Construction Of Targeting Gene Delivery System Based On Stem Cells Or Exosomes And Its Application In Ischemic Stroke Treatment

It is very critical to choose the drug carrier that can effectively target to the injury area of brain in the treatment of brain diseases.In recent years,synthetic drug carriers like liposomes and nanoparticles are commonly used.As the research moving on,biological carriers from the organism gradually began to be focused.Stem cell transplantation has been used for repairing tissue defects and enhancing functional recovery due to their potential of multi-directional differentiation and paracrine function.Moreover,stem cell-derived exosomes have been attracting attention because exosomes serve as the important tool of factors secretion.Ischemic stroke is a kind of neurological diseases with high mortality and high disability.It is necessary to design a targeting carrer to deliver drugs to the brain to increase survival rate and further reeonstruct the nerve function in the treatment.It is becoming the focus to establish a targeting therapeutic biological carrier based on stem cells or exosomes delivering gene or drugs for ischemic stroke treatment.It's expected to offer new treatment options for clinical application.This study aims to construct a novel targeting therapeutic biological carrier that is based on stem cell/exosomes therapy and genetic modification so as to the improvement of survival rate and nerve tissue repair after severe ischemic stroke injury.Brain-derived neurotrophic factor(BDNF)gene modified neural stem cells(NSCs)and exosomes secreted by NSCs after hypoxia culture are constructed to treat with ischemic stroke respectively.Firstly,we developed the reactive oxygen species(ROS)-responsive charge-reversal polymeric vector,which mediated efficient gene transfection of NSCs to express more BDNF,thereby enhancing their therapeutic effect in ischemic stroke treatment.Secondly,NSCs-derived exosomes were isolated and used as biological carrier.In order to achieve better neurological recovery,the NSCs were hypoxia eultured and exosomes seereted were eollected.The membrane protein and therapeutic gene in exosomes would be regulated.Through in vitro and in vivo studies,the ability of NSCs and exosomes to secrete and carry therapeutic substances was evaluated,and the improvement of survival rate and nerve recovery of cerebral ischemic injury was discussed.To develop a gene transfection system with high efficiency and safety for NSCs in infarct brain with high level ROS,we presented a ROS-responsive charge-reversal vector called poly[(2-acryloyl)ethyl(p-boronic acid benzyl)diethylammonium bromide](B-PDEA),that mediated efficient gene transfection in NSCs.The luciferase gene was used as a reporter gene to carry out conditional exploration and systematic study on gene transfection of B-PDEA on NSCs.The results displayed that B-PDEA can achieve two orders of magnitude higher gene transfection efficiency on NSCs comparing with other common non-viral vectors such as polyetherimide,Lipofectamine 2000 and so on.It also had lower toxicity on NSCs.The intracellular transport behavior of B-PDEA were studied and the results showed that B-PDEA was uptake through the clathrin pathway.Due to the boric acid groups in the structure,B-PDEA/plasmid polyplexes could be uptake by NSCs within 5 min,and escaped from the lysosome within 0.5 h,then released the plasmid into the nucleus for expression.Further investigation indicated that B-PDEA upregulated the transfection efficiency of NSCs by nearly 8 times with the increasing level of ROS in the transfected environment,which proved that B-PDEA was suitable for the transfection of NSCs in the ischemic injury site with therapeutic gene.The therapeutic ability of NSCs as a biological carrier for the treatment of ischemic stroke was examined.After transplanting NSCs into the middle cerebral artery occlusion(MCAO)model mice by intravenous injection through tail vein,NSCs could be gathered in the cerebral ischemic injury site.The modified NSCs with similar targeting ability could significantly increase the total amount of BDNF in the brain after transplantation.The survival rate of model animals could be greatly improved in 28 days,from 0%(without treatment)to more than 60%(transplanted with gene modified NSCs),which 1s much better than the transplantation with non-transfected NSCs group of about 20%.And the behavioral experiments showed that transfected NSCs transplantation promoted the recovery of motor and sensory function as well as regeneration of injuled tissue.As a novel biological