Intranasal Administration Of Engineered Exosome-Mediated BDNF In Myelin Regeneration

The myelin sheath of the central nervous system is formed by oligodendrocytes differentiated from oligodendrocyte progenitor cells(OPC)wrapped around neuronal axons.The myelin sheath is crucial for the normal functioning of nerve system.Multiple sclerosis(MS)is an autoimmune demyelinating disease of the central nervous system,which is one of the main causes of paralysis in young adults.The regeneration and repair of myelin sheath is the ultimate therapeutic goal of demyelinating disease.Currently,most of the therapeutic drugs for MS targeting the immune system,which can only slow down the development of the disease,but cannot promote the differentiation of OPC abundant in myelin injury region into mature oligodendrocytes to form new myelin sheath.The key problem of the treatment of demyelinating diseases of the central nervous system is to achieve efficient brain-targeted drug delivery across the blood brain barrier and promote the differentiation of endogenous OPC into mature oligodendrocytes.Brain-derived neurotrophic factor(BDNF)plays an important role in the regulation of OPC proliferation and the differentiation of OPC into mature oligodendrocytes,which is of great significance for myelin regeneration and repair.BDNF protein can not be directly used for the treatment of diseases due to its short half-life,high viscosity and poor permeability of blood-brain barrier.Exosomes,as nanoscale membrane vesicles secreted by cells,can be used as potential therapeutic drugs and drug delivery vectors for central nervous system diseases.Loading BDNF into exosomes can effectively overcome the disadvantage of its difficulty in becoming a clinical drug,and making BDNF accessible for disease treatment.However,exosomes are mostly taken up by the liver after intravenous administration,and have a low efficiency of entering the brain through the blood-brain barrier.The intranasal administration is a brain-targeted delivery route that can bypass the blood-brain barrier and directly enter the brain.The study of efficiency of brain targeted engineered exosomes loaded with BDNF entering the brain after intranasal administration and the therapeutic effect on demyelinating mice is of great clinical value for efficient targeted drug delivery and treatment of central nervous system diseases.Firstly,we constructed brain-targeted RVG-lamp2B-HA fusion protein and BDNF overexpression lentiviral vectors,then obtained stable RVG-lamp2B-HA and BDNF overexpression cell lines by infecting cells with lentivirus particles.By expanding culture of stable cell lines and collecting the cell culture supernatant,exosomes with RVG brain-targeted peptide and high level of BDNF(BDNF protein and mRNA)were obtained.Proving that brain targeted modification and enrichment of specific cargos in exosome can be realized by genetic engineering of cells.The enrichment of BDNF in exosomes can effectively overcome the disadvantage of its difficulty in becoming a clinical drug,and also provide a new idea and research basis for the application of other cellular active factors with important physiological functions.Secondly,engineered exosomes were used to coculture with neural progenitor cells(NPC),oligodendrocyte precursor cells derived from NPC differentiation(N-OPC)and primary OPC to investigated the regulatory effects of the brain-targeted engineered exosome-mediated BDNF on oligodendrocyte differentiation.The results showed that brain-targeted engineered exosome-mediated BDNF promoted the activation of MAPK/Erk1/2 signaling pathway,and significantly promoted the differentiation of N-OPC,OPC and NPC into mature oligodendrocytes.Thirdly,We investigated the efficiency of intranasal and intravenous administration of engineered exosomes into the brain and the effect of brain-targeted modifications on the efficiency of exosomes into the brain.The results showed that exosomes administered through the caudal vein are heavily trapped by the liver,and only a small amount of exosomes could cross the BBB and enter the brain.Excitingly,most of the exosomes entered the brain except for a small amount of retention in the nasal cavity after intranasal administration;Brain-targeted modification of exosomes can significantly increase the efficiency of their entry into the brain after drug administration.Exosome intranasal administration is a brain targeted route of exosome delivery,which avoids systemic circulation and liver first pass effect of intravenous administrationand.Brain targeting modification of exosome further improves the efficiency and precision of brain targeting for exosome intranasal administration,significantly improves the efficiency of exosomes entering the brain,and provides a more effective and efficient drug delivery strategy for the treatment of central nervous system diseases.Finally,we investigated the therapeutic effects of brain-targeted engineered exosomes mediated-BDNF intranasal administration on myelin regeneration of central nervous system demyelinating mice.The demyelination mouse model of the central nervous system was constructed by feeding mouse with rodent chow containing 0.2%Cuprizone(Bisoxaldihydrazone,CPZ)for 6 weeks.Then CPZ mice were intranasally administrated with engineered exosomes for 2 weeks.Brain-targeted engineered exosome-mediated BDNF was found to significantly improved the motor coordination ability of CPZ mice through by rotoard test and pole test.Detection of brain tissue samples showed that Brain-targeted engineered exosome mediated BDNF significantly promoted the number increase of OPC,the differentiation of mature oligodendrocytes and remyelination in the corpus callosum region of CPZ model mice,and effectively promoted myelin regeneration in animal models of demyelinating disease of central nervous system.In this study,brain-targeted modification of exosomes and intracellular loading of BDNF were achieved through engineering modification of cells.Intranasal delivery of brain targeted engineered exosome mediated BDNF was highly effective targeted delivery to the central nervous system,and had a significant therapeutic effect on myelin regeneration in CPZ model mice.These findings provide a research basis for efficient targeted drug delivery and treatment of central nervous system diseases.