 mu L of the target gene fragment after the enzyme digestion and 0.5 mu L T4 DNA ligase is established with the target gene fragment, and the reaction is carried out for 1h at 37 ℃. Subsequently, the glycerol cryopreserved Stbl3 strain was inoculated into a streak plate and cultured overnight at 37 ℃ in an inverted state. Picking the monoclonal into a test tube containing 3mL LB, shaking at 37 ℃ and 220rpm for 12h, sucking 1mL of the bacterial liquid into a 1.5mL centrifuge tube, centrifuging at 4 ℃ and 12000g for 3min, discarding the supernatant, and adding 400 μ L of precooled CaCl₂Resuspending the pellet, centrifuging at 12000g for 3min, discarding the supernatant, adding 200. mu.L of precooled CaCl₂Resuspend the pellet again and place on ice overnight. The above recombined 20. mu.L reaction solution was added to 200. mu.L competent bacteria, and placed on ice for 1h, heat-shocked at 42 ℃ for 90sec, quickly placed on ice for 5min, 600. mu.L LB medium preheated at 37 ℃ was added, shaken at 37 ℃ for 1h at 220rpm, centrifuged, and then applied to LB plate containing 100. mu.g/mL Amp, and cultured overnight at 37 ℃ in an inverted manner. Randomly pick 4 monoclonals into a tube containing 3mL LB culture solution of 100. mu.g/mL Amp, shake at 37 ℃ for 4h at 220rpm, take 100. mu.L for centrifugation, take the cell pellet, use 50. mu.L ddH₂And (3) resuspending, carrying out boiling water bath for 5min, centrifuging, taking 1 mu L of supernatant as a template, carrying out PCR identification, taking 10 mu L of supernatant after the reaction is finished, carrying out 1.0% Agarose electrophoresis identification, and carrying out sequencing verification on clone identified as positive by colony PCR. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 120s, 25 cycles, final extension at 72 ℃ for 3min, and reaction at 4 ℃.

Finally, 3 plasmid vectors which can correctly express 24 amino acid FKBPL polypeptides of interest by transfected processed cells are obtained by screening, and are named as FK1, FK2 and FK3 plasmids.

2) Cellular expression of FKBPL polypeptides

The above three plasmids, FK1, FK2 and FK3, were used to transfect human embryonic kidney cell line 293T. First, 293T cells were seeded into 6-well plates, and when the cell confluence reached 70% -80%, plasmid transfection was performed according to the kit procedure of Lipo3000 transfection reagent (Thermo Fisher Science, USA), the transfection system included well mixed 125. mu.L of Opti-MEM medium and 3.75. mu.L of Lipo3000 reagent (A tube); mu.L of Opti-MEM medium, 2.5. mu.g of plasmid DNA (plasmid containing protein of interest/corresponding empty plasmid) and 5. mu. L P3000 of reagent (tube B) were mixed well. And adding the content in the tube B into the tube A, gently mixing uniformly, incubating at room temperature for 10min, dropwise adding into a six-well plate containing 1.75mL of serum-free and antibody-free culture medium, and replacing 2mL of complete culture medium after 6h of transfection to continue culturing for 48 h. Subsequently, the cells were collected, lysed by adding tissue lysate (RIPA, pica, china) and protease inhibitor (PMSF, solibao, china), and then antibody against FKBPL polyclonal antibody (Proteintech, china) was added to verify the polypeptide expression level of 293T cells after plasmid transfection by Western blot. The images were collected using an Image Lab and analyzed with corresponding adjustments using a BIO-RAD exposure apparatus (ChemiDOCTM XRS +, BioRAD, USA).

As shown in FIG. 2A, the target gene sequence fragment is cloned into PCDNA3.1 plasmid vector by enzyme digestion reaction, and the target short peptide expression vector of FKBPL gene is successfully constructed. The sequencing results in FIG. 2B show that three gene fragments of the full-length FKBPL gene (FK1), 85 amino acids (FK2) and 150 amino acid gene fragment (FK3) were correctly cloned into the PCDNA3.1 plasmid expression vector via Kpn1/BamH1 cleavage sites. FIG. 2C shows agarose gel electrophoresis experiments, which shows that target gene fragments with different sizes are obtained by Kpn1/BamH1 double digestion. The construction of plasmid expression vector is the precondition of the expression of FKBPL gene target short peptide, and the constructed FK1, FK2 and FK3 three kinds of plasmid vectors can be successfully expressed to obtain the target short peptide.

