Nerve Guidance Conduit Loaded With NT-3 MRNA Delivering Exosomes Promotes The Regeneration Of Peripheral Nerves

BackgroundPeripheral nerves system widely distributes in human body.In China,peripheral nerve injury（PNI）is a common disease in clinical practice,accounting for about 5%of trauma patients.Natural disaster and traffic accident are the main causes of PNI.PNI secondary to tumor,bone malformation and diabetes is very common,in addition,iatrogenic PNI also occurs,such as brachial plexus injury caused by childbirth.PNI often leads to motor and sensory dysfunction,and some patients even suffer from incurable neuropathic pain,which in turn leads to an increased psychological burden and a declined quality of life.At the same time,it also has a tremendous impact on social development and medical resources investment.Therefore,finding effective clinical treatment strategies for PNI is of great significance.At present,autologous nerve transplantation is still the gold standard for clinical treatment.However,autologous nerve transplantation has several limitations,for example,the sacrifice of donor nerve function,neuromas formation and low survival rate in the poor blood recipient area.Recently,with the rapid development of tissue engineering and biomaterials,the application of engineered nerve guidance conduit（NGC）to repair peripheral nerve defect has become a promising treatment strategy.Studies have shown that after PNI,nerve axons express a large number of neurotrophic factor receptors.Neurotrophin-3（NT-3）interacting with its tyrosine kinase C（Trk C）receptor,has great potent in promoting axon regeneration.Maintaining a high concentration of NT-3 environment at PNI area could enhance axon extension to break through the glial scar,bridge the defect,and promote the recovery of regenerated nerve.It has been reported that the application of engineered NGC to peripheral nerve defects can significantly accelerate nerve regeneration and improve its function recovery.However,NT-3 delivery pose difficulties in maintaining its stability,retention duration,sustainable release,therefore,to develop a novel NGC functionalized NT-3 delivery efficiently and effectively is worthy of consideration.Exosomes are lipid bilayer nanovesicles derived from cells,with a diameter of 30-160nm.Exosomes could carry bioactive substances,such as mRNAs,proteins,mi RNAs,and other substances,and transmit the cargos to adjacent or distant cells,which plays a crucial role in intercellular exchange of information and materials.Besides,exosomes are characterized by its low immunogenicity,low biological toxicity,and high targeting.Therefore,exosomes have referred as a promising drug delivery strategy and have broad clinical application prospects.However,realization of the potential of exosome-mediated therapeutic mRNA delivery awaits optimal strategy to improve its delivery efficiency.Sodium alginate is a natural polysaccharide with hydrophilicity,biocompatibility and biological inertness.The structure of sodium alginate hydrogel is similar to natural extracellular matrix,it is beneficial to cell growth,intercellular information exchange,and delivery of various growth factors.Therefore,sodium alginate becomes an ideal biomaterial for regenerative medicine.Studies have reported that by using sodium alginate hydrogel to load therapeutic exosomes,the biological activity and stable release of exosomes can be maintained,and then they can play a role in promoting tissue regeneration and repair.This study aims to:（1）To engineered exosomes to load and deliver NT-3 mRNA effectively;（2）To construct a nerve guidance conduit loaded with sodium alginate-engineered exosomes hydrogel to verify whether it can promote the regeneration of peripheral nerve defects;（3）To improve the efficiency of therapeutic RNA loading of exosomes,an exosome loading system is constructed based on Protection aptamer and Decoy aptamer to provide an optimized strategy.MethodsPart one:construction of engineered exosomes loaded with NT-3 mRNA and verified its delivery efficiency in vitro.Engineered exosomes:NT-3 expressing vector was constructed by molecular cloning technology.The vector was transfected into ADSCs.The exosomes derived from ADSCs(Exo^NT-3)were collected and analyzed the size,morphology and gene and protein expression.Real-time quantitative PCR was used to detect the loading efficiency of NT-3 mRNA in engineered exosomes.Delivery efficiency of engineered exosomes in vitro:DiI-labeled exosomes were co-cultured with rat primary Schwann cells,the endocytosis of exosomes by primary Schwann cells was observed.Real-time quantitative PCR was used to detect NT-3 mRNA in Schwann cells.NT-3 protein expression in Schwann cells were detected