Transfection Of BDNF-PEG-PLGA Nanoparticles Into Neural Stem Cells For Treatment Rats Of Cerebral Ischemia-Reperfusion Injury

Objective: Construct nano-gene microspheres(PEG-PLGA-BDNF-NPs)with the function of express nerve repair protein.Explore the effects of transfection on exogenous neural stem cells(NSCs)on their biological functions,and the therapeutic effect of exogenous NSCs capable of expressing BDNF with neurorestorative function on cerebral ischemia-reperfusion injury in rats loaded with PEG-PLGA-BDNF-NPs.Methods: PEG-PLGA nanoparticles transfected with BDNF gene plasmids were prepared by double emulsion-solvent evaporation method.The morphology of nanoparticles was observed by scanning electron microscopy and transmission electron microscope.The particle size and zeta potential of nanoparticles were measured by laser particle size analyzer.The entrapment efficiency of PEG-PLGA nanoparticles and the drug loading of BDNF gene plasmids were examined.The in vitro release plasmid of PEG-PLGA nanoparticle transfection complex was tested.The biocompatibility of the nanoparticle transfection complex and the effect of the nanoparticle complex on the morphology of NSCs was detected by CCK-8.The secretion of neural stem cells after transfection of nanoparticles transfected complex was observed by inverted fluorescence microscope.Western blot and quantitative RT-PCR were used to detect the secretion of BDNF.Ischemia-reperfusion model of rats was built by middle cerebral artery occlusion and reperfusion(MCAO/R,occluded 90min)method.Rats were randomly divided into sham operation group(SHAM group),phosphate buffer(PBS)control group,NSCs transplantation group,and PEG-PLGA-BDNF-NPs transfected NSCs transplantation group.Then at the 1st,3rd,14 th and 28 th days after stroke and transplantation,the neurological functions were assessed by m NSS score.Rat head MRI scan on the 28 th day after transplantation and calculation of cerebral infarction volume.The neurological reconstruction of the cerebral ischemic area of MACO rats was evaluated by double-label immunofluorescence staining with frozen sections.Results:(1)The gene-loaded PEG-PLGA nanoparticles were prepared.The encapsulation efficiency and drug loading were high,which had a certain sustained release effect.(2)The neural stem cells with the ability to proliferate and express the nestin protein are isolated and cultured,and can be induced to differentiate into neurons and glial cells.PEG-PLGA nanoparticles containing BDNF gene were highly transfected into neural stem cells,and transfection did not cause significant cytotoxicity.Western-blot and RT-PCR showed that BDNF was abundantly expressed in these stem cells.(3)The rats in the control group and the experimental group all had neurological recovery,while the experimental group was more significant.The difference of m-NSS scores was statistically significant(P<0.05).The cerebral infarction volume of the experimental group was smaller than that of the control group,and the difference was statistically significant(P<0.05).Conclusion:The PEG-PLGA-BDNF nanospheres were prepared by W/O/W double emulsion solvent evaporation method,the rat neural stem cells were successfully extracted and cultured,and the PEG-PLGA-BDNF-NPs were prepared.An exogenous neural stem cell line loaded with PEG-PLGA-BDNF-NPs was first constructed to express BDNF protein with neurorestorative function.And the transplantation of NSCs loaded with PEG-PLGA-BDNF-NPs was more effective in promoting the repair of neurological function after cerebral ischemia-reperfusion injury in rats.