Application Of Exosomes Derived From Mesenchymal Stem Cell Modified By MiR-675 And Hydrogel

Exosome is an extracellular vesicle originates from intracellular multivesicular body that can be released by almost all cells with a diameter of 30-100 nm.Studies have shown that exosomes,as an important extracellular secretion,play an important role in intercellular communication,cellular function and signal transduction.In recent years,stem cell derived exosomes have been used to treat myocardial infarction and ischemic injury.Compared with its parent cells,exosomes not only have similar beneficial functions,but also have the advantage of not triggering immune response and reducing the potential risk associated with stem cell transplantation.Therefore,exosome based therapy has become a promising treatment strategy for ischemic cardiovascular disease.However,exosome therapy also has some shortcomings such as low targeting rates to specific cell types or tissues after intravenous injection,short tissue retention time after in situ injection,and low content of specific functional molecular.In this study,we used in situ injection of exosomes to verify the therapeutic effect of exosomes.Moreover,in order to improve the therapeutic effect,we modified exosomes by increasing the in situ retention time and the functional molecules contents,to promote future clinical application of stem cell-derived exosomes in the treatment of ischemic injury.In our study,we found that ischemic injury and aging can induce similar molecular and downstream pathway changes.As a key upstream molecule,miR-675 plays an important role in regulating vascular dysfunction induced by aging and ischemic injury.Upregulation of miR-675 can promote cell proliferation and reduce aging-related phenotypes.Moreover,miR-675 can inhibit aging and injury-induced vascular dysfunction by targeting the TGF-?1/smad2/3 signaling pathway.Unfortunately,the expression of miR-675 in the exosomes of human umbilical cord mesenchymal stem cells is low,so we transfected miR-675 mimic into human umbilical cord mesenchymal stem cells.The collected exosomes are enriched in miR-675,and the modified exosome was used to treat ischemia hindlimb.We also used silk fibroin hydrogel to improve the retention time of exosomes in situ.The results showed that the miR-675 exosome can significantly improve the function of ischemic hindlimb,which can be further improved by miR-675 exosome encapsulated in silk fibroin hydrogel.Recent studies have shown that silk sericin hydrogel does not induce immune response.Therefore,we further explore the feasibility of delivering exosomes by silk fibroin and sericin composite hydrogel to tissue in situ.We extracted the silk fibroin and sericin from the silk by separate extraction(high temperature alkaline water extraction for sericin,mild LiBr extraction for fibroin)and combined extraction(mild LiBr method).Four layers of sterile gauze,silk fibroin hydrogel sponge and silk fibroin hydrogel filled with exosomes solution were respectively inserted into the whole skin excision wound with a diameter of 1cm in the back of the mouse.The results showed that the immune reaction caused by silk fibroin and sericin composite hydrogel did not exceed that of four layers of sterile gauze.Moreover,silk fibroin and sericin composite hydrogel could promote the growth of skin and hair at the wound site,and the effect is more significant when exosomes were encapsulated in composite hydrogel.The results indicate that silk fibroin and sericin composite hydrogel sponges can be used as excellent alternatives to serve as wound dressing and can be also used as a potential material for delivery and sustained-release of exosomes.Our results showed that up to 40%of the encapsulated exosomes could be released in the first 8 days in vitro.In order to ensure the sustained release rate of exosomes,we designed a new functional peptide hydrogel.The cardioprotective peptide GHRP6 was added to the C terminal of self-assembled amphiphilic polypeptide with controllable degradation ability.The sequence of the peptide was C16-Gly-Thr-Ala-Gly-Leu-Ile-Gly-Gln-Gly-Gly-His-DTrp-Ala-Trp-DPhe-Lys-Ser(PA-GHRPS).The peptide could be self assembled into hydrogel and to encapsulate exosomes with the assistance of strong self-assembled polypeptide NaPFF.The release exosomes and GHRP6 can be achieved through controllable degradation.The results showed that cardiac function was improved by injecting 10-3M PA-GHRPS hydrogel into the peri-infarct region and the therapeutic effect was more significant when exosomes were added to the hydrogel.Conclusion:Our results confirmed that transfection of functional miR-675 into stem cells can yield specific functional exosome enriched in miR-675,which proved the concept that the molecular composition of exosomes can be modified at the cellular level.In addition,we showed that silk fibroin hydrogel and peptide hydrogel can promote the sustained release of exosome.Silk fibroin hydrogel has excellent mechanical properties,while peptide hydrogel can guarantee the sustained release of exosome due to its smaller pore size.Therefore,different hydrogel can be selected to meet different requirement.In addition,we also verified that silk fibroin and sericin composite hydrogel can be used as a good wound dressing and play an important role in delivering exosomes.In conclusion,we use external hydrogel encapsulation and internal molecular modification to improve the therapeutic effect of exosomes via in situ injection,which is an important step toward clinical application of exosomes.