Studyon The Role Of Engineered Exosomes In Promoting Diabetic Wound Healing And Preliminary Exploration Of Improving Its Production

Background and purposes:Diabetic chronic wounds such as diabetic foot and diabetic ulcer are difficult to heal,mainly due to the prolongation of the inflammatory phase and abnormal angiogenesis.At present,there is a lack of effective strategies to treat diabetic wounds in clinical practice.Exosomes are vesicle-like bodies secreted by cells,have a phospholipid bilayer membrane structure,mediate intercellular communication,play an important role in many physiological and pathological processes,and have the potential to be used as drugs or drug carriers for treating diseases.Mesenchymal stem cells(MSCs)have a strong ability in tissue regeneration,and the exosomes derived from them have achieved good effects in various disease models.However,the anti-inflammatory and angiogenesis-promoting components in natural exosomes are relatively limited.Studies have shown that miR-126 is involved in the regulation of inflammation and has the effect of promoting angiogenesis,but it is unstable and easily to be degraded in vivo.In the past ten years,exosome-related research has grown exponentially,but the number of exosome-related clinical trials is limited.One of the big challenges is that the yield of exosomes is very low,which is difficult for clinical translation.Therefore,this study aimed to load miR-126 in exosomes derived from hUC-MSCs and study its role in diabetic wound healing,as well as to explore ways to increase exosome production preliminarily.Methods and Results:1.Extraction and identification of primary hUC-MSCsThe hUC-MSCs were extracted by tissue block adherence method from umbilical cord,and the primary hUC-MSCs were identified by light microscopy,flow cytometry and trilineage differentiation assay.The results showed that,the hUC-MSCs could adhere to plastic,and had a high expression of positive markers CD105,CD90 and CD73(>99%)and lacked the expression of negative markers CD34 and CD45(0.28%and 0.09%,respectively).The hUC-MSCs also had the ability of osteogenic,adipogenic and chondrogenic differentiation in vitro.2.Preparation and identification of engineered exosomes(E-Exos)Exosomes were extracted from the conditioned media of hUC-MSCs by differential ultracentrifugation combined with tangential flow ultrafiltration and miR-126 was introduced into exosomes by electroporation.The morphology of exosomes was determined under transmission electron microscope,the size distribution was observed by nanoparticle tracking analysis,the protein makers were detected by Western blotting,and the loading rate of miR-126 in E-Exos was determined by RT-qPCR.The results showed that the exosomes had a hemispherical shape with a concave side and the particle size was around 130 nm.The exosomes had a high expression of TSG101,CD63,and CD81 and lacked the expression of calnexin.There were no significant changes in the morphology,size and marker proteins of exosomes before and after electroporation.The content of miR-126 in E-Exos was 1.63 times that of Exos.3.Study on the role of E-Exos in promoting diabetic wound healingA full-thickness splinted skin wound healing model was constructed in db/db diabetic mice,and the wound area during wound healing was recorded by taking pictures,and the effect of E-Exos on skin wound healing in diabetic mice was evaluated by H&E,Masson and immunofluorescence staining.The results showed that the E-Exos could significantly promote wound closure rate,epidermal growth,collagen deposition,and could significantly promote angiogenesis as well.4.Preliminary exploration of improving exosome productionEGFP-CD63-MSCs were constructed by lentivirus to produce exosomes labeled with fluorescence,and the amount of exosome secretion was characterized by measuring the fluorescence value in conditioned media,so as to screen for small molecule compounds that can promote exosome secretion.Norepinephrine and N-methyldopamine showed the most significant effect in promoting exosome secretion of hUC-MSCs,with an increase in the yield of exosomes up to 2 times.Conclusions:1.A miRNA delivery system based on exosomes derived from hUC-MSCs was successfully constructed and the E-Exos prepared by introducing miR-126 into it was proved to be able to promote diabetic wound healing in vivo by promoteing angiogenesis,epidermis regeneration,granulation tissue formation and ECM remodeling.2.Five small compounds that can promote exosomes secretion of hUC-MSCs were screened.Norepinephrine and N-methyldopamine showed the most significant effect in promoting exosome secretion of hUC-MSCs,with an increase in the yield of exosomes up to 2 times.