Preparation Of Electrospun Fibers With Sustained Release Of Cytokine Protein And Its Feasibility Study On Tissue Repair

Electrospun fibers exhibit a unique advantage in the preparation of tissue engineering scaffold because of their three-dimensional network structures similar to extracellular matrix（ECM）,which can provide the micro-environment required for cell growth.Tissue-engineered fibers that maintain the effective concentration of cytokine proteins inducing directional differentiation of stem cells at repairing sites may provide a better bionic environment for tissue regeneration treatment.However,to realize this goal,there are still a series of tough technical challenges that needs to be solved.For example,the long-acting sustained release cytokine protein encapsulated in the tissue engineered fibers needs to maintain its natural conformation without denaturation and production of harmful antibodies.At the same time,the scaffold is supposed to maintain its original shape and sufficient mechanical strength with a surface compatible with peripheral tissues.According to all these challenges mentioned above,in this study,nerve growth factor（NGF）and vascular endothelial growth factor（VEGF）were selected as model proteins to prepare electrospun fibers that could stabilize the bioactivity and sustain the release of cytokine proteins based on inventions previously developed in our lab from materialogy（electrospun fibers encapsulated protein,microcrystalline cross-linking of polyvinyl alcohol）to biopharmaceutics（preformulation of protein based on aqueous phase-aqueous phase emulsification）.First,we prepared NGF-dextran nanoparticles and VEGF₁₆₅-dextran nanoparticles of smaller sizes and more uniform distribution by condition optimization.And then according to the structural characteristics of electrospun fibers,particle size and their physio-chemical characteristics,coated beaded electrospun fibers loading particles above were prepared under conditions of different parameters followed by detection of pharmacokinetic characteristics of cytokine protein sustained release,stability of protein conformation,and the repairing effects in peripheral nerve defect model.The optimum electrospinning conditions of PLLA beaded fibers were at the concentration of 55mg/ml,the voltage of 10kv,the distance of 20cm and the flow rate of 1ml/h;PVA fibers had the best cross-linking effect after wetting for 48h followed by 4 frozen-thawed cycles.Based on that,PVA fibers were used to coat PLLA beaded fibers loading NGF and the composite fibers produced was found to have ideal surface characteristics and mechanical strength compared with tissue engineered fibers that are produced by other methods.PLGA electrospun fibers loading with VEGF₁₆₅ were prepared by the previous method and the ELISA kit was used to detect the in vitro release quantities of VEGF₁₆₅ in three different kinds of fibers.The results showed that dextran could maintain protein bioactivity and achieve sustained release profile.The nanoparticle composite fibers with coating membrane can achieve better sustained release compared with the nanoparticle composite fibers and the W/O emulsion electrospun fibers.The release percentage of the three fibers（nanoparticle composite fibers with coating membrane,nanoparticle composite fibers and W/O emulsion electrospinning fibers）were 96.17%,92.44%and 84.52%（n=3）respectively after 30 days.The total release of the nanoparticles composite fibers was more than 50000pg（n=3）.At the same time,PLGA beaded fibers loading FITC-BSA dextran nanoparticles was prepared.The results of laser confocal microscopy showed that most of the nanoparticles were evenly distributed in the bead,which verify the idea that the bead structure can control burst release,improve the entrapment rate and maintain the protein activity from the physical structure.In addition,the changes of protein activity of NGF during the preparation of nanoparticles and fibers were investigated by cell proliferation assay.The results showed that NGF protein had little activity loss in the process of preparation of dextran nanoparticles and electrospun fibers and the method could protect protein activity well.Finally,the rat sciatic nerve defect model was established.The NGF-dextran nanoparticles coated fibers were rolled into conduits and then sutured,and the nerve regeneration ability was observed for 8 weeks.The results of HE staining and toluidine blue staining showed greatrepairing effects of nanoparticle beaded fibers and nanoparticle coated beaded fibers group,which were even comparable to autologous nerve graft group,promising great application prospects of them in peripheral nerve regeneration.