Manipulation Of Schwann Cell Migration Across The Astrocyte Boundary By Polysialyltransferase-loaded Superparamagnetic Nanoparticles Under Magnetic Field

Spinal cord injury(SCI)usually causes severely damage for patient with life long disability,which is a heavy burden for his family and society.It is urgent to promote the treatment efficacy after SCI.Schwann cell(SC)transplantation is an attractive strategy for spinal cord injury,however,SCs are glial cells that are responsible for myelination in the peripheral nervous system(PNI)and are not normally found in the central nervous system(CNS).SCs have been shown to secrete neurotrophic molecules and multiple animal studies have demonstrated the ability of SCs to stimulate axonal growth in the CNS,they have been shown to be able to survive and remyelinate axons in the CNS.However,the effcacy of SC transplantation has been limited by the poor migratory ability of SCs in the astrocyte-rich CNS environment and the inability to intermingle with the host astrocyte.This limitation strongly affect the repair effcacy of SC transplantation.Polysialic acid(PSA)is a linear homopolymer of ? 2,8-Nacetylneuraminic acid,which is synthesized on neural cell adhesion molecule(NCAM)by polysialyltransferase(PST)and sialyltransferase X(STX).Many studies report that gene modification of SCs with PST has induced overexpression of polysialylation of NCAM(PSA-NCAM),which is a useful strategy to enhance their integration and migration in the injured CNS.Superparamagnetic iron oxide nanoparticles(SPIONs)have become increasingly well recognized as not only promising non-virus gene transfection vectors but also an attractive approach to drive cell migration in a desired direction under an applied magnetic feld(MF).In this study,we frst attempted to improve the migration of SCs by transferring PST into them,using SPIONs as gene vectors.After magnetofection,a static MF was applied to activate the magnetic vectors to generate cellular tension,in turn orientating the migration of cells along a specifc direction.This study sought to establish a new strategy to improve the integration of SCs into the astrocyte-rich CNS,thus promoting the effcacy of SCs in the treatment of SCIPart ?: Characterizations of PEI-SPIONs and PEI-SPIONs/PST complexes and cytotoxicity analysis of PEI-SPIONs.Objective: To exiamine the characterizations of PEI-SPIONs and PEI-SPIONs/PST complexes by transmission electron microscopy(TEM)and zeta potential/nanometer particle size analyzer.To evaluate the cytotoxicity of PEI-SPIONs and select a optimal transfection concentration.Methods: The morphology,distribution,and average size of PEI-SPIONs were analyzed by transmission electron microscopy.The zeta potential and size distribution of the PEI-SPIONs were evaluated using a zeta potential/nanometer particle size analyzer.Cell viability and cytotoxicity were analyzed using the Cell Counting Kit 8 and live–dead assays.Results: The average diameter of PEI-SPIONs examined by TEM was 28.1±3.6 nm.Results examined by Zeta PALS particle size analyzer was PEI-SPIONs had an average diameter of 186.5±23.4 nm and a positive charge of 19.20±3.2 m V,PEI-SPIONs/PST complexes had an average diameter of 378.8±109.1 nm and a positive charge of 17.51±2.4 m V.The percentage of dead cells was ~3%–5% at concentrations of PEI-SPIONs ranging from 0 to 4 ?g/m L both 24 h and 72 h after magnetofection.The percentage of dead cells increased to 6.4%±1.03% and 6.6%±1.16% when the concentration was increased to 8 ?g/m L at 24 and 72 h after magnetofection,respectively.At 24 and 72 h after magnetofection,the cytotoxicity of PEI-SPIONs was evaluated by CCK-8 assay.No significant difference of cell viability was found between PEI-SPIONs concentrations of 0,1,2,and 4 ?g/m L at 24 and 72 h after magnetofection.However,cell viability was significantly decreased by 19% and 22% when PEI-SPIONs concentration was increased to 8 ?g/m L at 24 and 72 h after