The expression level of the FKBPL gene in 293T cells transfected by the three plasmids was detected by Western blot, and the results showed that the full-length FKBPL gene (FK1) was successfully expressed, and FK2 and FK3 were short peptide chains and could not be detected by Western blot, so the bands were not significantly different from the control group (fig. 2D), but mass spectrometry showed that the desired FKBPL polypeptide was successfully expressed and contained in higher amount after 293T cells were transfected by FK2 and FK3 plasmids as compared with 24 chemically synthesized FKBPL polypeptide standards (see example 3).

3) Preparation of exosome peptides

FKBPL exosome peptide preparation was performed using 293T cells transfected with the FKBPL polypeptide gene. The transfected 293T cells were harvested and all cells were placed at 37 ℃ in 5% CO₂At saturation humidity. When the cells reach 70-80% fusion, the old culture medium is discarded, a proper amount of PBS is added for rinsing, the old culture medium is discarded, and the steps are repeated twice. Digesting cells with pancreatin containing EDTA, immediately adding serum to stop digestion when cells become round and bright under microscope, collecting liquid, centrifuging at 4 deg.C at 500g for 10min, and discarding supernatant. The cell pellet was resuspended with 1mL PBS, 10 μ L of cell suspension was taken, stained with trypan blue dye, and cell counted by cell counter. Take 2X 10⁷The cells were placed in a fresh 15mL sterile tube, washed with an appropriate amount of PBS, centrifuged at 500g for 10min at 4 ℃ and repeated twice to remove residual cell debris, dead cells and cell culture medium. The cells were resuspended in sterile PBS containing protease inhibitors to a cell concentration of 5X 10⁶mL, cells should be used immediately within 1 h. Since cells contain a large amount of Protease, the homeostasis of cells is unbalanced during the extrusion process, and endogenous Protease may degrade membrane surface proteins on the exosomes of cell membranes, we added 1 × Protease inhibitor (Protease inhibitor cocktail, 04693132001, Roche, switzerland) to PBS.

10 μm, 5 μm and 1 μm polycarbonate membrane filtration membranes (Nuclecore; Whatman Inc., USA) were taken and cells were continuously extruded through the filtration membranes of different pore sizes. First, a 10 μm membrane was placed in a micro-extruder (Avanti Polar lipids, uk), the prepared cell suspension was added, and extruded 5 times using the extruder. Subsequently, the collected solution was sequentially passed through 5 μm and 1 μm polycarbonate membrane filtration membranes and extruded 5 times using a micro-extruder, respectively. The liquid collected above contains cell-derived exosomes and larger cell debris, and therefore further purification is required to obtain exosomes of uniform size.

The collected exosomes were further purified by density gradient centrifugation using an ultracentrifuge. Iodixanol (OptiPrep) was used^TMSigma-Aldrich, usa) as density gradient centrifugation media, 10% and 50% iodixanol solutions were prepared, respectively. 1mL of 50% iodixanol solution, 2mL of 10% iodixanol and 7mL of collected sample were sequentially added to an ultracentrifuge tube (the sample was collected at the uppermost layer) and centrifuged at 100,000g for 2h at 4 ℃. Cell exosomes enriched between the 10% and 50% iodixanol levels were recovered and centrifuged at 100,000g for 2h at 4 ℃. The supernatant was discarded, 100. mu.L PBS was added for resuspension, and the samples were stored in a-80 ℃ freezer.

Example 2 characterization of exosomes

1. Transmission Electron Microscopy (TEM) identification of exosome morphologies

Samples with appropriate concentrations were dropped on an electron microscope copper mesh, incubated for 2min, washed with distilled water, and then negatively stained with 2% uranium acetate (SPI, usa) for 2min, and after the samples were dried, morphological characterization was performed using a transmission electron microscope (Tecnai G2 Spirit Bio Twoic, Fei, usa).

