Construction Of Selenium Nanomaterials For The Treatment Of Spinal Cord Injury

Background:Spinal cord injury(SCI)is caused by the spine fracture after trauma and violence,which result in sensory,motor,autonomic dysfunction,neurogenic bladder disorders,etc.This has seriously affected the patients'quality of life,as well as placing a heavy burden on the family,society,and medical care system.Currently,surgical decompression is often performed in order to decompress the spinal cord compression and stabilize the biomechanical alignment in clinic.However,the effect of treatment is dependent on various conditions,such as,the degree of injury,the time slot between the injury moment and the time to arrive at the hospital,and patients'tolerance.Spinal surgeons often administrate high-dose methylprednisolone during the acute phase of SCI.Previous studies have shown the dubious curative effect of high-dose methylprednisolone,and it is often accompanied with complications such as infection,gastrointestinal bleeding,and pulmonary embolism.Therefore,the application of high-dose methylprednisolone for SCI still has great limitations.It is urgent to explore effective methord to treatment of SCI in clinic.The poor therapeutic effect of SCI may be related to the dynamic complex cascade pathological process.This complex pathological process mainly includes the following four aspects:first,the spinal cord hemorrhage,ischemia,hypoxia,and thrombosis;second,the blood-spinal cord barrier destroyed after SCI,which facilates the infiltration of inflammatory cells(neutrophils,lymphocytes,macrophages)into the lesion site,and these inflammatory cells release pro-inflammatory cytokines,such as IL-1?and TNF-?;third,inflammatory cells and Fenton reactions produce excess reactive oxygen species(ROS),which will break the redox balance,and then damage nucleic acids,proteins and lipids,leading to the apoptosis of neurons and oligodendrocytes;fourth,the apoptotic product further recruiment inflammatory cell and inflammatory cell secrete proinflammatory cytokines,aggravating the inflammatory response and worsening the microenvironment of lesion site.Based on the above-mentioned complex pathological processes,the treatment of SCI mainly focused on neuroregeneration and neuroprotection.Neuroregeneration effect tends to use transplanted stem cells(marrow mesenchymal stem cells,embryonic stem cells,etc.)or promote the regeneration of local stem cells.However,the poor microenvironment after SCI is not favorable to the survival of transplanted stem cells or local stem cell regeneration,which limits its therapeutic effect and clinical application.Neuroprotection effect mainly tends to inhibit or ameliorate the complex pathological process of SCI,to further ameliorate the poor microenvironment and improve the tolerance of neurons and glial cells,finally promote functional recovery.Objective:With the rapid development of nanomaterials and board application to clinical medicine,nanomaterials have been studied extensively in neuroprotection.The nanomaterials have good biocompatibility,as well as effective ROS scavengeing ability and inhibition of inflammation,which is widely studied in SCI treatment.Herein,we firstly designed and synthesized Se-CQDs with ROS scavenging ability,and biocompatibility,antioxidant properties,inflammation inhibition and neuroprotection effect of Se-CQDs were systemically investgated.Moreover,we have further optimized and designed HA-Se nanoparticles with both scavenging ROS ability and targeting properties and investigated the protective effect of HA-Se nanoparticles in SCI treatment in vivo and in vitro.Methods:(1)Water-soluble Se-CQDs were prepared by hydrothermal treatment of L-selenocystine.The particle size,morphology and zeta potential of Se-CQDs were determined by dynamic light scattering(DLS),transmission electron microscope(TEM),Zetasizer Pro,respectively.The structure of Se-CQDs was characterized by¹H and ¹³C nuclear magnetic resonance(NMR),Fourier transform infrared,X-ray surface photoelectron,Ultra-Violet Ray and Fluorescence spectrophotometer.The free radical scavenging efficiency of Se-CQDs was detected by DPPH.In vitro study,the biocompatibility of Se-CQDs(6.25,12.5,25,50,100,200?g/m L)was evaluated through MTT assay against astrocytes,PC12,and N2a cells.Then,protective effects of Se-CQDs on astrocytes,PC12,and N2a cells were determined under an oxidative stress simulated by addition of H₂O₂(250?M).In addition,the inhibitory effect of Se-CQDs on the expression of inflammatory factors(IL-1?,IL-6,TNF-?)in LPS-stimulated BV2 cells was investigated by the ELISA method.In vivo study,the spinal cord contusion model was established with standard spinal cord percussion apparatus.After the contusion,saline or different concentrations(250?g/m L,1000?g/m L)of Se-CQDs were given in subdural via Hamilton syringe.Experiments are grouped into saline,Se-CQDs(250?g/m L),Se-CQDs(1000?g/m L)seperately.The whole animal protein from the injured spinal cord was extracted 24 hours after the operation,and western blot was performed to evaluate the expression of Caspase-9,Cleaved Caspase-3,Bcl-2,and Bax in vivo.Five days after the operation,the injured spinal cords(1 cm)were collected and immunofluorescence staining(CD68,activated microglia)was used to evaluate the anti-inflammation effect of Se-CQDs in vivo.The motor function was evaluated with BBB score every week.On 8 weeks after operation,the rats were sacrificed.The protective effect of Se-CQDs on SCI was evaluated by general imaging of the spinal cord,bladder function,hematoxylin and eosin(H&E)and Masson trichrome stained bladder,H&E and LFB stained spinal cord as well as immunofluorescence staining labeling astrocyte,glial scar and neuron(CS56&GFAP and Neu N&NF200).