magnetofection.Conclusion: These results suggest that dose-dependent cytotoxicity of PEI-SPIONs when used as a transfection reagent was safe and nontoxic for transfection at concentrations,8 ?g/m L.Therefore,PEISPIONs at 4 ?g/m L were used in the remaining experiments of this study.Part ?: Expression of PST and PSA-NCAM in Schwann cell induced by PST magnetofection.Objective: To evaluate the expression of PST and PSA-NCAM in Schwann cell induced by PST magnetofection.Methods: The morphology of magnetofected SCs and the localization of PEI-SPIONs in SCs was observed by SEM and tem.The expression of PST in Schwann cell induced by PST magnetofection was examined by RT-PCR.The expression of PSA-NCAM in Schwann cell induced by PST magnetofection was examined by Western blot and PSA-NCAM immunofluorescent staining.Results: After the magnetofection of SCs(PEI-SPIONs,4 ?g/m L),particles were observed by SEM on the surface of SCs,while those particles were not seen on normal control SCs.A cluster of particles was observed to localize within the cytoplasm of SCs.The m RNA level of PST was signifcantly enhanced when the concentration of PEI-SPIONs increased from 1 to 4 ?g/m L.Additionally,with the increase in weight ratio of PEI-SPIONs/PST plasmids from 1:2 to 1:4,the m RNA level of PST also increased notably.Western blot analysis and the immunocytochemical results demonstrated that PSA-NCAM protein was highly expressed in PEI-SPIONs/PST/SCs but not in normal control SCs.Conclusion: These results obtained from SEM,TEM,PCR,Western blot analysis and immunocytochemical staining suggest that PST was successfully transferred into SCs and that the exogenous PST enhanced the expression of PSA-NCAM in SCs.Part ?: Migration of schwann cells across the astrocyte boundary facilitated by PST-loaded superparamagnetic nanoparticles under magnetic feld..Objective: To investigate whether exogenous PST and PEI-SPIONs can be exploited to enhance the migration of SCs in astrocyte-rich environment and to manipulate the migration direction of SCs on application of an external MF.Methods: In the inverted coverslip migration assay the maximum distance of the migrated SCs from the marked edge was measured,and the number of migrated SCs at specifc migrating distance was counted within 500?m rows.Meanwhile,the angle? between the long axis of each migrated SC and the direction of MF outward were also measured.In the confrontation assay the total number of SCs that crossed the cell boundary and migrated into the astrocyte domains was counted and averaged over six randomly chosen areas per condition.Results: SCs magnetofected with PEI-SPIONs/PST(PEI-SPIONs,4 ?g/m L;PEI-SPION/PST,1:4)were subjected to an MF,the migration capacity of SCs was further enhanced,and SCs migrated preferentially along the axis of the magnetic force on the astrocyte monolayer.The orientation index in the PEI-SPIONs/PST/SC+MF group was signifcantly higher than that of the PEI-SPIONs/PST/SC and SC+MF groups.Additionally,the highest maximum migration distance was achieved by the PEI-SPIONs/PST/SC+MF group(968.89±161.76 ?m).It was also found that,in the presence of an MF,the number of PEISPIONs/PST/SCs(56.8±9.72 cells/500 ?m)that migrated away from the edge of the coverslips was signifcantly higher than that in the absence of an MF(46.3±6.47 cells/500 ?m).PEI-SPIONs/PST/SCs and PEI-SPIONs/DNA/SCs broke the astrocyte boundaries and migrated preferentially along the axis of an applied MF,Furthermore,the number of SCs that crossed the boundaries and migrated into astrocyte domain was highest in the PEI-SPIONs/PST/SC+MF group(44.67±6.18)and was 1.85 times higher than PEI-SPIONs/DNA/SCs.Conclusion: These fndings indicate that PST magnetofection is able to promote migration capacity,and SPIONs with an applied MF are capable of directing the migration of SCs.When PEI-SPIONs/PST and an MF were applied simultaneously,the migration of SCs can be enhanced and directed in astrocyte-rich environment.