carrier,the therapeutic function of stem cells is partly derived from secretory.Exosomes,the vesicles that deliver informations between cells retain the superiority of stem cell therapeutic ability as a biological earrier,and also are safe and can be modified.Exosomes can prevent uncontrollable risk of stem cells migration and differentiation in vivo and break through the limitation of rough disease microenvironment to the therapeutic function.In this study,exosomes secreted by NSCs(EXOs)were isolated by gel exclusion method,identified and analyzed.EXOs would concentrate in the vicinity of brain lesion after injected into the MCAO model mice.The outcomes indicated a similar brain targeting ability as NSCs.To further improve the targeting ability of EXOs,some modifications were manipulated.Firstly,the targeting peptide was inserted to the surface of the exosomes,but the targeting ability was not improved.Secondly,NSCs were incubated with iron oxide nanoparticles or hypoxic cultured before exosomes isolation.The results illustrated that the expression of receptor-associated receptor CXCR4 on the surface of exosomes secreted by NSCs incubated with iron oxide nanopartieles didn't change significantly.The expression of CXCR4 on the surface of exosomes secreted by hypoxic NSCs(H-EXOs)can be increased significantly comparing with EXOs.There was no apparent change in shape.However,the targeting ability was not improved.Preliminary sequencing analysis revealed that part of miRNA in H-EXOs was significantly up-regulated or down-regulated compared with EXOs.The related miRNA regulates the pathways related to nerve regeneration,synapse formation,cell proliferation and tissue regeneration.It illustrated that the membrane surface proteins could be shifted while preserving its inherent morphology through the hypoxia culture of NSCs.As a matter of fact,the gene pharmaceutical components and its amounts would also be indirectly changed.To investigate the treatment effect of exosomes as a novel biological carr1er,EXOs and H-EXOs were transplanted into MCAO model mice separately by intravenous injection through tail vein.The results showed that the survival rate in EXOs group(70%)was higher than NSCs group,but the recovery and final scores in behavioral experiments were not as good as NSCs.H-EXOs combined the advantages of EXOs and NSCs,the survival rate was further improved to about 90%,meanwhile ensuring the reconstruction of exercise and sensory functions.It promoted the regeneration of ischemic brain tissue and reduced the lesion area compared with EXOs.After treatment,the recovery condition in EXOs group was comparable to NSCs group and the survival rate was improved to about 90%.Finally,considering the safety of stem cell therapy,the toxicity and tumorigenicity of NSCs were investigated.When a single intravenous injection of NSCs at a dose of 1×103 cells or 1×106 cells,there was no significant toxicity in the whole period of the 4-week toxicity observation period.The observation indexes showed no abnormalities and the body weight enhanced continuously,which didn,t have significant difference to negative control group.After observation,the heart,lung,liver,spleen,kidney and brain were examined.The results showed no obvious toxicity and the weight of each organ had no significant difference compared to the negative control group.For the tumorigenicity of NSCs,after subcutaneous administration of NSCs at a dose of 1×106 cells or 5×106 cells,no tumor was produced in the 10-week observation pernod,and the body weight had no significant difference compared to the negative control group.There was no tumor cell generated in all major organs,and no tumorigenic effect was observed.These results indicated that NSCs is safe for transplantation therapy.This study constructed stem cells and exosomes as novel targeting therapeutic bio-vector for therapeutic gene targeting delivery and treatment of ischemic stroke.The non-viral gene transfection system of NSCs was studied.The membrane surface of NSCs derivate exosomes was modified and therapeutic gene carried was regulated as well.It was constructed the method for enhancing the drug carrying capacity and therapeutic function of biological carrier.This study examined the recovery of ischemic stroke model animals after transplantation and gradually revealed the function of NSCs,BDNF gene-modified NSCs,EXOs and H-EXOs in the repair of cerebral ischemic injury.This study provides a promising strategy for stroke treatment as well as successful examples for research of construction of biological targeting gene delivery system.