Preparation And Evaluation Of Multilayer Alginate Chitosan Microspheres Loading VEGF And Vancomycin

In the clinical，for the treatment of large bone defect, the center of the tissueengineering bone often appears the problems, for example limitation of bloodsupplyment and poor nutrition; complex fractures is often accompanied by bonedefect and infection. Therefore, construction of the tissue engineering bone, at thesame time, the promotion of small vascular ingrowth and the reconstruction of cellsand materials, and control the local microbial infection of the defect, become thecurrent problem to be solved.In recent years, multilayer drug-loading system become research focus, it isdifferent from ordinary single layer drug-loading system, it possesses the advantagesof various kinds of drug-loading、low of the burst release rate、good effect ofsustained-release. Among them, the most widely used is LBL, it is a technique basedon the interaction of positive and negative charge of polyelectrolyte. Because it hasthe advantages of simple、quick、high efficiency、mild preparation conditions、variouskinds of drug-loading、controllable and intelligent response to the release of drugs, itbecomes an important method of building multilayer drug controlled release systemat present.This thesis mainly studies that alginate and chitosan are considered as carrier,according to LBL, to prepare multilayer sustained-release microspheres systemloading VEGF and vancomycin, in order to achieve the effect of promotion ofangiogenesis and the control of microbial infection, so that it can play an importantrole in promoting the healing of bone and regeneration in tissue engineering.Objective:To prepare sodium alginate/chitosan multilayer microspheres loading vascularendothelial growth factor (VEGF)/vancomycin (VAN) and to study the features such as the size distribution, morphology, component, drug content, controlled release ofVEGF/VAN.Methods:The sodium alginate was crosslinked with calcium ion by using theneedleextrusion/external gelation method, forming calcium alginate core microsphereloading vascular endothelial growth factor (VEGF), then, in turn,CS、SA and CS werecoated on the surface of the core microspheres via LbL self-assembly technique,vancomycin(VAN) were loaded by chitosan, forming multilayer drug-loading system;the effect of the concentration of sodium alginate、calcium ion and chitosan onembedding rate and drug loading was investigated with orthogonal design; the surfaceand cross section morphology and particle size of multilayer microspheres wereobserved by scanning electron microscope; the self assembled of sodium alginate andchitosan was detected by FTIR; the embedding rate、drug loading and in vitro releaseof VEGF and VAN were detected by the method of ELISA double mono-clonedantibody and UV-Vis spectrophotometry separately.Result:The result of orthogonal design indicated that the optimal concentration ofsodium alginate, calcium chloride and chitosan were4%、15%、1%respectively, thenthe microspheres were smooth、rounded and homogeneous, the diameter of themicrospheres were estimated to be about500to1000um.Infrared spectra showscharacteristic absorption peak, it indicates that sodium alginate and chitosan react toachieve layer-by-layer self-assembly of multilayer microspheres. The embedding rateof VEGF of optimal group was61.31%, drug loading was0.59ug/g;the embeddingrate of VAN was3.48%，drug loading was12.47mg/g. In vitro release experimentsdemonstrated that the release of VEGF slowly, it sustained about21d, accumulativedrug releasing ratio was93%; the release of VAN quickly, the phenomenon of suddenrelease was obvious, it sustained about7d, accumulative drug releasing ratio was91%.The drug-loading multilayer microspheres had the potential of controllinginfection and promoting angiogenesis, so it was expected to be used in the basic research and clinical practice of tissue engineering bone.Conclusion:Sodium alginate/chitosan multilayer microspheres loading VEGF/VAN had thehigh embedding rate, it could realize the procedural, controllable release of the twodrugs, reduce the burst release of drug and delay the release of drug.