ObjectiveThe injury of vascular endothelial cells caused by continuous high glucose environment results in angiogenesis disorder,which is the main reason for the prolonged and unhealing of diabetic wounds.As an important paracrine substance,exosomes have been proved to have the function of promoting angiogenesis and wound repair,but natural exosomes have limitations.Through the transformation of engineering means,the function can be further improved and the application can be optimized.The aim of this study was to investigate the therapeutic effect and pro-angiogenesis effect of engineered exosomes loaded with HIF-1αstabilizer VH298 released from photo crosslinked gelatin methacryloyl(GelMA)hydrogel on wounds in diabetic mice.Methods1.Preparation and identification of engineered exosomes loaded with VH298.Exosomes(EXOs)were extracted from the culture supernatant of epidermal stem cells(Ep SCs)by ultracentrifugation;The optimal method for encapsulating VH298 into EXOs was selected from 37°C co-incubation method,cyclic freeze-thaw method,and electroporation method.The concentration of loaded VH298 was measured by high performance liquid chromatography(HPLC).Natural EXOs and VH298-loaded EXOs(VH-EXOs)were characterized by transmission electron microscopy(TEM),Western blot and nanoparticle tracking analysis(NTA),respectively.The rate of internalization of EXOs and VH-EXOs by vascular endothelial cells(HUVECs)was observed by confocal microscopy.2.In vitro experiments verified that the loaded VH298 enhanced the pro-angiogenesis effect of EXOs and explored its mechanism.The HUVECs cultured in vitro were treated with PBS,VH298,EXOs,and VH-EXOs,respectively.The cell proliferation ability was analyzed by CCK-8 and Ed U.The cell migration ability was analyzed by scratch test and transwell test,and the angiogenesis ability was analyzed by matrigel tube formation test.The expression levels of angiogenesis-related proteins HIF-1α and VEGFA in cells were detected by western blot experiment.Small molecule RNA interference technology was used to knock down HIF-1α in HUVECs,and then the HUVECs before and after knockdown were treated with EXOs and VH-EXOs,respectively.The protein expression levels of HIF-1α and VEGFA were verified.3.Synthesis of GelMA hydrogels containing VH-EXOs(Gel-VH-EXOs)and characterizing the basic properties and sustained release of VH-EXOs.After curing under the irradiation of 405 nm wavelength light source,the swelling rate and degradation rate of GelMA hydrogel with different concentrations in PBS were tested.Spatial structure.GelMA hydrogels with different concentrations were thoroughly mixed with VH-EXOs,and the sustained release efficiency of VH-EXOs in PBS was determined respectively.After selecting the concentration,the solidified Gel-VH-EXOs were scanned under a confocal microscope to form a 3D image to determine the uniform distribution of VH-EXOs in the interior,and to verify the sustained release effect of Gel-VH-EXOs in vitro and in vivo experiments.4.In vivo experiments to verify the effects of Gel-VH-EXOs on wound healing and angiogenesis in diabetic mice.Wounds of full-thickness skin defects were established on the backs of diabetic mice.PBS,VH-EXOs,and Gel-VH-EXOs were applied to the wounds respectively,and the wound healing rates of mice in different treatment groups were observed and recorded.Laser Doppler flow imaging perfusion instrument and stereomicroscope were used to analyze the blood perfusion and the distribution of new blood vessels on the wound surface.HE staining and Masson staining were used to analyze the degree of wound epithelium and collagen deposition at the tissue level,and immunohistochemical methods were used to detect the vascular markers.The expression levels of CD13,HIF-1α and its downstream protein VEGFA were further evaluated in vivo experiments to further evaluate the angiogenesis of wounds and the expression changes of HIF-1α pathway.Results1.Compared with the cyclic freeze-thaw method and electroporation method,the TEM results showed that the 37℃ co-incubation method had the smallest change in the morphology of EXOs,and the NTA test showed the least loss of EXOs before and after drug loading.The37°C co-incubation method was selected,and the HPLC results showed that when the concentration of EXOs was constant,the loaded drug concentration gradually increased with the drug incubation concentration until saturation.The NTA results showed that the median diameters of EXOs and VH-EXOs were 117.6 nm and 136.7 nm,respectively.Exosome marker proteins CD63,CD9,and TSG101 can be detected in EXOs and VH-EXOs.There was no difference in the efficiency of internalization of EXOs and VH-EXOs by HUVECs.2.VH298,EXOs and VH-EXOs can enhance the proliferation,migration and angiogenesis of HUVECs,among which VH-EXOs has the most obvious effect on the function of HUVECs.Western Blot results showed that HIF-1α and VEGFA had the highest expression in HUVECs treated with VH-EXOs group.Compared with normal HUVECs,in HUVECs with HIF-1α knockdown,the differences in the promotion of proliferation,migration and tube formation between VH-EXOs and EXOs were reduced.The increase in VEGFA expression levels was also reduced.3.With the increase in concentration,SEM results showed that the porosity and pore diameter of GelMA hydrogel decreased,the tensile strength of GelMA hydrogel increased,the degradation time in PBS and the sustained release time of VH-EXOs was prolonged.Uniform distribution of VH-EXOs in Gel-VH-EXOs was observed by confocal microscopy.The PKH26-labeled EXOs were placed in the HUVECs culture system in the free form and in the form of Gel-VH-EXOs,respectively.The free EXOs became less and less in the HUVECs after 12 h,while the EXOs released by Gel-VH-EXOs accumulated in the HUVECs within 72 h gradually.In vivo sustained release experiments indicated that free EXOs accumulated at the injection site and were almost eliminated from the body within 4 days,while Gel-VH-EXOs could be continuously and uniformly released around the wound for more than4 days.4.The results of wound healing procedure showed that Gel-VH-EXOs group had the most efficient therapeutic effect.While VH-EXOs group was better than EXOs group,GelMA group and PBS group.Histological results showed that the degree of epithelialization and collagen deposition also conformed to this trend.In the analysis of angiogenesis results,the Gel-VH-EXOs group had the highest density of new blood vessels and the best wound blood perfusion,and the VH-EXOs group was better than the EXOs group.The results of immunohistochemistry showed that the expressions of CD31,HIF-1α and VEGFA were the highest in Gel-VH-EXOs group,and decreased in VH-EXOs group,EXOs group and GelMA group in turn,but were higher than those in the PBS group.ConclusionIn the present study,we found that Ep SC-EXOs could promote wound healing and improve angiogenesis.By using engineering means to load a HIF-1α stabilizer VH298 into EXOs,the ability of EXOs to accelerate wound healing and angiogenesis was further improved.At the same time,we mixed VH-EXOs into a photocurable cross-linked hydrogel GelMA to develop a novel wound dressing with appropriate mechanical and biological properties to optimize the delivery method of exosomes to achieve sustained release of VH-EXOs and Improve bioavailability and provide better therapeutic effect for promoting the healing of diabetic wounds. |