Construction Of 3D Nano Drug Delivery System For Nerve Regeneration And Its Repair Application In Spinal Cord Injury

Spinal cord injury（SCI）is a highly destructive and disabling neurological disease caused by vertebral fracture or dislocation,often accompanied with sensory and/or motor defects of varying degrees.Nowadays,clinical treatment can only delay the injury process,reduce the secondary injury,and save the remaining neurons and nerve tissue,preserving the residual axon and nerve function,these treatments cannot reunite the severed nerve fibers.Hence,there is an urgent need for developing effective clinical treatment for SCI.In this paper,nerve cell deletion,inhibitory microenvironment and neural nutrition factor deficiency after SCI are concerned.The study is centered on stem cell tissue engineering,attempt to realize the good combination within seed cells,biological scaffolds and growth factors.Using GelMA as the biological scaffold,and loaded with TG2-EMSCs,SA-TPGS-MS mixed micelles and neural regeneration factor（SHH+NT3+NGF）,the bionic spinal cord scaffold was fabricated by micro-extrusion3D Bio-printing though‘one-step’printing technique.The highly bionic spinal cord scaffold was used for the repairment of spinal cord injury in SD rats.The main content of this paper includes the following five parts.1.Neuroprotection and anti-inflammatory effects of syringic acid,a natural small molecule drugAfter spinal cord injury,the accumulation of inhibition factors（ROS and inflammatory factors）would suppress the regeneration of neurocytes,hence,it is of great significance to explore natural small molecules with neuroprotective and anti-inflammatory effects.In this part,the neuroprotection and anti-inflammatory effects of syringic acid（SA）were evaluated by H₂O₂-induced toxicity in PC₁₂ cells and LPS-stimulated BV2 microglia.Results demonstrated that SA can significantly improve cell survival rates,reduce the LDH leakage,restrain the ROS accumulation.SA can preserve the cell membrane integrity though the inhibition of Ca²⁺influx and MMP decline.Moreover,SA can substantially improve the cell antioxidant capacity,and reduce the lipid peroxidation levels.The removal of ROS by SA ultimately inhibited apoptosis caused by oxidative stress.On the other hand,SA substantially suppressed the NO production,and regulated the inflammatory cytokines（IL-1β,IL-6,TNF-αand PGE2）.Collectively,SA can be a promising natural small molecule drug for the treatments of spinal cord injury.2.Construction and characterization of SA loaded slow-release drug delivery systemLow solubility and rapid elimination of SA hampers the application of SA.In this part,TPGS/F127/F68 mixed polymeric micelles was developed for the delivering SA,in vitro characterization,cellular uptake pharmacokinetics,and neuroprotection of SA-F127/F68/TPGS mixed polymeric micelles（SA-TPGS-Ms）were investigated.Results demonstrated that encapsulation efficiency of SA-TPGS-Ms was 94.39±0.24%,while drug loading was 4.42±0.29%.SA-TPGS-Ms can self-assembly into nano mixed micelles at a lower CMC value（0.00198 mg/m L）.SA-TPGS-Ms mixed micelles were nano-sized（21.18±0.44 nm）,spherically-shaped and homogeneously-distributed（PDI=0.134±0.002）nanoparticles.The high stability of SA-TPGS-Ms was affirmed by testing the robustness to dilution and p H changes.No obvious changes in nanoparticles size and EE were observed during the storage period（45 days）.In vitro release profiles showed that SA-TPGS-Ms realize a steady and sustained release,the release mechanism indicated that SA diffused from the SA-TPGS-Ms via a non-Fickian diffusion phenomenon.In vitro cellular uptake studies revealed that SA-TPGS-Ms substantially promoted cellular uptake with excellent biocompatibility.An effective UPLC-MS/MS method was developed for the determination of SA in bio-samples.Methodological validation demonstrated the high specificity,good linearity（10-2000ng/m L,r~2>0.999）,high precision and accuracy of the established method,which is suitable for the fast and accurate quantitative analysis of SA in bio-samples.The effect of drug concentration on cellular uptake were investigated by Michaelis-Menten function,fitting results showed that SA-TPGS-Ms substantially increased the dynamics parameters(K_mand V_max).The results of endocytosis inhibitors and efflux protein inhibitors showed that the endocytosis was mainly mediated by cage proteins.Cellular uptake pharmacokinetics demonstrated that SA-TPGS-Ms enhanced the drug uptake rate（71.355±6.460 vs.48.027±7.978 ng/h）,delayed the drug elimination rate(0.299±0.053 vs.0.364±0.106 h^-1).In vitro pharmacological studies in H₂O₂-induced toxicity on PC₁₂ cells and LPS-stimulated BV2 microglia showed that SA-TPGS-Ms significantly improved cell survival rates,reduced the LDH leakage,restrained the ROS accumulation,compared to the free drug.Additionally,SA-TPGS-Ms can inhibit the NO production and TNF-αrelease,demonstrating the excellent neuroprotection.Collectively,slow-released SA-TPGS-Ms could be a promising nanocarrier in bionic spinal cord scaffold for the treatments of spinal cord injury.3.Construction and evaluation of self-crosslinking protein long-release systemIn this part,self-crosslinking protein（TG2）long-release system was developed based on EMSCs,and cell biology and transplantation safety of TG2-EMSCs were investigated.Results demonstrated that Ad-TG2-GFP recombinant adenovirus was constructed by pYr-ad Shuttle-8 and Adenovirus Expression Clone system.Agarose gel electrophoresis results and BLAST sequence comparing analysis confirmed the consistency with the target gene（TG2）.Virus titer of Ad-TG2-GFP