Study On The Biological Toxicity Of Water-soluble CdSe/ZnS Quantum Dots And Its Mechanism Of Inducing Cellular Oxidative Damage

With the rapid development of nanotechnology over the past decades,nanomaterials have been extensively used in the fields of energy,environment,electronics,and biomedicine.Quantum dots?QDs?,as a functionalized nanomaterial,have been deemed to a new-type fluorescent probe and widely applied to biosensing,bioimaging,drug delivery,and cancer diagnostics.However,QDs may eventually enter the environment.During the weathering process,residual QDs may release toxic metal ions into the environment,which may cause certain effects on fungi,bacteria,large invertebrates,and even humans.Therefore,understanding the migration and transformation mechanisms of QDs is critical to the toxicity in biomedical and biological applications such as diagnostics,treatment,and imaging.However,due to the inherent physicochemical properties,environmental conditions and the complexity of the analytical methods,it is difficult to assess the overall environmental impact of QDs which were currently reported.The synthesis methods and surface modification of QDs will greatly affect its physicochemical properties and interaction of QDs with cell membranes and subsequent uptake in cells.At the same time,the systematic cytotoxicity assessment of QDs is also essential for practical biological and biomedical applications.This project is aimed at the above problems,which systematically studied the biological toxicity and the caused oxidative damage mechanism in cells of water-soluble CdSe/ZnS QDs.The specific research work and achievements of this paper include the following five parts:The first part described the uptake and accumulation of water-soluble CdSe/ZnS QDs in Phanerochaete chrysosporium.In order to explore the effects of various physicochemical properties of water-soluble CdSe/ZnS QDs on their own cellular uptake and the causing toxic effects,this study used inductively coupled plasma optical emission spectrometry?ICP-OES?and confocal laser scanning microscopy.Four types of CdSe/ZnS QDs?COOH CdSe/ZnS 525,COOH CdSe/ZnS 625,NH₂CdSe/ZnS 525,and NH₂ CdSe/ZnS 625?were studied the uptake and accumulation in P.chrysosporium.The results showed that within the test concentration range?10-80nM?,four types of CdSe/ZnS QDs accumulated in mycelia and caused oxidative stress in P.chrysosporium.In addition,the cellular uptake and cytotoxicity are related to the physicochemical properties of QDs,such as particle size and surface charge.The smaller size of negatively charged CdSe/ZnS QDs was more easily taken up by P.chrysosporium,so small-sized CdSe/ZnS QDs were more cytotoxic to P.chrysosporium.On the other hand,small negatively charged CdSe/ZnS QDs caused greater cytotoxicity than large negatively charged CdSe/ZnS QDs.The second part focused on the biological toxicity of water-soluble CdSe/ZnS QDs in P.chrysosporium.In this work,CdSe/ZnS QDs with extremely small particle diametersandhighluminescencewerecharacterizedbyfluorescence spectrophotometer?PL?,UV-vis spectrophotometer?UV-vis?,dynamic light scattering?DLS?and transmission electron microscope?TEM?,respectively.Scanning electron microscopy?SEM?was used to observe the morphological changes of P.chrysosporium mycelia before and after exposure.Confocal laser scanning microscopy was used to evaluate the bioaccumulation of CdSe/ZnS QDs in P.chrysosporium.The cellular activity and intracellular ROS levels of P.chrysosporium were also measured.The SEM results showed that the mycelial structure of P.chrysosporium was destroyed after CdSe/ZnS QDs exposure.The confocal laser scanning microscopy results showed that a large amount of CdSe/ZnS QDs accumulated in the mycelium and induced the ROS production,presenting direct toxicity to P.chrysosporium.With the increase of initial concentration of CdSe/ZnS QDs,the cellular