Engineered extracellular vesicle composition targeting P-selectin as well as preparation method and application of engineered extracellular vesicle composition

A selectin, engineered technology used in the field of biomedicine to achieve the effects of high biosafety, low immunogenicity and long blood half-life

Pending Publication Date: 2021-11-09
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no visual diagnosis and treatment strategy targeting P-selectin for the diagnosis and treatment of endothelial cell injury-related diseases

Method used

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  • Engineered extracellular vesicle composition targeting P-selectin as well as preparation method and application of engineered extracellular vesicle composition
  • Engineered extracellular vesicle composition targeting P-selectin as well as preparation method and application of engineered extracellular vesicle composition
  • Engineered extracellular vesicle composition targeting P-selectin as well as preparation method and application of engineered extracellular vesicle composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] A method for extracting extracellular vesicles secreted by human placenta-derived mesenchymal stem cells is as follows:

[0055] 1) Before the experiment, normal fetal bovine serum (FBS) was centrifuged at 120,000g for 18 hours at 4°C to remove extracellular vesicles (EVs) in fetal bovine serum, and filtered through a needle filter of 0.22 μm to remove Fetal bovine serum (EV-freeFBS) of EVs; and use the above-mentioned EV-free FBS to configure the complete medium without EV (comprising 10% EV-free FBS, 1% double antibody, 1% glutamine, 1% non-essential amino acids, 87% DMEM / F12 medium), used to cultivate human placenta-derived mesenchymal stem cells (hP-MSCs);

[0056] 2) When normally cultured at 75cm 2 When the confluence of hP-MSCs in the cell culture flask reaches 80%, discard the old medium, add 10 mL of EV-free complete medium, continue to culture for 24 hours, and then collect the conditioned medium for gradient centrifugation to separate EVs;

[0057] 3) Centr...

Embodiment 2

[0062] The method of using nitric oxide hydrogel to culture human placenta-derived mesenchymal stem cells and loading endogenous functional components in their extracellular vesicles is as follows:

[0063] 1) Coat the nitric oxide hydrogel with a concentration of 2 μg / μL on a surface of 75 cm 2 The bottom of the cell culture flask should be placed in a cell culture incubator at 37°C for more than 2 hours;

[0064] 2) hP-MSCs were cultured in cell culture flasks coated with nitric oxide hydrogel, and the EVs secreted by hP-MSCs cultured on the nitric oxide hydrogel were isolated and extracted according to the extraction method in Example 1;

[0065] 3) The endogenous functional components loaded in the isolated EVs were detected and confirmed by ELISA and real-time fluorescent quantitative PCR technology.

Embodiment 3

[0067] The method of loading exogenous functional component doxorubicin in extracellular vesicles by co-incubation is as follows:

[0068] 1) Collect the conditioned medium of the MDA-MB-231 cells, and separate and extract the EVs secreted by the MDA-MB-231 cells according to the extraction method in Example 1;

[0069] 2) Add 1 mmol / L doxorubicin (Dox) to the isolated and extracted EVs, mix well and place in a cell culture incubator at 37°C for 30 min;

[0070] 3) Transfer the EVs / Dox mixture after co-incubation into an ultracentrifuge tube, add 40 mL of sterile PBS to wash once, centrifuge at 100,000 g for 120 min, and the final pellet is the Dox-loaded MDA-MB-231 cell-derived EVs;

[0071] 4) To confirm the loading efficiency of Dox in EVs by detecting the fluorescence signal of Dox at 590nm wavelength.

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Abstract

The invention discloses an engineered extracellular vesicle composition targeting P-selectin. The engineered extracellular vesicle composition comprises extracellular vesicles, ligand molecules specifically targeting P-selectin, a molecular imaging contrast agent and endogenous or exogenous functional components carried in the extracellular vesicles, wherein the engineered extracellular vesicle is used for specifically recognizing and combining the P-selectin on the surface of an injured endothelial cell, the expression quantity of the P-selectin is indicated through the signal intensity of the molecular imaging contrast agent, and carried endogenous or exogenous functional components are delivered to an injured part. According to the engineered extracellular vesicle composition targeting the P-selectin as well as a preparation method and the application of the engineered extracellular vesicle composition, the engineered extracellular vesicle takes the P-selectin as a target spot to target injured endothelial cell, so that the purpose of targeting diseases related to endothelial cell injury is realized; and the blank that in the prior art, no universal technical means for targeted diagnosis and treatment of diseases related to injured endothelium exists is filled.

Description

technical field [0001] The invention relates to the field of biomedical technology, in particular to an engineered extracellular vesicle composition targeting P-selectin, its preparation method and application. Background technique [0002] Endothelial cells are flat cells on the inner wall of blood vessels, located between plasma and vascular tissue, they can not only complete the metabolic exchange of plasma and interstitial fluid, but also synthesize and secrete a series of vasoactive substances to participate in various physiological and pathological processes. Under physiological conditions, endothelial cells can regulate the tension of blood vessel walls, and have protective effects such as anti-inflammatory cell adhesion and anti-platelet adhesion activation. When it is stimulated by inflammatory factors, thrombin, hyperglycemia, reactive oxygen species, etc., endothelial cells are damaged, which leads to pathological changes such as changes in vascular tone, inflamma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/69A61K47/54A61K9/50A61K47/46A61K45/00A61P9/00A61P35/00A61P9/10A61P9/14A61P37/02A61P29/00A61K49/00
CPCA61K47/6901A61K47/549A61K45/00A61K49/0002A61P9/00A61P35/00A61P9/10A61P9/14A61P37/02A61P29/00A61K9/5068A61K49/0091A61K49/005
Inventor 李宗金张凯悦
Owner NANKAI UNIV
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