2. Nanoparticle Tracking Analyzer (NTA) for identifying exosome size

Use of

Nanoparticle tracking analyzer (

Particle metric, germany) analyzed the Particle size distribution and number of exosomes. After NTA calibration with standards, exosome samples were diluted to appropriate fold with PBS and pumped into assay. The ZetaView camera records the scatter of individual particles as a 60s video to determine average velocity and diffusivity. Finally, use

Software was used for instrument control and data analysis.

3. Western blot analysis of exosome protein markers

CD9 and CD63 were selected as surface markers for the identification of exosomes. Tissue lysate (RIPA, cloudiness, china), protease inhibitor (PMSF, solibao, china) in 100: 1: the exosome lysate is prepared according to the proportion of (1). And adding 10-20 mu L of lysis solution into the exosome precipitate collected by centrifugation, and obtaining an exosome protein sample after the exosome is fully disintegrated. Adding SDS-PAGE Loding Buffer (New Saimei, China), mixing, and denaturing at 99 deg.C for 10 min. Equal mass protein samples were added to each lane of the PAGE gel, electrophoretically separated, and transferred to a 0.22 μm PVDF membrane. After adding Anti-CD9 antibody (1:1000, ab223052, Abcam, USA) and CD63(1:1000, ab68418, Abcam, USA) and incubating overnight at 4 deg.C, horseradish peroxidase-labeled goat Anti-rabbit IgG (H + L) (Byunyan, China) was added and incubated at room temperature for 1H. The images were collected using an Image Lab and analyzed with corresponding adjustments using a BIO-RAD exposure apparatus (ChemiDOCTM XRS +, BioRAD, USA).

As shown in fig. 3, the TEM photograph of fig. 3A clearly shows the vesicle-like structure of the exosomes, whose morphology is consistent with that of the exosomes without the polypeptide loaded. The mean particle size of FKBPL exosome peptides captured by NTA was 141.16 + -4.44 nm (FIG. 3B-B), while that of exosomes not expressing FKBPL was 109.68 + -2.37 nm (FIG. 3B-a), consistent with the expected exosome characteristics. Western blot detection in fig. 3C showed that CD9 and CD63 were expressed in high abundance in the exosome extracts, while no expression was detected in the cells. These results indicate that the present invention obtains an exosome peptide with an intact exosome structure.

Example 3 cellular expression and identification of target FKBPL Polypeptides in exosome peptides

And identifying the target FKBPL polypeptide in the exosome peptide prepared from the 293T cell transfected by the plasmid by adopting a chromatography-mass spectrometry method. The standard substance used for mass spectrometric identification is a chemically synthesized 24-amino acid human endogenous FKBPL polypeptide (QIRQQPRDPPTETLELEVSPDPAS), in which the amino acid at position A is a heavy standard amino acid.

First, a quantitative internal standard FKBPL polypeptide was dissolved in a 0.1% aqueous formic acid solution (formic acid, water; V: V,1:1000) prepared in advance to give a concentration of 1 pM. An intermediate diluted solution was prepared by dilution with 0.1% formic acid solution to a final concentration of 100 fM.

Secondly, adding a proper amount of 0.1 percent pre-prepared formic acid aqueous solution into a filter membrane with 100KD and 10KD, centrifuging for 5 minutes at the temperature of 4 ℃ and at the rpm of 3000, and discarding the liquid in the sleeve. Mu.g of the target FKBPL polypeptide standard of chemical synthesis or the FKBPL-containing exosome disrupted by ultrasonication was aspirated into a 100KD ultrafiltration membrane, 380. mu.L of a 0.1% formic acid aqueous solution was added thereto, and after well mixing, the mixture was centrifuged at 13000rpm for 20 minutes at 4 ℃, and this step was repeated twice. And (3) putting the liquid in the ultrafiltration membrane sleeve into a 10KD ultrafiltration membrane, centrifuging at 13000rpm for 20 minutes at 4 ℃, adding a proper amount of 0.1% formic acid aqueous solution into the 10 kDa ultrafiltration membrane after the liquid in the 100kDa ultrafiltration membrane sleeve is used up, fully mixing uniformly, centrifuging at 13000rpm for 20 minutes at 4 ℃, and repeating the step twice. The liquid retained in the 10KD ultrafiltration membrane cartridge was transferred to an EP tube and concentrated in a vacuum concentrator. Then reconstituted with 0.1% formic acid solution to 25. mu.L. Redissolving the dry powder after concentration in the step into 25 mu L by using 0.1 percent formic acid solution; taking 10 mu L of redissolved sample and 10 mu L of internal standard intermediate diluent, wherein the final concentration of the internal standard is 50 fM/mu L, and the protein concentration is 1 mu g/mu L.

