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A kind of slow-release exosome composite material and its preparation method and application

A composite material and exosome technology, applied in the field of biomaterials and biomedical engineering, can solve the problems of easy loss of exosomes and inability to play a long-term effect, so as to achieve long-term effective treatment methods without destroying the integrity of the membrane structure, The effect of prolonging the duration of action

Active Publication Date: 2022-05-27
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The present invention aims at the problem that exosomes are easy to lose and cannot function for a long time. TG2 is used to cross-link exosomes with biological materials containing glutamine residues or lysine residues to prepare composite scaffold materials, so that exosomes Long-term stable physical load on the scaffold and achieve sustained release effect, improve its application efficiency in tissue engineering, and exert a better therapeutic effect; the biological materials include silk fibroin, collagen, gelatin, etc.; the exosomes include Exosomes secreted by functionalized adipose-derived mesenchymal stem cells, exosomes secreted by bone marrow mesenchymal stem cells, exosomes secreted by umbilical cord mesenchymal stem cells, and exosomes secreted by M2 macrophages one or a mixture of

Method used

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  • A kind of slow-release exosome composite material and its preparation method and application
  • A kind of slow-release exosome composite material and its preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0013] 1) Preparation of scaffold material: 7% gelatin and 0.25% glutaraldehyde mixture were placed at -20 °C for reaction for 24h, transferred to a freeze dryer for freeze-drying for 24h, and gelatin porous scaffolding with porous structure was obtained;

[0014]2) Acquisition of exosome suspension: culture of fat mesenchymal stem cells to the third generation, add 20ng / mLIFN-γ stimulate cells to obtain inflammatory mesenchymal stem cells for 48h, replace serum-free medium for 24h to collect cell culture supernatants; remove cell debris and larger vesicles by gradient centrifugation; use ultracentrifugation method 100 000g centrifugation for 70min, discard supernatant, add PBS again 100 000g centrifugation for 30min, discard supernatant, add 200 μL PBS resuspends the pellet i.e. exosome suspension;

[0015] 3) Mix the exosome suspension with 2% (w / v) TG2 enzyme and inject it into the complex scaffold to saturation, and the reaction at 37 °C for 30 min can load the exosomes into t...

Embodiment 2

[0017] 1) Preparation of scaffold material: Weigh the silk dissolved in 9.3M lithium bromide solution to prepare a 20% (w / v) silk solution, placed in a water bath pot at 60 °C for 4h, using a 3500D dialysis bag dialysis for 48h; subsequently, replace the 20% g / mL PEG solution to continue dialysis for 6-10h to obtain a concentrated silk protein solution; use 10000rpm, 4 °C, centrifuge for 20 minutes to remove impurities, and determine the concentration of concentrated silk protein by weighing method. After adding an appropriate amount of distilled water, the concentration is 6% and after adding 0.25% glutaraldehyde, it is placed at -20 °C for 24h, transferred to a freeze dryer for freeze-drying for 24h, and a gelatin porous scaffold with a porous structure is obtained;

[0018] 2) Culture bone marrow mesenchymal stem cells to the third generation, culture cells in a 2% oxygen environment for 48 h, replace serum-free medium culture for 24 h to collect cell culture supernatant; remov...

Embodiment 3

[0021] 1) Scaffold material preparation: 2% collagen and 0.135 μM hydroxide nano-mixed liquid placed at -20 °C reaction for 24 h, transferred to freeze dryer for 24 h, to obtain collagen porous scaffold with porous structure;

[0022]2) Acquisition of exosome suspension: culture of fat mesenchymal stem cells to the third generation, add 20ng / mLIFN-γ stimulate cells to obtain inflammatory mesenchymal stem cells for 48h, replace serum-free medium for 24h to collect cell culture supernatants; remove cell debris and larger vesicles by gradient centrifugation; use ultracentrifugation method 100 000g centrifugation for 70min, discard supernatant, add PBS again 100 000g centrifugation for 30min, discard supernatant, add 200 μL PBS resuspends the pellet i.e. exosome suspension;

[0023] 3) Mix the exosome suspension with 2% (w / v) TG2 enzyme and inject it into the complex scaffold to saturation, and the reaction at 37 °C for 30 min can load the exosomes into the porous scaffold.

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Abstract

The invention discloses a slow-release exosome composite material and its preparation method and application, which is characterized in that exosomes are chemically linked to biological The stent material serves the purpose of long-term stable and slow release. The preparation method includes the preparation of the scaffold material, the acquisition of the exosome suspension, and the loading of the exosome. The composite scaffold avoids damage to the body caused by chemical cross-linking agent residues. The loading process will not affect the biological activity of exosomes, and will not damage the structural integrity of the exosome membrane. It lays the foundation for the guarantee of the therapeutic effect of exosomes and can be released slowly. Exosomes prolong the action time in the body and provide long-term effective treatment for cases that cannot be transplanted repeatedly.

Description

Technical field [0001] The present invention relates to a method of sustained release exosomes, specifically the use of glutamine transaminase 2 (TG2) to crosslink the exosomes secreted by cells with proteins in the material, thereby achieving the purpose of sustained release exosomes, belonging to the field of biomaterials and biomedical engineering. Background [0002] Exosomes are small vesicles that can be secreted by a variety of cells, containing a large number of biologically active factors, such as nucleic acids, proteins, etc., which play an important role in cell communication and play a role in physiological and pathological processes. The transplantation of exosomes into biological scaffold materials to sites of injurious and defective diseases shows a good ability to promote tissue regeneration and regulate the host immune environment. However, exosome yields are low, so the technology that enables exosomes to be slowly released in the scaffold material is crucial. T...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61L27/38A61L27/22A61L27/24A61L27/50A61L27/54A61L27/56A61K35/28A61K35/15A61K47/64A61P19/08C12N5/0775C12N5/0786
CPCA61L27/3834A61L27/3852A61L27/227A61L27/24A61L27/222A61L27/50A61L27/54A61L27/56A61K35/28A61K35/15A61K47/64A61P19/08C12N5/0667C12N5/0663C12N5/0665C12N5/0645A61L2300/602A61L2300/412A61L2430/06C12N2509/00C12N2509/10C08L89/00
Inventor 王淑芳齐春晓刘语菲李承乾王彪祁玉婷
Owner NANKAI UNIV
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