| Nerve growth factor(NGF)is a kind of typical neurotrophic factor,which has been confirmed to promote nerve cell proliferation and induce axon growth.However,NGF has a short half-life,poor stability and it is easy to denature,to achieve a long time sustained release of NGF and to repair the nerve defect,this paper has selected polylactic acid(PLA)and graphene oxide(GO)to build a PLA/GO/NGF electrospinning sustained release drug delivery system.To protect NGF and achieve the sustained-release effect,NGF was coated in BSA nanoparticles by crosslinked with genipin,then drug was encapsuled in emulsion electrospun fibers Therefore,loading drug into PLA/GO/NGF sustained release system is likely to achieve the purpose of repairing peripheral nerve injury.Firstly,the preparation and characterization of PLA/GO electrospun fibrous membrane were discussed.Furthermore,the best electrospinning process conditions were screened by orthogonal experiment.The results showed that temperature was 25 ?,relative humidity was 50%,and the concentration of spinning fluid was 12%,voltage was 15 kV,feeding speed was 0.04 mm/min,and the distance was 15 cm.The morphology of the PLA/GO electrospun fibers were examined by scanning electron microscope and transmission electron microscope.The structure and composition of membrane were characterized by fourier transform infrared spectroscopy and Raman spectroscopy.The electrical conductivity of the PLA/GO electrospun fibrous membrane were investigated by Four-Point probe method.The porosity of electrospun fibrous membrane was measured by the method of pycnometer.The results indicated that graphene oxide could be dispersed highly into PLA nanofibers.The conductive property was increased when the right amount of GO was mixed with PLA.Along with adding GO higher,the greater the porosity of the fibrous membrane.The elongation rate of fiber achieved 125.4±1.46% by mechanical properties testing,which was higher than pure PLA.In addition,with addition of GO,degradation rate of fiber membrane to reduce.Secondly,the performance of sustained release drug delivery system was studied.NGF/BSA nanoparticles were wrapped in emulsion electrospun fibrous membrane.NGF release properties in vitro were discussed by enzyme-linked immunosorbent assay.The results show that load contents of NGF was(48±1.12)ng and the release period of NGF was about 35 days,the cumulative release rate was about 85.2%.NGF could achieved sustained release.The emulsion electrospun fibers are very promising in the application of peripheral nerve to achieve the effective and sustained release of NGF.Finally,to investigate the biological function of PLA/GO/NGF sustained release drug delivery system,the method of CCK-8 was selected to evaluate the cytotoxicity of electrospun fibrous membrane.The RSC96 rat schwann(RSC96)cells and bone marrow mesenchymal stem cells(BMMSCs)were cultured with PLA/GO/NGF sustained release drug delivery system to evaluate cells adhesion and proliferation by the immunofluorescence staining and scanning electron micrograph.The PC12 cells were used for the bioactivity of NGF.The immunofluorescence staining and calculation of stretch ratio were employed to study the differentiation of PC12 cells.The drug delivery system has low cytotoxicity and was conducive to the RSC96 cell and the adhesion or growth of BMMSCs cell.The PC12 cells were used for the bioactivity test of the released NGF and were co-cultured with the sustained release liquid for 1,7,14 days,it could differentiate into neuronal phenotype.The results showed that the PLA/GO/NGF sustained release drug delivery system contribute to protect the activity of NGF and it also has good electrical conductivity.Above studies indicated that PLA/GO/NGF drug delivery system could extended the release period of NGF and has a good electrical stimulation effect.In addition,it could effectively promote nerve regeneration and has potential application prospect in the field of neural tissue engineering. |
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