阅读说明：本技术 一种缓释外泌体的复合材料及其制备方法与应用 (Composite material for slowly releasing exosome and preparation method and application thereof ) 是由 王淑芳 齐春晓 刘语菲 李承乾 王彪 祁玉婷 于 2020-05-26 设计创作，主要内容包括：本发明公开了一种缓释外泌体的复合材料及其制备方法和应用,其特点在于利用天然存在的酶促反应(谷氨酰胺转氨酶TG2催化的蛋白连接)将外泌体化学连接在生物支架材料上起到长期稳定缓慢释放的目的。制备方法包括支架材料的制备,外泌体悬液的获取,以及外泌体的负载。该复合支架避免化学交联剂残留对机体的损伤,负载过程不会对外泌体生物活性产生影响,不破坏外泌体膜结构完整性,对于外泌体治疗效果的保证奠定基础,可缓释外泌体,延长体内的作用时间,对于不能反复手术移植病例提供了长期有效的治疗方法。(The invention discloses a composite material for slowly releasing exosomes and a preparation method and application thereof, which are characterized in that a naturally-existing enzymatic reaction (protein connection catalyzed by glutamine transaminase TG2) is utilized to chemically connect exosomes to a biological scaffold material to achieve the purpose of long-term stable slow release. The preparation method comprises the steps of preparing a scaffold material, obtaining an exosome suspension and loading exosomes. The composite stent avoids the damage of chemical cross-linking agent residue to the body, the loading process can not influence the bioactivity of exosome, the structural integrity of exosome membrane can not be damaged, the foundation is laid for the guarantee of the treatment effect of exosome, exosome can be slowly released, the action time in vivo is prolonged, and a long-term effective treatment method is provided for transplantation cases which can not be operated repeatedly.)

1. The composite material of the slow-release exosome is a composite scaffold material prepared by crosslinking an exosome and a biomaterial containing glutamine residues or lysine residues by using TG 2; the biological material comprises silk fibroin, collagen, gelatin and the like; the exosome comprises one or a mixture of several of exosomes secreted by adipose mesenchymal stem cells functionalized under specific conditions, exosomes secreted by bone marrow mesenchymal stem cells, exosomes secreted by umbilical cord mesenchymal stem cells and exosomes secreted by M2 type macrophages.

2. The preparation method of the composite material comprises the following steps:

1) preparing a scaffold material: preparing a biological material to be loaded into a proper concentration, adding a cross-linking agent, reacting at-20 ℃ for 24 hours, transferring to a freeze dryer, and freeze-drying for 24 hours to obtain a biological scaffold with a porous structure;

2) obtaining an exosome suspension: culturing the mesenchymal stem cells to the third generation, obtaining functional cells by 48h of inflammatory factors, medicaments, small molecules or hypoxia-stimulated cells, replacing a serum-free culture medium for culturing for 24h, and collecting cell culture supernatant; removing cell debris and larger vesicles by gradient centrifugation; centrifuging at 100000 g for 70min by ultracentrifugation, discarding supernatant, adding PBS, centrifuging at 100000 g for 30min, discarding supernatant, adding 200 μ L PBS to resuspend the precipitate, i.e. exosome suspension;

3) loading of exosomes: mixing the exosome suspension with 1-5% (w/v) TG2 enzyme, injecting the mixture into a composite scaffold until the composite scaffold is saturated, and reacting for 30min at 37 ℃ to load exosomes into the porous scaffold.

3. The composite scaffold catalyzed by TG2 has the function of slowly releasing exosome, can be used for long-term maintenance of treatment effect after in vivo transplantation of biological scaffold, and preferably solves the problem of burst release after loading exosome.

Technical Field

The invention relates to a method for slowly releasing exosome, in particular to a method for crosslinking exosome secreted by cells and protein in a material by using glutamine transaminase 2(TG2) so as to achieve the purpose of slowly releasing exosome, belonging to the field of biological materials and biomedical engineering.

