Study On The Biological Characteristics And Neural Tracing Application Of Red Fluorescent Carbon Dots

Peripheral nerve injury is one of the most common nervous system diseases in clinic,and its difficulty lies in the poor functional recovery after nerve injury,resulting in limb dysfunction and high disability rate.There are many factors affecting the repair effect of peripheral nerve after injury,including the injury site,pattern and degree of injury,timing and methods of surgical repair,microsurgical technique of the operator and age of the patients.At present,clinical methods evaluating the functional recovery of peripheral nerve mainly depend on motor function examination,sensory function examination,nerve conduction velocity measurement,electromyography and intraoperative electrophysiological assessments,but these methods are difficult to evaluate the recovery effect and prognosis of nerve function timely and accurately,often leading to delays in diagnosis and the missed optimal time for surgery.Therefore,there is an urgent need for a method to evaluate the level of nerve regeneration early,intuitively and accurately after nerve repair,so as to better judge the functional recovery effect of peripheral nerve.Especially for patients with poor nerve recovery,early surgical intervention can be carried out in order to shorten the treatment time and improve functional prognosis.Neural tracing technique is one of the most important methods in the study of nerve regeneration,and its theoretical basis is axon transport.Compared with neurobehavioral and electrophysiological examinations,neural tracing technique can evaluate the level of nerve regeneration more intuitively and accurately.Traditional neural tracers have obvious limitations,including tedious procedures and time-consuming immunohistochemical staining.Fluorescent dyes are widely used as neural tracers,which are simple and time-saving.However,traditional fluorescent tracers have common shortcomings,such as weak fluorescence intensity,poor photostability and photobleaching.Fluorescent nanomaterials are widely used in biomedical fields due to theirstable physical and chemical properties,and excellent optical properties,including cell labeling,molecular tracing,in vivo imaging,biosensors and so on.Carbon dots(CDs)is a new kind of carbon-based fluorescent nanomaterials with excellent biocompatibility.It has many advantages,such as stable physical and chemical properties,good optical properties,low toxicity and easy surface modification.Therefore,it has attracted wide attention in biomedical applications.Carbonized polymer dots(CPDs)are an important type of carbon-based nanomaterials.The newly synthesized red fluorescent CPDs have high quantum yield and good biocompatibility.Their unique optical properties have shown significant advantages for in vitro and in vivo imaging.In this paper,we systematically study the cellular behavior,blood-brain barrier permeability and biological safety of red fluorescent CPDs,and apply them to anterograde tracing of peripheral nerves.The main research contents are divided into four parts.Firstly,the cellular behavior of CPDs was studied.In this study,the cytotoxicity,uptake mechanism,intracellular distribution and exocytosis of CPDs were studied on neural cells in detail,so as to evaluate the cellular behavior characteristics of them.The results showed that CPDs have good biocompatibility and low cytotoxicity for neural cells;the cellular uptake of CPDs is dose-dependent and time-dependent;CPDs can enter the cells through active transport and passive diffusion,and primarily distribute in lysosomes and endoplasmic reticulum,but a small amount of them can enter the nucleus of cells;the internalized CPDs can be excreted from cells through exocytosis.Secondly,the blood-brain barrier(BBB)permeability of CPDs was studied.This study included cellular behavior of CPDs on glioma cells,BBB permeability of CPDs,and their application in glioma imaging,so as to evaluate their potential in imaging research of nervous system.The results showed that CPDs have good biocompatibility,low toxicity and high photostability;glioma cells exhibit high cellular uptake efficiency for CPDs,which is time-dependent and dose-dependent uptake,and the internalized CPDs primarily distribute in lysosomes;in vitro and in vivo studies confirmed that CPDs have the ability to penetrate BBB.Furthermore,in vivo studies showed that CPDs could successfully accumulate in orthotopic glioma,so they can be used in preoperative localization diagnosis andfluorescence-guided tumor surgery.Thirdly,the biosafety of CPDs was studied.This study included the in vivo toxicity and in vivo distribution of CPDs,so as to evaluate the biosafety of CPDs,which is the basis of their application in biomedical imaging.Studies have shown that CPDs do not cause apparent in vivo toxicities even at high doses(50 mg/kg);in vivo distribution studies have shown that CPDs can be rapidly cleared through liver and kidney metabolic pathways,thus reducing the toxicities of CPDs in vivo.In conclusion,in vivo experiments of CPDs have proved that they have good biosafety,which provides a reliable guarantee and feasibility for further application in neural tracing technology in vivo.Fourth,the neural tracing application of CPDs in peripheral nervous system was studied.CPDs were used to label biotinylated dextran amine to create novel red fluorescent nano-neural tracers;their physical and chemical properties,biocompatibility,intracellular and in vivo distribution,in vitro and in vivo imaging were studied;finally,they were applied in neural tracing of peripheral nervous system.We found that this tracer could be taken up by the neurons of the peripheral nervous system,and transported anterogradely to the axonal terminals of the neurons.The results showed that this tracer has many advantages,including strong fluorescence intensity,deep tissue penetration,high photostability,good biocompatibility and low toxicity.Therefore,this new tracer is a reliable,simple and economical fluorescent anterograde nano-neural tracer,which has important clinical application value.In conclusion,in order to create novel nano-neural tracers,we have carried out a series of biological studies of the newly synthesized red fluorescent CPDs.First,the cellular behavior of CPDs was studied to evaluate the feasibility of their application in biological research.Then,the blood-brain barrier permeability of CPDs was studied to expand their applications in nervous system.Next,the biological safety of CPDs was studied,which was a reliable guarantee for their application in neural tracing in vivo.Finally,CPDs were applied in neural tracing,and the anterograde neural method based on CPDs was reported for the first time.This method has been successfully applied to the study of anterograde neural tracing of peripherial nerve,and provides research basis and reference for biology,materialsscience and methodology for the preparation and application of new in vivo nano-neural tracers,which has great significance to improve the overall repair level of peripheral nerve.