Study On The Biotoxicity Mechanism Of Nitrogen-doped Carbon Dots

Carbon dots(CDs)were sphere-like fluorescent carbon nanomaterials with a size of10 nm or less.They were suitable for applications in biochemical sensing,bioimaging,drug delivery and nanotherapeutics due to their low cost,simple fabrication,good optical properties,and ease of functionalization.CDs were mainly composed of carbon,hydrogen,and oxygen.Nitrogen is also commonly used in the synthesis of carbon dots,as the doping of nitrogen results in higher fluorescence quantum yields,red fluorescence emission peaks and higher chemical stability.As a result,many nitrogen-doped CDs were widely used in biomedical applications.The low cytotoxicity of CDs has long been cited by researchers as an indication of their good biocompatibility.However,more in-depth studies have revealed that CDs have toxic effects on cells,aquatic organisms and plants,and the mechanisms of toxicity were not fully understood,suggesting that conventional cytotoxicity tests were no longer sufficient to evaluate the safety of CDs.Therefore,it was necessary to further investigate the in vivo toxicity and mechanism of action of CDs before they were applied to humans.Furthermore,when CDs were applied to the human body and the environment,they will inevitably come into contact with substances such as metal ions and organic small molecules,and it is not clear whether this interaction will affect the toxicity of CDs.Based on this,the following work has been carried out in this thesis to assess the safety of nitrogen-doped carbon dots:1.Uncovering the mechanisms of toxicity of carbon dots exposure to zebrafish embryos through metabolomics and lipidomics.Zebrafish were commonly used as biological models for nanomaterial toxicity studies due to their high genetic homology with humans and their rapid reproduction and development.In this study,the toxic effects and mechanisms of nitrogen-doped carbon dots(PCDs)prepared from pphenylenediamine precursors were investigated and revealed using a combination of traditional toxicology,untargeted metabolomics and lipidomics approaches in zebrafish embryos.Traditional toxicological studies had found that high concentrations of PCDs cause a significant increase in embryonic malformations(p < 0.05),with toxic effects such as spinal curvature,pericardial oedema and yolk sac malformations,and a significant decrease in embryonic hatching rates(p < 0.05).PCDs also cause a significant decrease in embryonic hatching enzyme levels(p < 0.05),with specific toxic effects in the form of delayed hatching and retarded development.The antioxidant enzyme activity of the embryos also increased with increasing exposure concentrations..The metabolomic results indicated that PCDs exposure caused the greatest perturbation of lipid differential metabolites in zebrafish embryos,so we performed further lipid metabolism analysis.The combined metabolomic and lipidomic analyses showed that PCDs significantly interfered with embryonic lipid metabolism and amino acid metabolism,which in turn affected the tricarboxylic acid cycle and lipid transformation in the embryo,preventing the embryo from hatching and developing properly due to impaired energy supply.Moreover,PCDs were likely to cause disturbances in embryonic lipid metabolism through oxidative stress,and oxidative damage was responsible for malformations in the embryo.This study has provided new insights into the mechanism of toxicity of CDs leading to embryonic malformation and delayed hatching in zebrafish,and more emphasis should be placed on the assessment of the safety of CDs in the future.2.Interaction of carbon dots with copper ions and their combined toxicity.Orange-yellow luminescent carbon dots(O-CDs)were obtained by a one-step hydrothermal method at high temperature using iron phthalocyanine and ophenylenediamine as precursors.The prepared O-CDs were uniform in size,rich in surface functional groups and have high stability.Cytotoxicity experiments showed that the O-CDs were biocompatible up to a concentration of 200 μg/m L.When studying the response of O-CDs to different metal ions,we found that copper ions could specifically enhance the fluorescence signal of O-CDs.Thus,we further investigated the interaction mechanism between O-CDs and copper ions in depth.The results show that copper ions readily bind to negatively charged O-CDs to form a rigid structure,and the aggregation restricts the non-radiative leap of O-CDs,leading to a significant enhancement of the fluorescence of O-CDs.Based on the specific response of O-CDs to copper ions,we speculate that this may affect the toxicity of O-CDs.The luminescent bacterial method has the advantages such as faster bacterial growth and reproduction,inexpensive test,easy preservation method and high sensitivity to environmental changes.Thus,this study conducted a preliminary evaluation of the combined acute toxicity of O-CDs and copper ions using luminescent bacteria.It was found that copper ions may synergistically enhance the toxicity of O-CDs,laying the foundation for further in vivo combined toxicity studies.In this paper,the toxicity of CDs was investigated in depth based on traditional toxicology and modern non-targeted metabolomics and lipidomics approaches,and the safety of nitrogen-doped carbon dots for biomedical applications was investigated.Then,the interaction between nitrogen-doped carbon dots and heavy metal copper ions was investigated,and the mechanism of fluorescence enhancement of O-CDs by copper ions was clarified.The combined toxicity of O-CDs and copper ions was further investigated using luminescent bacteria.This study clarifies the potential developmental toxicity mechanism of CDs and suggests the health risks associated with their application in the biomedical field.In addition to the examination of the toxic effects and toxicity mechanisms of CDs themselves,the process and environment in which carbon dots actually act should be considered,and the combined toxicity study is of great significance for the practical application of CDs.