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Short chain polypeptide EXP (Extracellular Vesicle Binding Peptide), medicine delivery system based on short chain polypeptide EXP and EVs (Extracellular Vesicles) recovery kit

A delivery system and drug delivery technology, applied in the direction of blood/immune system cells, animal cells, vertebrate cells, etc., can solve the problems of limited binding efficiency, exosome non-binding binding efficiency, etc., and achieve high stability

Active Publication Date: 2019-12-31
TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the binding efficiency of CP05 to exosomes is relatively limited, and it does not bind or binds to exosomes with no or low CD63 expression

Method used

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  • Short chain polypeptide EXP (Extracellular Vesicle Binding Peptide), medicine delivery system based on short chain polypeptide EXP and EVs (Extracellular Vesicles) recovery kit
  • Short chain polypeptide EXP (Extracellular Vesicle Binding Peptide), medicine delivery system based on short chain polypeptide EXP and EVs (Extracellular Vesicles) recovery kit
  • Short chain polypeptide EXP (Extracellular Vesicle Binding Peptide), medicine delivery system based on short chain polypeptide EXP and EVs (Extracellular Vesicles) recovery kit

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0131] Experimental example 1. Detection of binding ability of CP05 and different short peptides to exosome.

[0132] 1.1 The binding ability of CP05 and different short peptides to exosome was detected by flow cytometry.

[0133] (1) Mix 0.06 μg of FAM-labeled CP05 and different short peptides with 10 μg of exosomes from different sources (dC2C12, CDC, serum), respectively, and make up DPBS to a volume of 200 μL.

[0134] (2) Rotate and incubate on a vertical shaker at 4°C for 2-4hrs.

[0135] (3) Detection of binding efficiency by flow cytometry.

[0136] The result is as figure 1 (A) shown.

[0137] 1.2 Detection of Exosome-mediated ability of CP05 and different short peptides to enter cells.

[0138] (1) C2C12 cells were digested and counted, planted in a 24-well plate, 4×10 4 / hole. 5%CO 2 , grow at 37°C for 12h.

[0139] (2) Incubate the FAM-labeled CP05 and different short peptides for 4hrs according to the method in 1.1 above.

[0140] (3) Put the incubated EX...

experiment example 2

[0149] Experimental example 2, CP05 and EXP combined with exosom stability detection.

[0150] (1) C2C12 cells were digested and counted, planted in a 24-well plate, 4×10 4 / hole. 5%CO 2 , grow at 37°C for 12h.

[0151] (2) FAM-labeled CP05 and EXP (20 μg) were co-incubated with 10 μg DiI-labeled exosome respectively, and incubated at 4° C. for 4 hours with rotation.

[0152] (3) The above-incubated EXO CP05 and EXO EXP They were added to C2C12 cells respectively, and the culture medium was replaced with serum-free DMEM at 37°C to continue culturing.

[0153] (4) After culturing for different times (6hrs, 12hrs, 24hrs, 48hrs), the cells were washed with DPBS.

[0154] (5) 4% paraformaldehyde was fixed at room temperature for 30 min and then sealed.

[0155] (6) The colocalization efficiency and FRET fluorescence energy transfer of exosome (DiI) and short peptide (FAM) were observed under microscope at different time points.

[0156] The result is as figure 2 shown. ...

experiment example 3

[0157] Experimental example 3, detection of competitive binding ability between CP05 and EXP and exosome.

[0158] (1) AF750-labeled CP05 (0.06 μg) was incubated with FAM-labeled CP05 (0.06 μg, 0.3 μg) or EXP (0.06 μg, 0.3 μg) and 10 μg exosome, and supplemented with DPBS to a volume of 200 μl.

[0159] (2) Incubate with rotation at 4°C for 4 hours.

[0160] (3) Exosome positive percentage of FITC and APC-Cy7 channels detected by flow cytometry.

[0161] The result is as image 3 shown

[0162] Experimental example 4, EXP target detection verification.

[0163] (1) Mix 100 μg EXP or CP05 with 30 μL activated Flag magnetic beads, and incubate overnight at 4°C.

[0164] (2) PBST washes away short peptides not bound to the magnetic beads, and recovers the magnetic beads.

[0165] (3) C2C12 cells were lysed with non-denaturing tissue lysate, lysed on ice for 30 min, and centrifuged at 12000 rpm to recover the supernatant.

[0166] (4) Add the C1C12 lysed supernatant to the r...

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Abstract

The invention provides a short chain polypeptide EXP (Extracellular Vesicle Binding Peptide), a relevant medicine delivery system and an EVs (Extracellular Vesicles) recovery kit, and belongs to the technical field of biomedical engineering. The amino acid sequence of the short chain polypeptide EXP is shown as SEQ ID NO.1. Based on the short chain polypeptide EXP, the invention also provides themedicine delivery system, a targeted medicine delivery system, an enhanced medicine conveying carrier, conveying enhanced medicine, targeted medicine, the EVs kit, a disease diagnostic kit, an EVs purification method, and a purpose of the short chain polypeptide EXP to the field of pharmacy and diagnostic reagent preparation.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to a short peptide EXP and its related drug delivery system and extracellular vesicle recovery kit. Background technique [0002] Extracellular vesicles (EVs) are vesicles with a lipid bilayer structure secreted by a variety of cells and released into the extracellular space, which are mainly divided into exosomes (exosomes) and microvesicles ( Mocrovesicles, MV) two kinds. Extracellular vesicles play a very important role in biological processes such as information transmission and material exchange between cells. [0003] Exosome, also known as "exosome", is a biological vesicle secreted by cells with a diameter of 30-150nm, which is widely found in cell supernatant, blood, urine and other sources. Exosomes can carry endogenous proteins, mRNA, miRNA, and noncoding RNA during the formation process to mediate biological processes such as cell-to-cell material exchan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N5/078A61K47/64
CPCC07K7/08C12N5/0634A61K47/64C12N2509/00
Inventor 尹海芳冉宁林曹瑞高先军
Owner TIANJIN MEDICAL UNIV
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