Magnetic Virus-like Mesoporous Silica Nanoparticles Loaded With NEP1-40 Across The Blood-brain Barrier And Their Therapeutic Study In Spinal Cord And Cauda Equina Injury

Background:The spinal cord ends at the conus medullaris,and the nerve tract below is called cauda Equina.The spinal stenosis caused by trauma,infection,tumor,degeneration,or others may lead to their acute or chronic compression.Then Spinal Cord Injury?SCI?or Cauda Equina Injury?CEI?occurs,with symptoms including sensory,motor and autonomic nerve dysfunction.Most patients affected are young and suffer from long-term disabilities,which imposes a high psychological and financial burden.Surgical decompression is the main treatment procedure in clinic,with limited functional improvement for the patients with complete injuries.Recently,molecular therapy that can reverse their pathophysiological process and reconstruct nerve circuits has become the focus of research.The regeneration in central nervous system?CNS?is limited partly due to the myelin-derived inhibitors,which include myelin-associated glycoprotein?MAG?,oligodendrocyte myelin glycoprotein?OMgp?,and Nogo-A.All the three inhibitors can bind to the receptor Ng R to inhibit axon growth.NEP1-40,the competitive antagonist of Ng R,can promote axon regeneration and synaptic remodeling in SCI models,with significantly motor functional recovery.Additionally,blocking the Nogo-A/Ng R signaling has been shown to promote regeneration and restored sensory function after brachial cervical dorsal root crush in adult rats.Therefore,NEP1-40 can be a potential candidate for the treatment of SCI and CEI.The intrathecal application of NEP1-40 is widely used,but risks secondary damage and infection.Systemic administration is suitable for the clinical practice,however,challenged by?1?trans-vascular barrier delivery?such as Blood-Brain Barrier,BBB?,?2?targeting release at the injured site,and?3?sustained release of NEP1-40.Mesoporous Silica Nanoparticles?MSNs?,due to their good biocompatibility,the controllable size of particle and pore morphologies,and facile surface-modification,has gained increasing attention in the drug delivery field.Recent data have shown that MSNs could pass through the BBB in vitro and vivo.Given these advantages,the research aimed to constitute core-shell magnetic MSNs with a virus-like roughness surface?named PEG-Fe₃O₄@VSNs?as carrier across the BBB which characterizes with magnetic targeting and real-time biological tracking of drugs by magnetic resonance imaging.Further we evaluated their feasibility in the delivery of NEP1-40 in the rat SCI and CEI models.Methods:1.Magnetic targeting of Fe₃O₄@VSNs across the BBB in vivo.Fe₃O₄@VSNs were synthesized by two-stage method and their characteristics,such as morphology,particle size,were identified.They were further modified by polyethylene glycol?PEG?.Cellular toxicity and uptake of smooth and rough surface PEG-Fe₃O₄@VSNs were evaluated.These two kinds of nanoparticles were injected into rats via tail vein,then magnets were fixed on their heads for 30 min.Magnetic resonance imaging of brain was performed before,30min and 24h after injection respectively.To eliminate the influence of cerebrovascular granule deposition of nanoparticles,iron content of brain tissue was assessed by ICP-AES after cardiac perfusion in rats.2.Therapeutic study of PEG-Fe₃O₄@VSNs loaded NEP1-40 on spinal cord injury in rats.Rat spinal cord injury models were established by weight-drop method and grouped into:sham group,injury group,NEP1-40 treatment group and magnetic nanoparticles loaded NEP1-40 treatment group.BBB scores was evaluated in 3d,1w,2w and 4w after operation.The rats were sacrificed 4w after operation,and their injured and adjacent spinal cords were taken:HE staining was used to evaluate the tissue damage and repairment,TUNEL staining was used to evaluate the neuronal apoptosis,and immunofluorescence staining was used to evaluate the axons outgrowth in the injured site.3.Expression and effect study of Nogo-A in rats with acute cauda equina injury.Rat cauda Equina injury models were established by silicone piece compression method and grouped into sham and injury group.Behavioral,histological,and TUNEL analyses were conducted to assess the establishment of the model.The dynamic expression change of Nogo-A was evaluated using real time-q