Background

Exosomes are vesicles secreted by a variety of cells, containing a large number of bioactive factors, such as nucleic acids, proteins, etc., that play an important role in cellular communication and play a role in physiological and pathological processes. The exosome is loaded in the biological scaffold material and transplanted to the position with the injury and the defect diseases, which shows good capability of promoting tissue regeneration and regulating and controlling host immune environment. However, the production of exosomes is low, so the technology of enabling exosomes to be slowly released in the stent material is crucial. TG2 is a protein molecule with catalytic action commonly existing in human cells, and proteins and polypeptides containing two amino acid residues are connected by catalyzing the formation of covalent bonds of glutamine residues and lysine residues. The TG 2-mediated protein ligation allows a naturally occurring enzymatic reaction that does not affect protein activity, and the covalent bonds formed can be degraded by cell-secreted metalloproteinases (MMPs) to dissociate the ligated proteins. The invention utilizes the activity of TG2 catalytic protein connection to connect exosomes to a biological scaffold material containing glutamine residues and/or lysine residues so as to realize the chemical loading of exosomes and further realize the slow release of exosomes.

Disclosure of Invention

Aiming at the problems that the exosome is easy to run off and cannot play a role for a long time, TG2 is utilized to crosslink the exosome and a biological material containing glutamine residues or lysine residues to prepare a composite scaffold material, so that the exosome can be stably loaded on the scaffold for a long time and achieves a slow release effect, the application efficiency of the exosome in tissue engineering is improved, and a better treatment effect is played; the biological material comprises silk fibroin, collagen, gelatin and the like; the exosome comprises one or a mixture of several of exosomes secreted by adipose mesenchymal stem cells functionalized under specific conditions, exosomes secreted by bone marrow mesenchymal stem cells, exosomes secreted by umbilical cord mesenchymal stem cells and exosomes secreted by M2 type macrophages. The specific preparation method comprises the following steps:

1) preparing a scaffold material: preparing a biological material to be loaded into a proper concentration, adding a cross-linking agent, reacting at-20 ℃ for 24 hours, transferring to a freeze dryer, and freeze-drying for 24 hours to obtain a biological scaffold with a porous structure;

2) obtaining an exosome suspension: culturing the mesenchymal stem cells to the third generation, obtaining functional cells by 48h of inflammatory factors, medicaments, small molecules or hypoxia-stimulated cells, replacing a serum-free culture medium for culturing for 24h, and collecting cell culture supernatant; removing cell debris and larger vesicles by gradient centrifugation; centrifuging at 100000 g for 70min by ultracentrifugation, discarding supernatant, adding PBS, centrifuging at 100000 g for 30min, discarding supernatant, adding 200 μ L PBS to resuspend the precipitate, i.e. exosome suspension;

3) loading of exosomes: mixing the exosome suspension with 1-5% (w/v) TG2 enzyme, injecting the mixture into a composite scaffold until the composite scaffold is saturated, and reacting for 30min at 37 ℃ to load exosomes into the porous scaffold.

The composite scaffold catalyzed by TG2 has the function of slowly releasing exosome, can be used for long-term maintenance of treatment effect after in vivo transplantation of biological scaffold, and preferably solves the problem of burst release after loading exosome.

Compared with the prior art, the invention has the outstanding advantages that:

1) in the selection of materials, the biodegradable natural protein catalyst has better biocompatibility. The catalyst is free after finishing the catalytic action, does not participate in forming a product, can be completely washed from the stent material, and cannot damage the fabric due to the residue of the cross-linking agent.

2) In the preparation process, the exosome is covalently connected with the protein in the composite material by utilizing natural enzymatic reaction, so that the stable load of the exosome is realized, the bioactivity of the exosome is not influenced, the structural integrity of the exosome membrane is not damaged, and a foundation is laid for ensuring the treatment effect of the exosome.

3) The product is functional, the prepared composite stent material can slowly release exosome, prolongs the action time in vivo, and provides a long-term effective treatment method for transplantation cases which can not be repeatedly operated.

The specific embodiment is as follows: