Studies On The Impact And Mechanism Of CdTe Quantum Dots On Oxidative Stress, Cell Viability And Relative Effects

Quantum dots (QDs) are engineered semiconductor nanocrystals with the size of 2-100 nm, which are composed of the elements from group IIB-VIA (eg, CdSe, CdTe) or group ⅢA-ⅤA (eg, InP, GaAs). Due to their novel photophysical properties (narrow emission spectra, size-depending emission, high fluorescence intensity, broad absorption coefficients and high photostability) and excellent chemical process ability, quantum dots have been widely used as imaging agents or biomedical labeling in medicine, biology and chemistry. With the extensive exploration and application of QDs, many studies reported the toxicity of these nanoparticles, such as the interaction with biomacromolecules, the production of reactive oxygen species (ROS), the subsequent release of toxic heavy metals, cell death and the change of gene expression, which arouse wide concern of the public. Since nanoparticles can enter human bodies through lung and digestive system, it is significant to explore the toxicity of QDs to vital proteins and target organs. Firstly, we investigated the interactions of CdTe QDs with oxidative stress enzymes (catalase and copper-zinc superoxide dismutase), which might subsequently determine the response of cells or organs. Then we investigated the cytotoxicity of CdTe QDs to mouse primary hepatocytes and nephrocytes, because numerous studies have shown that liver and kidney are the main target organs for cadmium-based quantum dots. At last, we investigated the interactions of CdTe QDs with blood proteins (bovine serum albumin and bovine hemoglobin), which can impact the fate of CdTe QDs in organisms. In this research, we explored the toxicity of CdTe QDs from the molecular and cellular perspectives. The major works and results consist of the following four parts:In the first part, N-acetyl-L-cysteine-Capped CdTe QDs with fluorescence emission peak at 612 nm (QDs-612) was synthesized via one pot method, which was purified and characterized afterwards.In the second part, the toxic effect of QDs-612 on catalase (CAT) was investigated by fluorescence, fluorescence lifetime, synchronous fluorescence, ultraviolet-visible (UV-Vis) absorption and circular dichroism (CD) techniques. Binding of QDs-612 to CAT caused static quenching of the fluorescence and the change of the secondary structure of the protein. QDs-612 affected the microenvironment of tryptophan residues in CAT. The association constants K was determined to be K298K=7.21×105 L mol-1. The activity of CAT and was inhibited for the bound QDs-612.In the third part, the toxic effects of QDs-612 on copper-zinc superoxide dismutase (Cu/ZnSOD) was investigated by fluorescence, isothermal titration calorimetry (ITC), fluorescence lifetime, synchronous fluorescence, ultraviolet-visible (UV-Vis) absorption and circular dichroism (CD) techniques. QDs-612 bound to Cu/ZnSOD through hydrophobic interaction with association constants determined to be 3.28×105 L mol-1, and caused static quenching of the fluorescence and the change of microenvironment of tyrosine residues and secondary structure of the protein. The activity of Cu/ZnSOD was inhibited by adding QDs-612.In the fourth part, the cytotoxicity of QDs-612 to mouse primary hepatocytes and nephrocytes were preliminary analysed by assessment of cell viability performed using the Cell Counting Kit-8 (CCK-8) assay. The cell viability, SOD and CAT activity of mouse primary hepatocytes and nephrocytes decreased significantly after 2h exposure. In the presence of vitamin C, the cell viability of these two primary cells with higher concentrations of QDs-612 increased markedly compared with the experimental groups in the absence of vitamin C, which demonstrated that QDs-612 induces the oxidative damage of these primary cells. The cellular study also reflected that QDs-612 had a deeper influence on nephrocytes than hepatocytes.In the fifth part, the interactions of QDs-612 with bovine serum albumin (BSA) and bovine hemoglobin (BHb) were investigated by isothermal titration calorimetry (ITC), fluorescence, fluorescence lifetime, synchronous fluorescence, ultraviolet-visible (UV-vis) absorption and circular dichroism (CD) techniques. Fluorescence data of BSA-QDs and BHb-QDs revealed that the quenching was static in every system. While QDs-612 changed the microenvironment of tryptophan in BHb, the microenvironment of BSA kept unchanged. Adding QDs-612 affected the skeleton and secondary structure of the protein (BSA, BHb). The ITC results indicated that the interaction between the protein (BSA, BHb) and QDs-612 was spontaneous and the predominant force was hydrophobic interaction. In addition, the binding constants were determined to be 1.19×105 L mol-1 (BSA-QDs) and 2.19×105 L mol-1 (BHb-QDs) at 298 K. From these results, we concluded that CdTe QDs had a larger impact on the structure of BHb than BSA.In this research, we explored the impact of CdTe QDs on oxidative stress, cell viability and relative effects at molecular and cellular level, which provides valuable information to understand the toxicity of quantum dots in vitro and can be used to assist in the design of biocompatible and stable quantum dots.