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A method for modifying ligands on the surface of cell microvesicles

A surface modification and microvesicle technology, which is applied in the modification of cell membranes, biochemical equipment and methods, microorganisms, etc., can solve problems such as the influence of membrane structure and physical and chemical properties, the inability to modify cell microvesicles, and the limitations of research and application progress , to achieve the effect of reducing the uptake by phagocytes, saving the amount of ligand, and maintaining physiological activity

Active Publication Date: 2020-08-21
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Current methods include engineered donor cells and membrane insertion methods, both of which have many steps and are complicated and time-consuming
Among them, the method of engineering donor cells requires specialized biological techniques to process cells, cannot modify any type of ligand, and cannot modify cell microvesicles derived from body fluids
The method of membrane insertion may have a great impact on the original membrane structure and physical and chemical properties of cell microvesicles, the insertion efficiency is difficult to control, and the free ligand is difficult to remove
Therefore, research and application progress in related aspects are greatly limited

Method used

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  • A method for modifying ligands on the surface of cell microvesicles
  • A method for modifying ligands on the surface of cell microvesicles
  • A method for modifying ligands on the surface of cell microvesicles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1, RGD polypeptide modified on the surface of exosomes.

[0036] Such as figure 1 shown.

[0037] Step (a), the 1×10 12 Exosomes were resuspended in 1 mL pH7.4 PBS and added to a 1.5 mL centrifuge tube. Then 3 μM DBCO-sulfo-NHS was added. The particle number / mole ratio of exosomes to DBCO-sulfo-NHS is 10 12 : 3 nmol, the centrifuge tube was fixed on a rotary mixer, and reacted at room temperature for 4 hours.

[0038] In step (b), add the solution obtained in step (a) to the upper layer of two ultrafiltration tubes (pore size 100kD, capacity 0.5mL), and centrifuge at 10000g for 10 minutes. The lower layer filtrate was discarded, and the upper layer was supplemented with PBS at pH 7.4 to a volume of 0.5 mL. The ultrafiltration was repeated twice according to the same procedure to obtain 1 mL of DBCO-modified exosomes.

[0039] In step (c), the solution obtained in step (b) was placed in a 1.5 mL centrifuge tube. Add 0.3 μM azido-modified RGD polypeptide (th...

Embodiment 2

[0042] Example 2, EGFR antibody modified on the surface of microvesicles.

[0043] Step (a), the 1×10 13 The microvesicles were resuspended in 10 mL of PBS, pH 7.4, and added to a 15 mL centrifuge tube. Then add 8 μM DBCO-NHS. The particle number / mole ratio of microvesicles to DBCO-NHS is 10 12 : 8nmol, the centrifuge tube was fixed on the rotary mixer, and reacted at room temperature for 6 hours.

[0044] In step (b), add the solution obtained in step (a) to the upper layer of two ultrafiltration tubes (pore size 300kD, capacity 5mL), and centrifuge at 12000g for 10 minutes. The lower layer filtrate was discarded, and the upper layer was supplemented with PBS at pH 7.4 to a volume of 5 mL. The ultrafiltration was repeated twice according to the same procedure to obtain 10 mL of DBCO-modified microvesicles.

[0045] In step (c), the solution obtained in step (b) is placed in a 15mL centrifuge tube. Add 2 μM azide-modified EGFR antibody (the azide-modified antibody molecu...

Embodiment 3

[0048] Example 3, siRNA modified on the surface of extracellular vesicles.

[0049] Step (a), the 1×10 14 Extracellular vesicles were resuspended in 1 mL pH7.4 PBS and added to a 1 mL centrifuge tube. Then 1 mM DBCO-PEG4-NHS was added. The particle / mole ratio of extracellular vesicles to DBCO-PEG4-NHS is 10 12 : 10 nmol, the centrifuge tube was fixed on a rotary mixer, and reacted at room temperature for 12 hours.

[0050] In step (b), add the solution obtained in step (a) to the upper layer of two ultrafiltration tubes (pore size 100 kD, capacity 0.5 mL), and centrifuge at 12000 g for 8 minutes. The lower layer filtrate was discarded, and the upper layer was supplemented with PBS at pH 7.4 to a volume of 0.5 mL. Repeat the ultrafiltration three times according to the same procedure to obtain 1 mL of DBCO-modified extracellular vesicles.

[0051] In step (c), the solution obtained in step (b) was placed in a 1 mL centrifuge tube. Add 0.5 mM azido-modified siRNA (the azid...

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Abstract

The invention relates to a method for modifying ligands on the surfaces of cell vesicles. The method comprises the following steps of (a) connecting diphenyl cyclooctyne onto the surfaces of the cell vesicles; (b) removing free diphenyl cyclooctyne; (c) connecting azide-modified ligands onto the diphenyl cyclooctyne on the surfaces of the cell vesicles; (d) performing purification on the modified cell vesicles. Compared with the prior art, the method has the advantages that the time is saved; the process is simple and convenient; the reaction efficiency is high; the ligand types are slightly limited; meanwhile, the physiological activity of the cell vesicles can be maintained to the maximum degree. The treatment effect of the cell vesicles can be favorably enhanced.

Description

technical field [0001] The invention relates to a method for modifying ligands on the surface of cell microvesicles, belonging to the field of biotechnology. Background technique [0002] Cell microvesicles are vesicles with a lipid membrane structure and diameters ranging from tens of nanometers to several microns, which are automatically secreted and released by cells of humans, animals, plants or microorganisms or obtained after fragmentation. Including extracellular vesicles, microvesicles, exosomes, apoptotic bodies, shedding vesicles, and microparticles. Cellular microvesicles carry biologically active proteins, lipids, messenger RNA (mRNA), microRNA (miRNA), non-coding RNA (ncRNA), and DNA fragments. And it can deliver these active molecules to recipient cells to regulate the biological functions of target cells, tissues and organs. Certain types of cells, such as mesenchymal stem cells, produce cellular microvesicles containing a variety of active biomolecules that...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/00
CPCC12N5/0006
Inventor 田田高隽王静王梅张卉馨寇晓林
Owner NANJING MEDICAL UNIV
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