Thirdly, analyzing the obtained sample by a chromatography-mass spectrometry method. A chromatographic column: ACQUITY UPLC BEH C181.7 μm2.1 x 100mm (Waters); mobile phase a: 0.1% formic acid water (formic acid, water; V: V,1:1000), mobile phase B: 0.1% formic acid acetonitrile (formic acid, acetonitrile; V: V,1: 1000). Liquid chromatography method is 0-1.0 min, 10% phase B; 1.0-3.0 min, 10% -90% of phase B; 3.0-3.5 min, 90% phase B; 3.5-3.8 min, 90% -10% of phase B; 3.8-5.0 min, 90% -10% of phase B. The method comprises the steps of adopting positive ion pair data acquisition, electrospray voltage 5500V, the temperature of an ion transmission tube is 400 ℃, Gas1 and Gas2 are both 50arb, collision energy is both 30eV as mass spectrum conditions, and transferring b ions and y ions predicted by small peptides and internal standards to a mass spectrometer (QTRAP 6500) by using a skyline calculation method⁺SCIEX, usa) and data were collected and analyzed using Multiple Reaction Monitoring (MRM).

The specific method for detecting and calculating the content of FKBPL short peptide carried by the exosome peptide by the mass spectrometry is that a certain amount of exosome containing FKBPL short peptide is put into aqueous solution containing 1 volume percent of Triton X-100 (Biyunyan, China) and is subjected to ultrasonic disruption for 5 minutes, then formic acid solution with the total volume of 0.1 percent is added, and the mass spectrum peak intensity is detected and the content of the FKBPL short peptide is calculated according to the method. The results are shown in FIG. 4.

The method adopts a general detection method for qualitative and quantitative polypeptide, and according to the mass spectrum result of the positive synthesized re-labeled short peptide FKBPL, 5b ions and y ions with different molecular weights are analyzed in an experiment to be used as an identification internal standard substance of the FKBPL polypeptide to detect the target FKBPL polypeptide in the exosome peptide, and the method has excellent accuracy. As shown in FIG. 4A, consistent with the positive re-labeled short peptide FKBPL (Control in the figure), the characteristic peak appears at the mass spectrum retention time of 3.0s in the sample of FKBPL exosome peptide prepared from 293T cells transfected by three plasmids of FK1, FK2 and FK 3. The peak intensity of FK2 was greatest in 10000 samples of exosome peptides prepared by transfection of FK1, FK2 and FK3 plasmids. It is shown that after the FK2 plasmid is transfected into 293T cells, the content of the target FKBPL short peptide loaded by exosome is the highest. The content of FKBPL short peptide in the prepared exosome peptide is detected by a mass spectrum and internal standard method. As shown in fig. 4B, data on the content of FKBPL short peptides in increasing concentrations (1000, 2000, 4000, 8000, 16000, 32000 exosomes) of exosome peptide solutions showed that the FKBPL short peptide content increased from 55.27nM to 655.69nM and reached a plateau with increasing amounts of exosomes.