(2)Hyaluronic Acid(HA)as a stabilizer in an aqueous solution was used to synthesize HA-Se nanoparticles through a redox system.The particle size,zata potential and morphology of HA-Se nanoparticles were determined by dynamic light scattering(DLS),Zetasizer,transmission electron microscope(TEM),respectively.The structure of HA-Se nanoparticles were characterized by Fourier transform infrared,X-ray surface photoelectron spectroscopy.The free radical scavenging efficiency of HA-Se nanoparticles were detected by DPPH.In vitro study,the biocompatibility of HA-Se nanoparticles(3.125,6.25,12.5,25,50,100?g/m L)were evaluated with MTT assay against astrocytes,PC12 cells.Then,the anti-oxidative stress effect of HA-Se nanoparticles on astrocytes was determined in the presence of H₂O₂(100?M).In addition,the expression of inflammatory factors(IL-1?,IL-6,TNF-?)in BV2 cells stimulated by LPS alone or LPS combined with HA-Se nanoparticles was investigated by the ELISA method.In vivo study,the spinal cord contusion model was established with standard spinal cord percussion apparatus.Western blot was performed to evaluate the expression of CD44 between the SCI group and the sham group.Moreover,co-localization of immunofluorescence staining with CD44 was used to explore the cell type.To investigate the targeting efficiency of HA-Se nanoparticles in the injured spinal cord,NH₂-CY5 labeled HA-Se nanoparticles were administrated to localize the distribution of nanoparticles.Fluorescence in the collected tissue were imaged and analyzed by animal imaging system.To evaluate the anti-apoptois effect of HA-Se nanoparticles,the whole animal protein of the injured spinal cord was extracted,and western blot was performed to detect the expression of apoptosis-related protein(Cleaved caspase-3).To evaluate the anti-inflammation effect of HA-Se nanoparticles,the rats were sacrificed at 5 days after operation.Inflammatory cells in spinal cord were stained with CD68&Iba-1.Therapeutic effect of HA-Se nanoparticles for SCI was evaluated through BBB score every week.After that,the protective effect of HA-Se on SCI was evaluated by general imaging of spinal cord,H&E and LFB stained spinal cord,as well as immunofluorescence staining of neuron and axon(Neu N&NF200).Results:(1)Se-CQDs with good solubility and uniform size(34.5±3.3 nm)were prepared.Se-CQDs has limited cytotoxic effect on N2a cells,PC12 cells,and astrocytes at a concentration up to 200?g/m L.The clearance rate of DPPH with 62.5?g/m L,125?g/m L,250?g/m L,and 500?g/m L Se-CQDs were 56.4%,72.1%,91%,98%,respectively.Under simulated oxidative stress environment(250?M H₂O₂),Se-CQDs can effectively improve the survival rate of N2a,PC12 cells and astrocytes.Se-CQDs significantly inhibit LPS-activated BV2 cells to produce IL-1?and IL-6.In addition,Se-CQDs can effectively reduce the cavity area of the spinal cord,demyelination rate and inhibit inner membrane thicken and fibrosis of bladder as well as protect neurons in SCI rats when compared to saline group.At the same time,it can effectively inhibit the expression of Bax and Cleaved Caspase-3,Caspase-9,promote the expression of Bcl-2.BBB score and spontaneous urination recovery indicated that compared to saline,Se-CQDs improving hinder limb motor function and bladder function.(2)HA-Se nanoparticles had a core-shell structure with good water dispersibility and uniform size(95±2.3 nm).HA-Se nanoparticles have limited cytotoxicity to PC12 cells and astrocytes at a concentration up to 100?g/m L in vitro.Scavenging efficiency of DPPH situmated with 250?g/m L HA-Se nanoparticles at30 min,60 min,90 min,and 150 min were 0.8%,5.9%,9.7%,14.5%respectively.Scavenging efficiency of DPPH situmated with 1000?g/m L HA-Se nanoparticles at30 min,60 min,90 min,and 150 min were 14.6%,19.5%,25.7%,31.8%respectively.Under the 100?M H₂O₂simulated oxidative stress environment,HA-Se nanoparticles can effectively improve the survival rate of astrocytes.In addition,HA-Se nanoparticles significantly inhibit the production of IL-1?,IL-6,and TNF-?in LPS stimulated BV2.We demonstrated that the increased expression of CD44 was located at astrocytes and part of microglia after SCI.Meanwhile,stimulating astrocytes with LPS indeed increase the expression of CD44.HA-Se nanoparticles was demonstrated to target the injuired spinal cord tissue effectively,reduce the cavity area of spinal cord,inhibit demyelination,protect neurons,and inhibit inflammatory cell infiltration.In addition,it can effectively inhibit the expression of apoptotic proteins(Cleaved Caspase-3)and improve the motor function of the hinder limbs.Conclusions:(1)Se-CQDs have a good biocompatibility,can efficiently scavenge ROS,as well as reduce oxidative stress and inflammatory cell infiltration after SCI,leading to significant improvement of the neurological function after SCI.Se-CQDs is expected to become a promising SCI treatment strategy.(2)CD44 is highly expressed in astrocytes after SCI.HA-Se nanoparticles can specifically bind to CD44,which give HA-Se nanoparticles the ability to target the injuried spinal cord.Moreover,HA-Se nanoparticles can significantly inhibit inflammatory cell infiltration,reduce cavity area of spinal cord,promote neuronal survival,and greatly improve neurological function of SCI rats.In this study,we provide new strategy for the accurate targeting treatment of SCI.