AuNPs@PDA-PLGA Composite Materials Combined With Electrical Stimulation In Spinal Cord Injury Repair

BackgroundSpinal cord injury can lead to motor and sensory dysfunction below the injured level.How to effectively promote the recovery of motor function after spinal cord injury has been a hot topic in clinical research.Although the body will generate electric field in vivo after injury to stimulate the proliferation and differentiation of endogenous neural stem cells(NSCs)to start the in situ repair mechanism,the effect of endogenous repair is not obvious under the influence of adverse factors such as inflammatory microenvironment and oxidative stress.Electrical stimulation can activate signaling pathways in vivo through a variety of ion channels,regulate molecular transport as well as signal transduction in the body,and promote both repair and regeneration of various tissues and organs.After spinal cord injury,nerve cells die and the original nerve signal transmission pathway is destroyed,making it impossible for impulsive electrical signals to pass through the injured site to the other side.Using materials and external stimuli to enhance the effect of local electrical stimulation can further promote the reconstruction of nerve signal transmission pathway,which is conducive to the functional recovery after spinal cord injury.Polylactic-co-glycolic acid(PLGA)is a kind of high molecular compound with good histocompatibility and mechanical property,which has been widely used in tissue engineering,but PLGA alone is not enough to meet the demand for spinal cord injury repair.Gold nanoparticles(Au NPs),a kind of metal particles with good electrical conductivity and biocompatibility,can improve the physical and chemical properties of materials and enhance the therapeutic effect of electrical stimulation.Therefore,in order to maximize the therapeutic effect of materials and electrical stimulation on spinal cord injury,this study aims to construct a polydopamine coated PLGA multifunctional composite material loaded with gold nanoparticles(Au NPs@PDA-PLGA),which endow the material with both good electrical conductivity and biological properties,and the combined treatment with electrical stimulation maximizes the repair of spinal cord injury.PurposeThis study plans to construct a polydopamine coated PLGA composite loaded with gold nanoparticles,by regulating the physical and chemical properties and biological properties of the composite,the therapeutic effect of NSC on spinal cord injury is promoted.Cell experiments were conducted to verify the promoting effect of composite materials combined with electrical stimulation on the proliferation of NSCs and the directional regulation effect of NSCs on nerve cell differentiation.The composite material was transplanted in situ into the spinal cord injury site of rats,and combined with electrical stimulation to promote in situ nerve cell regeneration and pathway reconstruction,reduce inflammatory cell infiltration and glial scar formation,and achieve effective recovery of motor and sensory functions after spinal cord injury.Method1.Preparation of Au NPs@PDA-PLGA composite materials.A PLGA composite coated with Au NPs was constructed based on the reducing and adhesion of Polydopamine(PDA).Energy dispersion spectrometer(EDS)and X-ray diffractometry(XRD)were used to identify the composition of the PLGA composite.The surface morphology,hydrophilicity,conductivity and other physicochemical properties of Au NPs@PDA-PLGA composite were assess by scanning electron microscopy(SEM),atomic force microscope(AFM),contact angle measuring instrument,four probe conductance instrument and other instruments.2.Investigation on biological properties of Au NPs@PDA-PLGA composite materials.BCA kit and ROS kit were used to detect the protein adsorption capacity and reactive oxygen scavenging capacity of Au NPs@PDA-PLGA composite,respectively.NSCs were inoculated on the surface of the composite,and the cytotoxicity of Au NPs@PDA-PLGA composite was evaluated by CCK-8 and cell live-death staining.The Au NPs@PDA-PLGA composite material was implanted into the animals,and the inflammatory response and pathological changes of the organs following transplantation were evaluated by HE staining.3.The regulatory effect of Au NPs@PDA-PLGA composite materials combined with electrical stimulation on NSCs.NSCs were extracted from the hippocampus of fetal mice and inoculated in different materials for culture.CCK-8 and immunofluorescence staining were used to investigate the proliferation and cell activity of NSCs under different composite materials and electrical stimulation parameters.Immunofluorescence staining was used to investigate the differentiation and proliferation of NSCs under the effect of Au NPs@PDA-PLGA composite material and electrical stimulation.Finally,RT-PCR was used to validate the effects of Au NPs@PDA-PLGA composite materials and electrical stimulation on the differentiation trend of NSCs.4.Au NPs@PDA-PLGA combined with electrical stimulation on functional recovery of spinal cord injury.The