by Western blot.Part two:construction of the nerve guidance conduit loaded with sodium alginate-modified exosomes hydrogel and verify its effect on peripheral nerve regeneration in vivo.Construction of the nerve guidance conduit:2%sodium alginate hydrogel was prepared,and the modified exosomes or the control exosomes were loaded within the hydrogel respectively.The release of exosomes in hydrogel was quantitatively detected by BCA analysis.Then,then hydrogel was injected into the silicone catheter（10 mm at length,and1.57 mm at inner diameter）to construct the nerve guidance conduit.Transplantation of the nerve guidance conduit in vivo:A 10 mm long defection of rat sciatic nerve was build.The nerve guidance conduit loaded with sodium alginate hydrogel,sodium alginate-Exo^NT-3 hydrogel or sodium alginate-Exo^empty hydrogel was transplanted at the defect site respectively using microsurgery technology.The autologous nerve transplantation was set as control.The expression of NT-3 mRNA and protein in the regenerated nerve tissue were tested at 2 weeks after transplantation.The rat sciatic nerve injury index was examined at 12 weeks after surgery.After 12 weeks,the electrophysiological function of the regenerated nerves was tested,the cross-sections of the regenerated nerves were observed by HE staining,and NF-200 and S100 immunofluorescence staining.Cross-sectional of gastrocnemius muscle was stained by Masson staining to evaluate the difference in the ratio of muscle fibers and collagen fibers.Part three:promotion of exosome mediated NT-3 mRNA delivery by designed RNA aptamerDesign of NT-3 mRNA Protection aptamer and Decoy aptamer:Protection aptamer was designed containing MS2 stem loops and 17 nucleotides which is complementary paired with NT-3 mRNA adjacent to its initiation codon.Besides,MS2 stem loops linked with another 17nucleotides totally unpaired with NT-3 mRNA was also designed as a comparison.Decoy aptamer which complementary paired with Protection aptamer was also designed.Engineered exosomes:CD9-MCP fusion protein expressing vector was constructed by molecular cloning technology.CD9-MCP expressing vector,NT-3 expressing vector and Protection aptamer were transfected into HEK293T cells.The exosomes derived from donor cells(Exo^{NT-3 Pro})were collected.Decoy aptamer was also transfected into HEK293T cells and the exosomes(Exo^Decoy)were collected.The exosomes were characterized.Real-time quantitative PCR was used to detect the loading efficiency of NT-3 mRNA in Exo^{NT-3 Pro},and Decoy aptamer encapsulation in Exo^Decoy.Detection of exosomes mediated NT-3 mRNA delivery in vitro:DiO-labeled Exo^{NT-3 Pro}and DiI-labeled Exo^Decoy were co-cultured with Schwann cells.The endocytosis of exosomes by Schwann cells was observed.Real-time quantitative PCR was used to detect NT-3 mRNA expression in Schwann cells.Western blot was used to detect NT-3 protein expression.Results1.The constructed NT-3 expressing vector allowed NT-3 mRNA to be expressed in ADSCs and then encapsulated into ADSC derived exosomes.The modified exosomes could deliver NT-3 mRNA to Schwann cells and increase the NT-3 protein level in Schwann cells.2.The engineered exosomes could be stably released in sodium alginate hydrogel for at least2 weeks.Exo^NT-3-NGC increased the level of NT-3 in Schwann cells after its transplantation at sciatic nerve defect site in rats.12 weeks after transplantation,the number of axon regeneration was significantly higher than the control group,and the sciatic nerve injury index was significantly restored.The amplitude and latency of CMAP restored after 12 weeks.Gastrocnemius muscle atrophy was also relieved during nerve regeneration process.3.Optimized exosome-mediated delivery system by RNA aptamer can effectively improve the loading efficiency of NT-3 mRNA,and deliver NT-3 mRNA to recipient cells for stable expression.Conclusions1.Engineered exosomes could be used as an efficient and stable carrier for neurotrophic factors delivery.Combined with engineered nerve guidance conduit construction,engineered exosomes can effectively promote the regeneration of peripheral nerves.2.Construction of sodium alginate hydrogel nerve conduit carrying engineered exosomes to bridge peripheral nerve defects,could realize the stable release of Exo^NT-3 in the injured area,promoting nerve regeneration and myelination,and enhance the reestablishment of the regenerated nerve tissue and the target gastrocnemius muscle,accelerating the recovery of the motor function and atrophied gastrocnemius muscle.2.Optimized exosome-mediated delivery system by RNA aptamer designing could efficiently enrich and deliver the target RNA.This strategy has a promising application for therapeutic RNA delivery.