Example 4 anticancer Effect of exosome peptides on triple negative breast cancer

1. Uptake of FKBPL exosome peptides by breast cancer HCC1937 cells

The method for evaluating the uptake of FKBPL exosome peptides by HCC1937 cells was first to assay HCC1937 cells at 2X 10 per well⁵Individual cells were plated in 35mm dishes at 5% CO₂Was cultured overnight in an incubator at 37 ℃. Subsequently, a staining solution (PKH26, Sigma-aldrich, USA) was added to the FKBPL-containing exosome peptide solution, mixed well and ultracentrifuged at 100000g for 1h at 4 ℃ and washed 2 times. After resuspension, the cells were added to HCC1937 cells and incubated for 6h, and the culture dish was removed and fixed with 4% paraformaldehyde for 30 min. Adding 2mg/mL glycine (Biyunyan, Zhongzhong)Country), incubating for 5min in a shaker, adding 1mL of penetrant (Biyuntian, China) and incubating for 10min, and then adding a mixture of the penetrant and the penetrant in a volume ratio of 1:1000 DAPI staining solutions (Byunnan, China) were washed with PBS and photographed under a laser confocal microscope (LSM5 Live Carl Zeiss, Germany).

FKBPL exosome peptide and cell nucleus were stained red and blue with PKH26 staining solution and DAPI staining solution, respectively, and it was observed that exosome peptide was taken up in large amounts by HCC1937 cells, which are triple negative breast cancer cells, in a photograph taken by a laser confocal microscope (see FIG. 5A). The fact that the exosome peptide is used as a drug loading system is shown to remarkably improve the bioavailability of the FKBPL polypeptide.

2. Effect of exosome peptides on the proliferative differentiation of triple negative breast cancer

Influence of exosome peptides on proliferation and differentiation of triple negative breast cancer HCC1937 cells expression of Delta-like ligand 4(DLL4) and CD44 in HCC1937 cells on HEMA-coated culture plates was detected by flow cytometry. Specifically, low-adhesion culture dishes (NUNC, Thermo Scientific, USA) were coated with 1.2% poly-2-hydroxyethyl methacrylate (poly-HEMA, Merck, China), washed twice with PBS, and 1000 HCC1937 cells were added to each dish at 37 ℃ and 5% CO₂And culturing for 24 hours. Subsequently, exosome peptides containing FKBPL short peptides at 0, 100 and 500nM were added to the medium in the petri dish, respectively, and the culture was continued for 5 days. After the experiment was completed, the cells were digested with 1.5% trypsin and resuspended in 1ml PBS and incubated for 15min with DLL4(MA5-17068, Invitrogen, USA) and CD44 flow antibody (11-0441-82, Invitrogen, USA). Finally, the expression of DLL4 and CD44 was detected and counted by flow cytometry (BD FACS, usa).

Experimental results as shown in fig. 5B, after addition of exosome peptides equivalent to 100nM and 500nM FKBPL short peptides to HCC1937 cells, DLL4 expression of HCC1937 cells was significantly reduced to 57.4% and 25.4%, indicating that exosome peptides interfered with the maintenance of the sternness of tumor cells. At the same time, the expression of the tumor dryness marker CD44 is also significantly reduced to 32.7% and 28.4%.

The proliferation and differentiation of tumor cells are closely related to tumor angiogenesis, and the appearance of a large number of new blood vessels is always a prerequisite for malignant proliferation of tumors. The process of tumor neovascularization is often accompanied by increased expression of DLL4 and CD 44. The expression level of FKBPL protein of tumor cells is in a negative correlation with the angiogenesis of the FKBPL proteins, so that the FKBPL exosome peptide reduces the expression levels of DLL4 and CD44 of HCC1937 cells, and the exosome peptide is suggested to inhibit the angiogenesis of triple negative breast cancer.

3. HUVEC tube formation experiment

HUVEC cell tubulogenesis experiments can investigate the relationship between the exosome peptides and tumor cell angiogenesis. First, 50. mu.L of matrigel (Corning, USA) was added to each well of a 96-well plate, and incubated in a 37 ℃ cell incubator for 30min to form a gel. After adding 100. mu.L of cell suspension of LHUVEC (approximately 10000 cells) per well, exosome peptides corresponding to FKBPL short peptide contents of 0, 100 and 500nM, respectively, were added to the medium. After further incubation in the cell incubator for 24h, the cell morphology was photographed under an inverted microscope (Primovert, Carl Zeiss, germany).

