Cellular Behavior Study And Neuroanatomical Tracing Application Of Carbon Dots

Peripheral nerve injuries are reasonably common in the clinic and there is noeasily available formula for successful treatment. Functional recovery after surgicalrepair of peripheral nerve injury is often suboptimal, even fail to recover. Manyfactors determine the success of repair, including the pattern, location and degree ofinjury, the type and timing of surgical repair, the surgical technique, the age and wholecondition of patients. Clinically common methods for measuring the axonalregeneration and evaluating functional recovery of peripheral nerve mainly involvemuscle strength testing, sensory examination, electromyogram, Tinel’s sign and so on.However, the lack of timeliness, objectivity, accuracy and visualization is consideredto be the major limitation of these methods. Therefore, early and accurately assessingperipheral nerve regeneration is crucial for predicting outcomes of surgical repair.Neuroanatomical tracing technique is one of the most commonly used methods in thefield of neurobiology. Comparing with electrophysiological and behavioralexaminations, neural tracing technique is able to achieve direct and visual detection.Traditional neural tracers, including horseradish peroxidase, biotinylated dextranamine, fluorescent dyes and bacterial toxins, have several limitations, such astime-consuming immunohistochemical or immunofluorescent staining procedures,weak fluorescence signal and rapid quenching. Recently, fluorescent nanoparticleshave been widely used in biosensors, in vitro and in vivo bioimaging, and otherbiomedical applications. Carbon dots, including carbon nanodots (CNDs), polymerdots (PDs) and graphene quantum dots (GQDs), as a new class of carbon-basedfluorescent nanoparticles, have drawn immense attentions due to their good solubility,excellent optical properties, good chemical stability, low toxicity, resistance to photo bleaching, low cost as well as easy chemical modifications. Most importantly,comparing to currently widely used semiconductor quantum dots, which possesscertain limitations due to the heavy metal toxicity, carbon dots exhibit more promisingpotentials on the clinical application due to good biocompatibility andenvironmental-friendly properties. Therefore, this thesis will focus on three topics,including investigating endo/exo-cytosis and intracellular distribution, evaluating invivo toxicology, and developing the novel fluorescent neural tracers based on carbondots.First part is about cellular behavior study of carbon dots. CNDs, PDs and GQDswill be introduced respectively. To study the internalization mechanism of CNDs inneural cells, we cultured two neural cell types, including differentiated rat adrenalpheochromocytoma cells (PC12), derived from a neuroendocrine tumor of thesympathetic nervous system, and rat Schwann cells (RSC96), a type of neuroglialcells in peripheral nervous system. Confocal laser scanning microscopy (CLSM),fluorescence activated cell sorter (FACS) and atomic force microscopy (AFM) wereused to study the uptake kinetics and endocytic pathways. To further determine theintracellular trafficking and distribution, we assessed the co-localization of CDs withendocytic markers and organelles specific dyes by using CLSM and directly observedtheir existence in cellular ultrastructures by using transmission electron microscopy(TEM). The exocytosis of internalized CNDs was studied using CLSM and FACS. Wefound that CDs exhibit low cytotoxicity and highly efficient internalization in neuralcells. Cellular uptake of CDs is dose, time and partially energy-dependent along withthe involvement of passive diffusion. CDs are endocytosed via caveolae-mediated andclathrin-mediated pathways. Internalized CDs are dispersed in cytoplasm and nucleus,and most of them accumulate in endo-lysosomal structures and Golgi apparatus. CDswere actively transported to the cell periphery and exocytosed with a half-life of2h.Subsequently, similar methods were used to study the intrecellular uptake of PDs. PDspossess low cytotoxicity and highly efficient internalization and the cellular uptake ofPDs is time-/energy-dependent. PDs can be endocytosed via caveolae-mediated pathways and mainly accumulate in lysosomes without entering nucleus. GQDs wereused to investigate the application of in vitro and in vivo bioimaging. GQDs alsoexhibit to be low cytotoxic and the uptake is time-dependent. Internalized GQDs aresignificantly distributed in lysosomes. The major advantage of GQDs is the longerexcitation/emission wavelength which is much more suitable for in vivo imaging.Second part is about evaluation of in vivo toxicology of carbon dots. Currently,semiconductor quantum dots have been widely used in the field of bioimaging,however, the major drawback is the heavy metal toxicity which significantly restrainsits pre-clinical applications. Thus, the most fascinating advantage of carbon dotsrefers to the low toxicity and good biocompatibility. But many toxicologicalinvestigations of carbon dots focus on the cytotoxicity and in vivo toxicology is stilllack of studies. We use Wistar rats as the animal model and systematically evaluatethe in vivo toxicology of CNDs with highest quantum yield (ca.80%) and ultrasmallsize (1-5nm) at the dose (100mg/kg), which is the highest reported to date forfluorescent carbon-based materials. The body weight, blood serum biochemistryanalysis, hematological tests and histological examinations prove the low toxicity ofCNDs in the in vivo level. The biosafety of CNDs is an outstanding prerequisite forthe further application in neuroanatomical tracing.Third part is about development of carbon dots based neural tracing technique. Inthe first two chapters, we study the physiochemical properties, cellular behaviors, andbiosafety of CNDs. Based on these understanding, we use CNDs as a fluorescentbio-probe to label cholera toxin B subunit, which is a non-toxic traditional neuraltracer, to synthesize a novel fluorescent neural tracer: cholera toxin B-carbon dotsconjugate (CTB-CDs) via EDC/NHS coupling reaction. We find that CTB-CDs can betaken up and retrogradely transported by neurons in the peripheral nervous system ofthe rat. Our results show that CTB-CDs possess high photoluminescent intensity, goodoptical stability, a long shelf-life and non-toxicity. Tracing with CTB-CDs is a directand more economical way of retrograde labelling experiments. Therefore, CTB-CDsare a reliable fluorescent retrograde tracer. In conclusion, from cellular behavior study to neuroanatomical tracingapplication of carbon dots, we followed the easy-to-difficult principle to complete thisinterdisciplinary research. We deeply investigated the cellular uptake mechanism andintracellular trafficking of carbon dots, systematically evaluated in vivo toxicology ofcarbon dots, and firstly reported the synthesis and application of carbon dots-basedfluorescent neural tracer. This research will provide a novel idea for the developmentof next generation of neural tracer.