The Study On Sustained Release Of EGF-loaded Microspheres And Its Repair Effect On Diabetic Ulcer

The modified w/o/w extraction–evaporation technique was chosen to prepare epidermal growth factor (EGF) loaded microspheres using poly (lactic-co-glycolic) acid (PLGA). Bovine serum albumin(BSA) was used as molding drug. The emulsification concentration of inner aqueous phase, the mixture ratio of solvent, the emulsification concentration of outer aqueous phase, pH value, salt concentration was researched for the highest encapsulation efficiency. The microspheres were characterized for morphology by transmission electronic microscopy (TEM). The particle sizes of microspheres were determined by Particle Size Analyzer. The release performances of EGF loaded PLGA microspheres in vitro were studied, and EGF was measured by enzyme-linked immunosorbant assay (ELISA). The result showed that microspheres were smooth and spheriacal with a mean particle size 193.5nm, the index of polydispersity was 0.176, and the encapsulation efficiency was 85.6%..There was a linear relationship between the amount of accumulative release Q and time square root t^1/2 Q=46.80738+10.09451t1/2(R=0.94341). The release of EGF from PLGA microspheres can persist for 24h.The proliferation effects of microspheres on fibroblast were evaluated by MTT assay. We found that all of concentration of EGF loaded microspheres can accelerate the proliferation of fibroblast, however, there were some relationships between the concentration of microspheres and the proliferation effects. The result showed that 10μg/L was the optimal concentration for the proliferation. The proliferation effects of 10μg/L EGF loaded microspheres were much better compared with equal EGF. The proliferation effects of different concentration of blank microspheres on fibroblast were investigated. There was no toxicity on fibroblast with blank microspheres concentration range of 0.01μg/L to 100μg/L. All values were treated by statistical analysisThe diabetic ulcer model rats were used for researching the healing effects of EGF loaded microspheres. The change of ulcer areas visual displayed that there was significantly enhanced wound closure with EGF loaded microspheres from the 14th to 21th day compared with the control (EGF solution, the blank microspheres and PBS treatment) groups. Histological examination, the expression of the proliferative cell nuclear antigen(PCNA) and EGF receptor(EGFR), observation with electron microscope were used to observe the theraputic effects in microscopic view. Firstly, the results showed that the blank microspheres was non-toxic and no inhibited action. Secondly, EGF activity was well maintained by EGF loaded microspheres, which can release EGF slowly and stimulate fibroblast continuously. The healing effects of EGF loaded microspheres were more effective than traditional EGF solution treatment.This work offers evidences for treating the diabetic ulcer using EGF loaded microspheres in clinic.