Rapid growth of vascular endothelial cells is a prerequisite for angiogenesis, and HUVEC cells tend to form a circular vascular-like structure in culture plates. After 24h of culture on matrigel, HUVEC cells formed a rounded-like structure, indicating that they had good angiogenic ability (FIG. 5 Ca). Upon treatment with 100nM exosome peptide, HUVEC cells hashed lines with no loop structures present (FIG. 5 Cb). Whereas upon addition of 500nM exosome peptide, HUVEC cells aggregated in clumps and cell growth was severely affected (FIG. 5 Cc). It has been reported in the literature that the FKBPL gene affects tumor angiogenesis by affecting the Notch4 gene in the pathway. The data can verify that the FKBPL exosome peptides of the present invention have the same anti-angiogenic activity and thus have the potential to be anti-cancer by inhibiting angiogenesis.

Example 5 targeting of exosome peptides to mouse triple negative breast cancer

To label exosomes, 100 μ g of exosome peptide was mixed with 10 μ g of dye near-infrared lipid dye DiR (Invitrogen, usa) and after incubation for 1h at 37 ℃, free dye was removed by gel filtration using PBS hydrated exosome spin columns (3kDa MWCO, Invitrogen, usa). 1 female BALB/c mouse (weight 18g) of 5 weeks old was taken, right afterSubcutaneous dorsal vaccination 1X 10⁶And HCC1937 cells. Calculating the tumor size according to a formula when the tumor size reaches 100mm³In this case, the fluorescent-labeled exosome peptide (10 μ g/mouse) was injected into the tail vein, and after 24 hours, the excitation wavelength and emission wavelength were adjusted to 750nm and 780nm, respectively, in a small animal in vivo imaging system (IVIS Spectrum, PerkinElmer, usa), and the imaging effect was photographed, and as a result, as shown in fig. 6, it was found that the exosome peptide was accumulated in a large amount in the tumor site.

Comparative example

According to the restriction enzyme cutting site of the plasmid vector PCDNA3.1, FKBPL gene sequence fragments with 7 different amino acid lengths of FKBPL are cloned into the PCDNA3.1 plasmid vector to obtain 7 plasmid vectors, namely FK1 (1 st to 349 th amino acid), FK2 (1 st to 85 th amino acid), FK 3(1 st to 150 th amino acid), FK4 (1 st to 33 th amino acid), FK5 (1 st to 47 th amino acid), FK6 (1 st to 57 th amino acid) and FK7 (34 th to 57 th amino acid). And (3) transfecting the 7 plasmid vectors into a processing cell 293T for 48 hours respectively, collecting the processing cell, extracting the intracellular protein and polypeptide of the processing cell, and detecting the content of the 24-amino-acid target FKBPL polypeptide by using a mass spectrum. After alignment with the mass spectrum results of the FK bpl polypeptide standard of interest, as described above, among them FK1 (1 st to 349 amino acids), FK2 (1 st to 85 th amino acids) and FK 3(1 st to 150 th amino acids) correctly expressed the 24 amino acid FKBPL polypeptide of interest, FK4 (1 st to 33 th amino acids), FK5 (1 st to 47 th amino acids), FK6 (1 st to 57 th amino acids) and FK7 (34 th to 57 th amino acids) did not find expression of the bpfkl polypeptide of interest.

TABLE 1 amino acid sequence of FK1 plasmid

METPPVNTIGEKDTSQPQQEWEKNLRENLDSVIQIRQQPRDPPTETLELEVSPDPASQILEHTQGAEKLVAELEGDSHKSHGSTSQMPEALQASDLWYCPDGSFVKKIVIRGHGLDKPKLGSCCRVLALGFPFGSGPPEGWTELTMGVGPWREETWGELIEKCLESMCQGEEAELQLPGHSGPPVRLTLASFTQGRDSWELETSEKEALAREERARGTELFRAGNPEGAARCYGRALRLLLTLPPPGPPERTVLHANLAACQLLLGQPQLAAQSCDRVLEREPGHLKALYRRGVAQAALGNLEKATADLKKVLAIDPKNRAAQEELGKVVIQGKNQDAGLAQGLRKMFG