model of spinal cord injury in rats was constructed and the composite materials were implanted into the injured sites and electrical stimulation was given after operation.BBB score,gait analysis and electromyography were used to evaluate the recovery of motor and sensory functions after spinal cord injury.HE,LFB and transmission electron microscopy(TEM)were used to evaluate the spinal cord tissue recovery and myelin regeneration.Finally,immunofluorescence staining was used to observe the regeneration of nerve cells and myelin sheath after spinal cord injury,inflammatory cell infiltration and glial scar formation in the injured area,and to explore the potential mechanism of Au NPs@PDA-PLGA combined with electrical stimulation in spinal cord injury repair.Result1.The presence of gold element was found in the prepared composite by EDS and XRD analysis,which proved that Au NPs were successfully grafted on the surface of the material,and Au NPs@PDA-PLGA composite loaded with Au NPs was successfully prepared.The hydrophilicity and electrical conductivity of Au NPs@PDA-PLGA composite modified by Au NPs were significantly improved compared with PLGA and PDA-PLGA.The coarse surface morphology of Au NPs@PDA-PLGA composite material was observed in SEM and AFM detection,which promoted electrical stimulation effect and was conducive to cell adhesion growth.2.The results of cell survival and death staining showed that NSCs inoculated on Au NPs@PDA-PLGA composite material grew well,and no obvious pathological changes were observed in HE staining of the rat organ tissue implanted with the composite material,which proved that Au NPs@PDA-PLGA composite material had good biological compatibility.It lays a foundation for the successful implementation of subsequent cell experiment and animal experiment.The results of BCA protein adsorption experiment showed that PDA could improve the protein adsorption capacity of the material,and Au NPs could further improve the protein adsorption capacity of the material.ROS assay results showed that,compared with PLGA and PDA-PLGA,Au NPs@PDA-PLGA can effectively remove reactive oxygen species and significantly reduce the level of reactive oxygen species in cells.3.Nestin immunofluorescence staining proved that NSCs were successfully extracted from the hippocampus of fetal mice.The results of cell proliferation experiments showed that NSCs grown on the surface of Au NPs@PDA-PLGA composite and under the intervention of electrical stimulation significantly increased the activity and proliferation ability of NSCs.Meanwhile,the combined application of Au NPs@PDA-PLGA and electrical stimulation could further promote the proliferation of NSCs.Immunofluorescence staining and RT-PCR results showed that Au NPs@PDAPLGA combined with electrical stimulation could target the differentiation of NSCs,promote the differentiation of NSCs toward nerve cells,and inhibit the differentiation of NSCs to astrocytes.The combination of the two can promote the growth of nerve cell axons and accelerate the establishment of connections between neurons.4.By constructing the model of spinal cord injury in rats and giving corresponding treatment,we found that Au NPs@PDA-PLGA combined with electrical stimulation can significantly improve the motor function score of the rats’ hind limbs,and the gait footprint of the hind limbs is most similar to that before surgery.The results of electromyography showed that Au NPs@PDA-PLGA combined with electrical stimulation could shorten the latency of nerve electrical signal conduction and increase the amplitude.HE,LFB and TEM staining showed that Au NPs@PDA-PLGA combined with electrical stimulation could significantly promote tissue regeneration and myelin sheath growth in the injured area.The results of immunofluorescence staining showed that the combined treatment of composite material and electrical stimulation could significantly promote the regeneration of nerve cells and the growth of axons in the injured area,inhibit the infiltration of inflammatory cells and the accumulation of glial scar,which promote the recovery of spinal cord injury.ConclusionIn this study,Au NPs@PDA-PLGA composite materials with hydrophilic,electrical conductivity and rough surface structure were successfully constructed.This composite material can effectively remove reactive oxygen species,promote protein adsorption,and create a suitable microenvironment for nerve cell growth.Au NPs@PDA-PLGA and electrical stimulation can play a synergistic role in promoting the proliferation of NSCs in vitro,regulating the directional differentiation of NSCs to nerve cells,while inhibiting the formation of astrocytes.It has been demonstrated that Au NPs@PDA-PLGA combined with electrical stimulation can promote the regeneration of nerve cells and axon growth in the injured area,reduce inflammatory cell infiltration,inhibit glial scar aggregation,and promote the recovery of motor and sensory functions after spinal cord injury in rats.This innovative combination therapy will provide new ideas for the treatment of spinal cord injury.