Microsphere-based Silk Fibroin3D Scaffold For Localized Insulin Release

Skin ulcer is one of the most common complications in diabetes mellitus. It is anurgent problem in the field of skin ulcer to find an effective method to promot skin woundrepairs. Topical insulin enhanced wound healing by shortening the needed time andimproving the healing quality, meanwhile it had little effects on the concentration of bloodglucose. But insulin readily hydrolysed by the protease in blood, leading to the loss of itsactivity within a short time. Moreover, a high concentration insulin is hazardous to health.Therefore, it is crucial to search for a suitable drug release carrier. In this study,insulin-loaded silk fibroin(SF) microspheres were prepared with electrostaticdifferentiation technology, and the surface morphology and internal structure ofmicrospheres was investigated. At the same time, the release characteristics of insulin andits promotion for the migration and proliferation of HaCaT cells and EA.hy926cells. Thenthe microspheres were embedded into SF scaffolds for localized insulin release.Firstly, insulin-loaded SF microspheres were fabricated by coaxial high-voltageelectrostatic differentiation technology and freeze-drying method. The morphology wereobserved by SEM, the results indicated that the mean diameter was about93μm, when thesolution concentration, voltage and flow rate were1.75wt.%,13kV and0.4ml/h,respectively, the porous microspheres contained numerous nanoparticles which connectedby nanofibers. The diameter of nanoparticles were in the range of a few hundrednanometers to one micrometer. The nanoparticles disappeared as the solution concentrationincreased, resulting that the internal structure of microspheres depent on the solutionconcentration. The second structure of microspheres was studied by FTIR and XRDspectra. The results indicated that the second structure contents of insulin remained aboutthe same. The CD spectra showed that released insulin has the same spatial conformationas native insulin.Secondly, the insulin release behavior was determined by fluorescence labeling. Theinsulin-loaded microspheres prepared by absorption had an initial burst release, most insulin released within8h. While the insulin-loaded microspheres prepared by coaxial andblending methods exhibited a relative slow release rate, and could sustained release for15d. The morphology of microspheres released for28d were observed by SEM. The resultsshowed that the surface micropores gradually increasing as insulin released. Then,microspheres-embedded scaffolds was prepared by soaking, blending, layered blendingand layer-by-layer assembling methods. The SEM observation showed that soakingmethod could embedded the microspheres on the scaffold surface only. Blending methodmade a lot of microspheres fall off from scaffold after freeze-drying. Layered blending andlayer-by-layer assembling method could embedded microspheres into scaffold evenly.Microspheres loading insulin were prepared by penetration-absorption, coaxial andblending methods. The microspheres-embedded scaffold had a more stable sustainedrelease.Finally, the effect of released insulin on the migration and proliferation of HaCaT andEA.hy926cells were respectively studied. The results indicated that insulin inducedHaCaT and EA.hy926cells migration and proliferation which were determined byscarification and CCK-8, respectively.In this paper, porous insulin-loaded SF microsphere was prepared by coaxialhigh-voltage electrostatic differentiation technology and freeze-drying method, and thenthe microspheres were embedded into SF scaffolds. The results indicated that releasedinsulin was bioactive. This may provide an effective drug delivery system for localizedinsulin release to promote wound healing.