activity of P.chrysosporium gradually decreased,and the intracellular ROS levels of P.chrysosporium gradually increased,indicating that the bioaccumulation and toxicity of CdSe/ZnS QDs were dose-dependent and time-dependent.The third part emphasized the biological toxic difference of water-soluble CdSe and CdSe/ZnS QDs in prokaryotic and eukaryotic microorganisms.This study focused on the cellular uptake and cytotoxicity of Cd²⁺,CdSe QDs,and CdSe/ZnS QDs in Escherichia coli and P.chrysosporium,respectively.Two types of QDs were characterized by TEM and DLS.The average physical diameters of CdSe QDs and CdSe/ZnS QDs were 6.78 nm and 7.26 nm,respectively.The ICP-OES results showed that the uptake amount of CdSe QDs by E.coli and P.chrysosporium was larger than that of CdSe/ZnS QDs due to the smaller particle size and less negative surface charges of CdSe QDs.Plasma membrane fluidity and membrane H⁺-ATPase activity of E.coli and P.chrysosporium decreased gradually with the increase concentrations of Cd²⁺,CdSe QDs,and CdSe/ZnS QDs.The cell viability and intracellular ROS level results indicated that the induced-cytotoxicity decreased as follows:CdSe QDs>CdSe/ZnS QDs>Cd²⁺.These findings suggested that the cytotoxicity of QDs is not only attributed to their heavy metal components,but also the induced particle-specific toxicity due to their nanosize effects.Furthermore,the ZnS coating could greatly prevent the releasing of Cd²⁺from CdSe/ZnS QDs,leading to the reduced cytotoxicity of CdSe/ZnS QDs to E.coli and P.chrysosporium.The forth part investigated the mechanism of cell oxidative damage induced by water-soluble CdSe/ZnS QDs in P.chrysosporium.Four types of water-soluble CdSe/ZnS QDs were used as different types of poisons to study their induced physiological responses in P.chrysosporium.The lipid peroxidation product of malondialdehyde?MDA?content,superoxide radical?O₂·^-?level,superoxidation dismutase?SOD?activity,catalase?CAT?activity and glutathione?GSH?content were respectively determined.CdSe/ZnS QDs enter cells via endocytosis and are oxidized in the intracellular environment,releasing free Cd²⁺,and CdSe/ZnS QDs and free Cd²⁺accumulate intracellularly and induce a large amount of intracellular ROS,leading to oxidative stress in P.chrysosporium.With the increase initial concentration of CdSe/ZnS QDs,MDA content,O₂·^-level,SOD activity and CAT activity in P.chrysosporium also gradually increased,and the content of GSH gradually decreased.These findings provide valuable theoretical guidance for revealing the toxicity mechanism of CdSe/ZnS QDs in living cells.The last part explored that different sulfide sources alleviated the biological toxicity and detoxification mechanism of water-soluble CdSe/ZnS QDs.The potential toxicity of QDs has caused large challenges in clinical tests and biomedical applications due to their heavy metal components and nanosize effects.Hence,we comprehensively investigated the physiological responses of P.chrysosporium to four types of CdSe/ZnS QDs with either an inorganic sulfide NaHS or an organic sulfide cysteine as antidote.The results indicated that the cell viability of P.chrysosporium increased with the assistance of NaHS or cysteine when exposed to CdSe/ZnS QDs.NaHS and cysteine have assisted P.chrysosporium to alleviate oxidative damage against CdSe/ZnS QDs by regulating lipid peroxidation and O₂·^-production.What's more,NaHS and cysteine have also stimulated P.chrysosporium to produce more antioxidant enzymes?SOD and CAT?,which played significant roles in the defense system by relieving CdSe/ZnS QDs-induced oxidative stress.In addition,NaHS and cysteine were used as sulfide sources to promote the GSH biosynthesis to defense the toxicity of CdSe/ZnS QDs.In conclusion,this work indicated that sulfide sources?NaHS and cysteine?exerted a strong positive effect in P.chrysosporium against the toxicity induced by CdSe/ZnS QDs.