TABLE 2 amino acid sequence of FK2 plasmid

METPPVNTIGEKDTSQPQQEWEKNLRENLDSVIQIRQQPRDPPTETLELEVSPDPASQILEHTQGAEKLVAELEGDSHKSHGSTS

TABLE 3 amino acid sequence of FK3 plasmid

METPPVNTIGEKDTSQPQQEWEKNLRENLDSVIQIRQQPRDPPTETLELEVSPDPASQILEHTQGAEKLVAELEGDSHKSHGSTSQMPEALQASDLWYCPDGSFVKKIVIRGHGLDKPKLGSCCRVLALGFPFGSGPPEGWTELTMGVGP

Sequence listing

<110> Jiangsu province national hospital (the first subsidiary hospital of Nanjing medical university)

<120> exosome peptide and preparation method and application thereof

<130> XSQ-hzh(21)048

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Met Glu Thr Pro Pro Val Asn Thr Ile Gly Glu Lys Asp Thr Ser Gln

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Ile Gln Ile Arg Gln Gln Pro Arg Asp Pro Pro Thr Glu Thr Leu Glu

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Leu Glu Val Ser Pro Asp Pro Ala Ser Gln Ile Leu Glu His Thr Gln

50 55 60

Gly Ala Glu Lys Leu Val Ala Glu Leu Glu Gly Asp Ser His Lys Ser

65 70 75 80

His Gly Ser Thr Ser Gln Met Pro Glu Ala Leu Gln Ala Ser Asp Leu

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Trp Tyr Cys Pro Asp Gly Ser Phe Val Lys Lys Ile Val Ile Arg Gly

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His Gly Leu Asp Lys Pro Lys Leu Gly Ser Cys Cys Arg Val Leu Ala

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Leu Gly Phe Pro Phe Gly Ser Gly Pro Pro Glu Gly Trp Thr Glu Leu

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Thr Met Gly Val Gly Pro Trp Arg Glu Glu Thr Trp Gly Glu Leu Ile

145 150 155 160

Glu Lys Cys Leu Glu Ser Met Cys Gln Gly Glu Glu Ala Glu Leu Gln

165 170 175

Leu Pro Gly His Ser Gly Pro Pro Val Arg Leu Thr Leu Ala Ser Phe

180 185 190

Thr Gln Gly Arg Asp Ser Trp Glu Leu Glu Thr Ser Glu Lys Glu Ala

195 200 205

Leu Ala Arg Glu Glu Arg Ala Arg Gly Thr Glu Leu Phe Arg Ala Gly

210 215 220

Asn Pro Glu Gly Ala Ala Arg Cys Tyr Gly Arg Ala Leu Arg Leu Leu

225 230 235 240

Leu Thr Leu Pro Pro Pro Gly Pro Pro Glu Arg Thr Val Leu His Ala

245 250 255

Asn Leu Ala Ala Cys Gln Leu Leu Leu Gly Gln Pro Gln Leu Ala Ala

260 265 270

Gln Ser Cys Asp Arg Val Leu Glu Arg Glu Pro Gly His Leu Lys Ala

275 280 285

Leu Tyr Arg Arg Gly Val Ala Gln Ala Ala Leu Gly Asn Leu Glu Lys

290 295 300

Ala Thr Ala Asp Leu Lys Lys Val Leu Ala Ile Asp Pro Lys Asn Arg

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Ala Ala Gln Glu Glu Leu Gly Lys Val Val Ile Gln Gly Lys Asn Gln

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Asp Ala Gly Leu Ala Gln Gly Leu Arg Lys Met Phe Gly

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Met Glu Thr Pro Pro Val Asn Thr Ile Gly Glu Lys Asp Thr Ser Gln

