Experimental Study On The Effect Of Magnetic Forces Mediated By Fluorescent-magnetic Bifunctional Fe₃O₄·rhodamine 6G@PDA Superparticles On The Repair And Regeneration Of Peripheral Nerves

Currently,peripheral nerve injury is a disease of worldwide concern for which there are few options for successful treatment,and it often causes loss of function and poor outcomes,which cause a heavy burden for patients and society.The incidence of peripheral nerve injury is approximately 13-23/100,000,with tens of thousands of new cases occurring annually in Europe and the United States alone.Despite more than 100 years of intense laboratory and clinical investigations,the results of nerve repairs are somewhat discouraging,with only 50%of patients regaining useful function.The regeneration of peripheral nerves exemplifies the plasticity that exists within the nervous system.Unfortunately,unsuccessful nerve regeneration is often the result in practice.The main reasons for poor functional recovery after peripheral nerve injury are inaccuracy and a delay in axonal regeneration.Research on these two factors is key to solving the above described clinical problems and is also one of the top priorities in the field of neuroscience.The importance of mechanical factors in the nervous system has been appreciated only recently.Recently,it was demonstrated that the external application of mechanical tension is sufficient to direct axonal outgrowth and to stimulate axonal elongation.For this purpose,delivering accurate force to direct axonal growth cones in vivo appears to be a challenge that must be overcome.Ideally,this would be achieved noninvasively,and the magnitude and duration of the force would be controllable.Magnetic nanoparticles（MNPs）are promising new tools that now offer the possibility to influence such axonal regeneration processes.Due to their magnetic properties,MNPs experience forces in inhomogeneous magnetic fields and hence can be manipulated through such fields.In this study,we propose an innovative approach for remotely guiding neuronal regeneration by incorporating MNPs into cells and transferring MNP-loaded cells into a magnetically sensitive environment,in which they will respond to external magnetic field gradients.This study will be described in the following four parts.Part 1:Synthesis and characterization of FMSPsWe designed and prepared novel fluorescent-magnetic bifunctional Fe₃O₄·Rhodamine6G@polydopamine superparticles（FMSPs）.Fe₃O₄ nanoparticles（NPs）and Rhodamine 6G fluorescent molecules were selected as the core materials to prepare Fe₃O₄·Rhodamine 6G superparticles（SPs）.Dopamine（DA）monomers can be oxidized and spontaneously polymerize to form a polydopamine（PDA）shell on the surface of Fe₃O_4·Rhodamine 6G SPs under alkaline conditions.These novel fluorescent-magnetic superparticles（FMSPs）possess the following characteristics.（1）Due to the high saturation magnetization of FMSPs,we can achieve precise and noninvasive remote manipulation of neuron regeneration under external magnetic fields.（2）Because of the excellent biocompatibility of the FMSPs and the introduction of the PDA shell,there was high uptake of the FMSPs by cells,which guaranteed that a sufficient number of FMSPs would be taken up by the cells to highly magnetize them even at low working concentrations.（3）The fluorescence properties of the FMSPs would allow the real-time dynamic observation of magnetically loaded cells.Part 2:Biological behaviors involved in the cellular uptake of FMSPsThrough investigation of the cellular uptake kinetics,cellular uptake pathways,intracellular distribution,and exocytosis of FMSPs,we comprehensively studied the biological behaviors involved in the cellular uptake of FMSPs,which are the foundation of their biomedical applications.Our study shows that the uptake of FMSPs by neural cells occurs mainly through caveolae-mediated endocytosis and micropinocytosis,which are energy-dependent active transport processes that conform to time-and concentration-dependent uptake.FMSPs are mainly distributed in the Golgi apparatus and mitochondria after being internalized by neural cells.In neuroglial cells（Schwann cells）,internalized FMSPs are primarily transported to lysosomes and mitochondria in RSC96 cells.We found that internalized FMSPs can be transported bidirectionally along the axons and tended to cluster in the growth cones.The internalized FMSPs are digested and decomposed by lysosomal degradation and are ultimately expelled by exocytosis.During the whole process of FMSP uptake and exocytosis,the cells maintain good activity.Part 3:Magnetic mechanical forces induce axonal outgrowth and cell migrationWe confirm that under the action of an external magnetic field,magnetic stimulation mediated by FMSPs can not only improve the rate and direction of neuronal axon regeneration but also affect the migration and distribution of Schwann cells.In our system,we demonstrate that internalized FMSPs in neural cells may generate a tension force on the order of pN over long time periods.Such magnetic stimulation can induce the stretching growth of axons and accelerate the regeneration of axons by stretching the axon cytoskeleton.At the same time,it can also guide the direction of axonal growth and the migration of Schwann cells through the directivity of the magnetic force.Part 4:Magnetic mechanical forces promote gene expressionTo further investigate the potential mechanism involved in the conversion of mechanical stimuli mediated by FMSPs into biochemical signaling for axon elongation and cell migration,we performed mRNA transcriptome sequencing and bioinformatics analysis on cell samples obtained under the action of nanomagnetic forces.The results show that magnetic stimulation mediated by FMSPs in neural cells could induce the upregulated expression of the key genes Cdh11,Csf1r and Ppp1r1c and finally promote the regeneration of neuronal axons by regulating the chemokine secretion process,central nervous system neuron differentiation and axonogenesis process as well as the mitotic cell cycle phase transition process.In neuroglial cells（Schwann cells）,magnetic stimulation mediated by FMSPs can induce the upregulation of the key genes Fdg2 and Podxl by regulating MAP kinase activity and integrin-mediated cell adhesion and finally promoting Schwann cell migration.This study focuses on the practical problems presented by peripheral nerve injury andrepair encountered in clinical practice.Using cutting-edge biological nanotechnology,Fe₃O₄·Rhodamine 6G@polydopamine superparticles（FMSPs）with fluorescent-magnetic dual functions are designed and synthesized according to research needs.By virtue of the excellent biocompatibility and ability to interact with neural and neuroglial cells,a sufficient number of FMSPs can be taken up by cells,which become highly magnetized.The magnetic mechanical forces generated by the interaction between FMSPs and magnetic fields provide remote,noninvasive,accurate,and controllable guidance and control of neuronal axon regeneration and glial cell migration.We expect that our study will provide new ideas and strategies for the repair and regeneration of peripheral nerves after injury.