PCR for 7 days after surgery.Immunofluorescence was used to evaluate the expression of Nogo-A in the DRG and cauda equina.Furthermore,other models were grouped into 4 groups:the sham group,the injury group,the NEP1-40 treatment group,and the JTE-013?the S1PR2 antagonist?treatment group.Behavioral assessments and western blotting were used to evaluate the preliminary therapeutic effect of cauda equina injury via blocking Nogo-A and its receptor.4.Therapeutic study of PEG-Fe₃O₄@VSNs loaded NEP1-40 on cauda equina injury in rats.Rat cauda Equina injury models were established and grouped into:sham group,injury group,NEP1-40 treatment group and magnetic nanoparticles loaded NEP1-40treatment group.Behavioral assessments were conducted before and 1-7d after operation.The rats were sacrificed 7d after operation,and their lumbosacral spinal cords,cauda Equinas and DRGs were taken:HE staining was used to evaluate the tissue damage and repairment of cauda equina,immunofluorescence staining was used to evaluate the axonal outgrowth and remyelination in cauda equina,and TUNEL staining was used to evaluate the retrograde apoptosis in ventral spinal cord and DRGs.Results:1.The Fe₃O₄@VSNs with 160nm diameter presented good dispersion and magnetization.The rough surface did not increase their cytotoxicity,but significantly increased the cellular uptake efficiency.Systemic administration of the particles showed BBB crossing quickly and effectively,making it an ideal trans-vascular barrier carrier.2.Systemic administration of NEP1-40 can promote the locomotor impairment in SCI models,and the effect can be enhanced by PEG-Fe₃O₄@VSNs delivery system.In the injury group,cavity formation was found,and it was surrounded by astrocytes with few axons grown into.The NEP1-40 treatment group showed reduced cavitation/astrogliosis and axonal sprouting?with transverse-direction?in the lesion site.The magnetic nanoparticles loaded NEP1-40 treatment group shown enhanced tissue repairment and axonal regeneration?rebuilt the connection between cranial and caudal axons?.The neuronal apoptosis rate decreased gradually in such pattern:the injury group>the NEP1-40 treatment group>the magnetic nanoparticles loaded NEP1-40treatment group.3.Tactile allodynia and heat hyperalgesia in the CEI model developed as soon as 1day after surgery and recovered to normal at 7 days,which was followed by the downregulation of Nogo-A in DRG neurons.However,the locomotor function impairment in the CEC model showed a different prognosis from the sensory function,which was consistent with the expression change of Nogo-A in the spinal cord.Immunofluorescence results also demonstrated that Nogo A-positive/Neurofilament-negative neurons and axons increased in the DRG and cauda equina 7 days after surgery.Surprisingly,Schwann cells,which myelinate axons in the PNS,also expressed considerable amounts of Nogo-A.Then,after blocking the Nogo-A/Ng R signaling pathway by NEP1-40,significant improvement of mechanical allodynia was identified in the first 2 days after the surgery.Western blotting suggested the NEP1-40 treatment group had lower expression of cleaved caspase-3 than the CEC and JTE-013 treatment group.4.Systemic administration of NEP1-40 can promote the sensory and locomotor impairment in CEI models,and the effect can be enhanced by PEG-Fe₃O₄@VSNs delivery system.The axon degeneration and demyelination can be found in cauda equina in the injury group.The NEP1-40 treatment group showed axons with Neurofilament-positive increased and the MBP signal around the axons was dense and uniform.The axonal regeneration and remyelination was further enhanced in the magnetic nanoparticles loaded NEP1-40 treatment group.The apoptosis rate decreased gradually in such pattern:the injury group>the NEP1-40 treatment group>the magnetic nanoparticles loaded NEP1-40 treatment group.Conclusion:This study has successfully developed PEG-Fe₃O₄@VSNs,which provide powerful magnetic vector for vascular barrier crossing.Applied magnetic field facilitated them accumulated in the targeting site,and they can be tracked in real time by magnetic resonance imaging.Systemic administration of NEP1-40 can promote the axonal outgrowth and remyelination,and decrease the neuronal apoptosis around the injured site in SCI and CEI models,which can be enhanced by delivery with our magnetic MNPs.