recombinant adenovirus was 3.435×10~9 PFU/m L.The optimal MOI was selected to be 125 according to the GFP expression strength and protein expression level of TG2.Stable,high expression and long-term release of TG2 protein was achieved in TG2-EMSCs within two weeks.Cell proliferation was not affected by the transfection of Ad-TG2-GFP,while migration capabilities of TG2-EMSCs was enhanced.Annexin V PE/7-AAD apoptosis FACS analysis and WB of apoptosis-related proteins（Caspase-3,Caspase-9,Bax and Bcl-2）showed that Ad-TG2-GFP had no obvious effects on the cell apoptosis.Moreover,positive expression of stem cell markers（CD133,SOX1,Vimentin,Nestin and Snail）confirmed the stemness of TG2-EMSCs.In addition,the toxic effects,tumorigenesis and tumorigenicity of TG2-EMSCs were investigated in nude mice.Results showed that there was no subcutaneous tumorigenesis and no venous tumorigenicity in TG2-EMSCs-transplanted mice within 8 weeks.Also,no obvious changes in heart function index（BNP）,liver function indexes（ALT,AST,ALP,GGT,TBA,and TBIL）,and kidney function indexes（UA,Cr,BUN,and LDH）,as well as no lesion were observed in the organs of TG2-EMSCs-transplanted mice,indicating the good biosafety of TG2-EMSCs,which could be the seed stem cell in bionic spinal cord scaffold for the treatments of spinal cord injury.4.Construction and characterization of 3D GelMA hydrogelIn this part,neuro-regeneration protein factors were screened by 3D inkjet bio-printing,while the bionic spinal cord GelMA hydrogel was fabricated by 3D micro-extrusion bio-printing,physical characterization and biocompatibility of 3D GelMA hydrogel were investigated.Results demonstrated that SHH+NT3+NGF promoted the cell proliferation and migration.Positive expression of nerve-related proteins（MAP2,NF200,Tuj1,Tau,and Synapsin）confirmed that the combination factor can induce EMSCs to differentiate into neurons,indicating SHH+NT3+NGF could be the neuro-regeneration protein factors in bionic spinal cord scaffold.GelMA polymers were successfully synthesized and confirmed by NMR and FT-IR spectrum.GelMA polymers were solidified by UV-crosslinking to form a hydrogel solid phase with suitable mechanical properties.The storage Modulus of 3D GelMA hydrogel was 266Pa,loss factor was 0.0929±0.0119,viscosity was 264.727±10.295 Pa.s.Porous and interpenetrating structures of 3D GelMA hydrogel were observed in TEM images,the pore size was about 60～80μm.The results of swelling properties show that moisture content was 87.69±0.06%,swelling rate was 5.59±0.11 g/g,porosity was 39.22±2.32%,indicating the abundant polyporous structure,which was beneficial to the spread of nutrients.Also,ECM can be simulated by GelMA hydrogel,which facilitated the growth and differentiation of seed cells.Biocompatibility results showed that EMSCs can fully stretched and attached in GelMA hydrogels with good condition.There was no cytotoxicity in GelMA hydrogels via the experiments of CCK-8,Live/Dead staining.cell proliferation and actin expression.The migration capabilities were also not affected by GelMA hydrogel,altogether indicating the excellent biocompatibility of GelMA hydrogels.Moreover,positive expression of stem cell markers（CD133 and Vimentin）confirmed the stemness of EMSCs in GelMA hydrogel.3D GelMA hydrogel could be the scaffold for the treatments of spinal cord injury.5.Repair effects of 3D spinal cord bionic hydrogel in SCI ratsUsing GelMA as the biological scaffold,and loaded with TG2-EMSCs,SA-TPGS-Ms mixed micelles and neural regeneration factor（SHH+NT3+NGF）,the bionic spinal cord hydrogels were fabricated by micro-extrusion 3D Bio-printing,and the repair effects of 3D-printed bionic GelMA hydrogels were investigated in SCI rats.Compared to the scaffold group,seed cell group,and SHH+NT3+NGF group,3D-printed bionic GelMA hydrogels showed obvious improvements in motion function of SCI rats.BBB scores of 3D-printed bionic GelMA hydrogels achieved 11.5±1.87 at 8^th week.Open-field trial showed that the movement of SCI rats was significantly increased and complicated.Hind limb photography and footprint analysis showed that SCI rats had significant recovery of hind limb support function.Treated SCI rats could walk continuously within a short distance,with relatively coordinated movement of front and rear limbs.Pathological examination showed that 3D-printed bionic GelMA hydrogels could fill the cavity formed by SCI,significantly restore the integrity of spinal cord tissue,reduce glial scar holes,and improve the arrangement of spinal cord nerve fibers.Luxol Fast Blue staining indicated that purple neurons were visible in the lesion and caudal area,while BDA staining showed that the 3D-printed bionic GelMA hydrogels could act as a“bridge”to re-connect nerve fibers.Results of immunofluorescence staining demonstrated that 3D-printed bionic GelMA hydrogels could reduce the expression of GFAP,which indicated the inhibition of astrocytes and the decrease of glial scars.NF200-positive expression areas passed through the glial scar at the lesion center,indicating that the nerve cells successfully crossed the glial scar and re-formed connections.In addition,the Tuj1-positive expression areas was not only present in the hydrogel transplant area,but also existed at the upper and lower ends of the truncation,and the Tuj1 positive area passes through the glial scar area,indicating that 3D-printed bionic GelMA hydrogels can significantly promote the regeneration of neuronal cells.Collectively,3D-printed bionic GelMA hydrogels realized excellent repair effects in SCI rats,which provides a new technique and new idea for spinal cord injury treatment.