1 5 10 15

Pro Gln Gln Glu Trp Glu Lys Asn Leu Arg Glu Asn Leu Asp Ser Val

20 25 30

Ile Gln Ile Arg Gln Gln Pro Arg Asp Pro Pro Thr Glu Thr Leu Glu

35 40 45

Leu Glu Val Ser Pro Asp Pro Ala Ser Gln Ile Leu Glu His Thr Gln

50 55 60

Gly Ala Glu Lys Leu Val Ala Glu Leu Glu Gly Asp Ser His Lys Ser

65 70 75 80

His Gly Ser Thr Ser

85

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<211> 150

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Met Glu Thr Pro Pro Val Asn Thr Ile Gly Glu Lys Asp Thr Ser Gln

1 5 10 15

Pro Gln Gln Glu Trp Glu Lys Asn Leu Arg Glu Asn Leu Asp Ser Val

20 25 30

Ile Gln Ile Arg Gln Gln Pro Arg Asp Pro Pro Thr Glu Thr Leu Glu

35 40 45

Leu Glu Val Ser Pro Asp Pro Ala Ser Gln Ile Leu Glu His Thr Gln

50 55 60

Gly Ala Glu Lys Leu Val Ala Glu Leu Glu Gly Asp Ser His Lys Ser

65 70 75 80

His Gly Ser Thr Ser Gln Met Pro Glu Ala Leu Gln Ala Ser Asp Leu

85 90 95

Trp Tyr Cys Pro Asp Gly Ser Phe Val Lys Lys Ile Val Ile Arg Gly

100 105 110

His Gly Leu Asp Lys Pro Lys Leu Gly Ser Cys Cys Arg Val Leu Ala

115 120 125

Leu Gly Phe Pro Phe Gly Ser Gly Pro Pro Glu Gly Trp Thr Glu Leu

130 135 140

Thr Met Gly Val Gly Pro

145 150

Claims (9)

1. An exosome peptide, characterized in that an exosome is a vector, which comprises a desired FKBPL polypeptide consisting of 24 amino acids whose amino acid sequence is QIRQQPRDPPTETLELEVSPDPAS.

2. A method for preparing the exosome peptide of claim 1, comprising the steps of:

1) preparing a sequence of an FKBPL gene or gene fragment comprising a sequence encoding an FKBPL polypeptide of interest and cloning into a plasmid vector;

2) transfecting a processing cell by using the plasmid vector, and collecting the processing cell expressed by the target FKBPL polypeptide after culturing;

3) the exosome is obtained by naturally secreting the processing cell, or the exosome of the processing cell is prepared by adopting an extrusion method to obtain the exosome peptide.

3. The method for producing an exosome peptide according to claim 2, wherein in step 1), the FKBPL gene is derived from human F-box protein 31.

4. The method for preparing an exosome peptide according to claim 2, wherein the plasmid vector contains the full length or gene fragment of the FKBPL gene, the full length FKBPL gene encodes amino acids 1-349 of the FKBPL polypeptide, and the cloned plasmid vector is denoted as FK 1; the FKBPL gene segment encodes amino acid 1-85 of FKBPL polypeptide, and the cloned plasmid vector is marked as FK 2; or the FKBPL gene segment encodes amino acid 1-150 of FKBPL polypeptide, and the cloned plasmid vector is marked as FK 3.

5. The method of preparing an exosome peptide according to claim 4, wherein the plasmid vector is FK2, comprising an FKBPL gene fragment encoding amino acids 1-85 of FKBPL polypeptide.

6. A method of producing an exosome peptide according to claim 2, wherein the processing cell is selected from 293T cells, or NK cells, T cells, macrophages or DC cells.

7. The method for preparing an exosome peptide according to claim 2, wherein the natural secretion is to transfect a plasmid vector into a processing cell, collect a culture solution of the processing cell, and centrifugally purify to obtain an exosome.

8. The method for preparing an exosome peptide according to claim 2, wherein the expression method is that after a plasmid vector is transfected into a processed cell, the processed cell is collected, a cell suspension of the processed cell is extruded by an extruder, and a cell filtrate is centrifuged and purified to obtain an exosome.

9. Use of the exosome peptide of claim 1 in the preparation of a medicament for the treatment of triple negative breast cancer.

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