Functionalized Collagen Ⅰ Scaffold Delivering Specific MicroRNA-enriched Exosomes For Spinal Cord Injury Repair

Spinal cord injury(SCI)is a severe neurological disorder resulting in sensory,motor,and autonomic deficits.Many factors such as the inflammatory microenvironment,cell apoptosis,and glial scar formation contribute to the pathogenesis of SCI and make the treatment extremely difficult To date,no efficient therapies are available for complete recovery from SCI.MicroRNAs(MiRNA)is a small single-stranded non-coding RNA molecule that contains about 22 nucleotides.Mature miRNAs can bind to the 3’ untranslated region(3’ UTR)of target mRNA,functioning mRNA silencing and post-transcriptional regulation of gene expression.Recent studies reported that SCI caused changes in the global miRNAs expression patterns,which indicates that miRNAs may play a pivotal role in the SCI pathophysiology.It has been reported that miR21 inhibits its targeting pro-apoptotic genes to reduce cells apoptosis and then exert neuroprotective effects in SCI.The expression of miR124 was decreased in SCI rats,and it may affect SCI severity.Increasing the expression of miR124 is beneficial to increase the number of neurons to SCI repair.Therefore,miRNA is a novel therapeutic target for the repair of SCI.However,miRNA-based therapies are hampered by the insufficient stability of miRNAs and their poor cellular uptake in vivo.The new efficient miRNA delivery methods need to be developed for the therapy of SCI.Exosomes are extracellular vesicles secreted by cells,containing diverse ingredients including mRNAs,proteins,miRNAs,lipids and metabolites.Exosomes were considered ideal carries for miRNAs delivery for their advantages of low immunogenicity,innate stability and excellent tissue/cell penetration.However,the practical applications of exosomes remain challenging due to their off-targeting,low capacity of specific miRNAs and short time retention in vivo.Therefore,innovative strategies and methods should be developed to deliver,target and entrap the exosomes to the lesion site to promote tissues repair and regeneration.Collagen is a major component of the extracellular matrix(ECM)with the properties of high biocompatibility,low immunogenicity,cell adhesion and biodegradability.Type Ⅰ collagen(Collagen I,Col-I)scaffolds have been used to fill the cystic cavity formed in SCI and promote neuronal differentiation for the repair of SCI.The collagen-binding domain(CBD),a polypeptide consisting of TKKTLRT amino acids,is derived from collagenase and has the excellent ability to bind to Col-I-based biomaterials.A recent study reported a novel dual bio-specificity peptide(BSP)containing a CBD at the N-terminus and a C-terminal peptide(AHLHNRS)that can bind to transferrin receptor(TfR)on the membrane of exosomes.This dual BSP can help to tether the paclitaxel-loaded exosomes to the collagen scaffold that was used to enhance neural regeneration in SCI repair.However,the excess exogenous polypeptides are difficult to be removed from the collagen scaffold,which may raise concerns regarding biosafety,and the binding efficiency may not meet the requirement of high enrichment of exosomes in the collagen scaffold.The aim of this study was to construct a new type of functional collagen-I-based biomaterials through genetic engineering.The scaffolds can enrich the exosomes containing specific miRNAs for the treatment of SCI.First,we constructed the fusion sequence of CBD and lamp2b(exosome-associated membrane protein)into the lentiviral expression vector,and 293T cell line(CBD-Lamp2b-293T)stably overexpressing CBD-Lamp2b was generated via lentiviral transduction.RT-PCR,gel electrophoresis and Western bolt experiments showed that CBD-Lamp2b-293T cells expressed high level of CBD-Lamp2b and secreted the exosomes with CBD-Lamp2b(CBD-Lamp2b-exosome,CBD-LP-EXO).Second,the expression plasmid of miR21 or miR124 was introduced into CBD-Lamp2b-293T cells using liposomes,so that miR21 or miR124 was transiently overexpressed in CBD-Lamp2b-293T cells.Those cells can produce miR21/miR124-loaded CBD-LP-EXO that was named CBD-LP-miR21-EXO/CBD-LP-miR 124-EXO.Nanoparticle tracking analysis,scanning electron microscopy,Western blot and RT-PCR showed that transient technology did not affect the characteristics of exosomes,and could make exosomes containing specific miRNA.Through dual-luciferase reporter assay,immunocytochemistry and Western blot,we confirmed that CBD-LP-miR21-EXO could deliver miR21 into cells,inhibit the apoptosis of PC 12 cells by inhibiting the expression of PDCD4/FASLG.CBD-LP-miR124-EXO delivered miR124 into neural stem cells(NSCs),inhibited the expression of PTBP1 and promoted the differentiation of NSCs into neurons.Finally,the CBD-LP-miRNA-EXO-Col scaffolds were generated by infusing and entrapping CBD-LP-miR21-EXO in the Col-I scaffold and used for SCI repair in vivo.The result of immunofluorescence(IF),TdT-mediated dUTP nick-end labeling(TUNEL)and fluorescence in situ hybridization(FISH)showed that the CBD-LP-miR21-EXO-Col could sustain release of secretes exosomes containing specific miRNAs by extending the retention time of exosomes in the scaffold,inhibited cell apoptosis,reduced the production of glial scars and promoted the survival of neurons in the lesion site.These results indicated that CBD-LP-miR21-EXO-Col promoted functional recovery after SCI.To sum up,our studies developed the novel functional CBD-LP-miRNA-EXO-Col scaffold biomaterials,which can promote SCI repair by the slow-releasing exosomes containing specific miRNAs through the adsorption of genetically engineered exosomes.