| Quantum dots (QDs), also known as semiconductor nanocrystals, are nanoscale particles with size ranging from 1 to 100 nm in three dimensions. Due to their unique advantages of broad excitation, narrow emission, high quantum yield, excellent photostability and anti-photobleaching, QDs have shown great prospect in clinical application. However, the biosafety of QDs has been a tough question to be answered because of their nano-size effect and heavy-metal components. In recent years, a great deal of concern has been raised about the potential hazards of QDs to human beings. However, little consensus on the biosafety of QDs has been reached. And several important problems need to be resolved urgently in related researches, such as the lacking of standardization methods, and unknown molecular mechanism and secondary effects of QDs. To solve these problems, studies on the biosafety of QDs should be carried out more deeply and systematically by introducing novel strageties and methodologies.Metallomics is a novel omics science, focusing on the amount, species, distribution, structure and function of metals in biological system. The effective platform for metallomics study presently is hyphenated techniques combining high-resolution separation technique (liquid chromatography, gas chromatography, gel electrophoresis and capillary electrophoresis etc.) with sensitive elemental specific detection techniques (eg. inductively coupled plasma mass spectrometry, ICP-MS) and molecular mass detection techniques (eg. electrospray ionization, matrix-assisted laser desorption ionization). Due to the metal composition of QDs, utilization of these hyphenated techniques would provide the information of the amount, species, distribution, transformation and metabolism of QDs in living cells and organism, which benefits the explanation of the mechanism of toxicity caused by QDs from molecular levels. Therefore, metallomics can be expected to be an important Strategy for the study of the biosafety of QDs. However, to our best knowledge, reaserches on the biosaftey of QDs based on metallomics have been rarely reported.The aim of this dissertation is to develop preliminary studies on the biosafety of CdSe/ZnS QDs from the point view of metallomics:to study the biokinetic behavior (uptake, distribution and elimination) and toxicity of CdSe/ZnS QDs in HepG2 cells by mass spectrometry-based hyphenated techniques and biochemical methods; to analyze the species of CdSe/ZnS QDs in HepG2 cells by mass spectrometry-based hyphenated techniques and related analytical methods; to obtain the deeper information of the species of Cd-metallothioneins by high resolution mass spectrometry, providing strategy and basic data for revealing the molecular mechanism of QDs toxicity. The major contents of this dissertation are described as follows:(1) The cellular uptake, elimination and toxicity of CdSe/ZnS QDs in HepG2 cells were comprehensively studied using ICP-MS and biochemical methods. ICP-MS analytical results showed that the uptake efficiency of CdSe/ZnS QDs by HepG2 cells was lower than that of Cd(II) and Se(IV), and the uptake was dose-and time-dependent. The uptake amount was related to the physicochemical properties of QDs, and NH2-QDs with smaller size were more easily taken up by cells. The toxicity of CdSe/ZnS QDs, Cd (II) and Se (IV) was conducted with several biochemical methods. Although Cd(II) above 8.9?M exhibited obvious toxicity to the cells, no obvious toxicity of four CdSe/ZnS QDs was observed within the tested concentration range (10-100 nM). However, QDs at concentration of 100 nM up-regulated cell stress related genes (MT1A and CYP1A1) to different extents, which was positively correlated with the intracellular amount of QDs. Thus, the potential risk of QDs to human beings cannot be completely excluded and long-term studies will be processed.(2) Speciation of four CdSe/ZnS QDs in HepG2 cells was carried out by using size exclusion chromatography (SEC) coupled with ICP-MS. On the basis of the signal of Cd, two kinds of chemical forms, named as QD-1 and QD-2, were observed in HepG2 cells incubated with CdSe/ZnS QDs. QD-1 was demonstrated to be a kind of QD-like nanoparticles, confirmed by chromatographic retention time, transmission electron microscopy (TEM) characterization, and fluorescence detection. QD-2 was demonstrated to be cadmium-metallothioneins complex (Cd-MTs) by reversed phase liquid chromatography synchronously (RPLC) coupled with ICP-MS and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) analysis. Meanwhile, speciation of QDs in HepG2 cells incubated with different conditions was analyzed. With the variation of QDs incubation concentration/time, and elimination time, the species of QD-1 and QD-2 were also observed without other obvious species, and both the amount of QD-1 and QD-2 increased with incubation concentration and time. The obtained results provide a novel strategy for understanding the molecular mechanism of QDs toxicity.(3) The RPLC-ICP-MS/ESI-Q-TOF-MS method was improved by optimizing the gradient elution procedure of RPLC and an effective separation among the MTs subisoforms was obtained. The improved method was used to analyze the detail components of QD-2. On the basis of Cd signal determined by ICP-MS, four species, Peakl, Peak2, Peak3 and Peak4, were found in QD-2. According to the result of ESI-Q-TOF-MS with/without post-column acidification, Peakl-Peak4 was confirmed to be Cd,Zn-MTs formed by different molar ratio of Cd/Zn binding with four MTs subisoforms, N-terminal acetylation MT2a (N Ac-MT2a), N-terminal acetylation MT1e (N Ac-MT1e), N-terminal acetylation MT1g (N Ac-MT1g) and MT1m, respectively. Meanwhile, the components of QD-2 in HepG2 cells incubated with different conditions were analyzed. With the variation of QDs incubation concentration/time, and elimination time, all of Peakl-Peak4 was observed in QD-2 and all the amount of Peak1-Peak4 increased with incubation concentration and time. However, a growing number of the species with different molar ratio of Cd/Zn in Peakl-Peak4 was obtained with the increase of incubation concentration and time. The detail information of Cd released from CdSe/ZnS QDs was obtained, greatly benefiting the understanding of metabolism and toxicity of QDs in living cells. |
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