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Cadmium selenide quantum dots on human fibroblast epithelial cells Toxicology

Author: ZhangChen
Tutor: YangJun
School: Zhejiang University
Course: Health Toxicology
Keywords: Quantum dots γH2AX DNA damage MTS ROS
CLC: R114
Type: Master's thesis
Year: 2011
Downloads: 130
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Abstract

[Objective] semiconductor nanoparticles - quantum dots (Quantum dot, Qdot) is a novel nano-materials, its radius is less than or close to the exciton Bohr radius of nanoparticles, also known as semiconductor nanocrystals (semiconductor nanocrystal) (1) . Quantum dots has been the materials, electronics, physics and other disciplines of research hotspot in recent years due to its unique spectral characteristics, good biocompatibility, coupled with adaptability and high-throughput molecular recognition and other characteristics, in the life many areas of science has made breakthrough progress (2-5). Superior fluorescence quantum dot so that it can be used as a fluorescent marker and then type the desired biological fluorescent probes. In the biomedical field, the specific application of quantum dots mainly: imaging at the cellular level can be observed in various states organelles (5); multicomponent biological molecules can be the determination of the interference elimination impurity molecules have a good effect ; can be marked on a variety of proteins to detect protein-protein interactions and so on. Of course, the quantum dot application in biological systems is far more than that, it can be applied to bio-chip solution matrix, medical imaging, etc. (6). You can see, biological applications of quantum dots based on quantum dots are basically harmless to biological cells assumptions above. Currently on the biological effects of quantum dots and their pharmacokinetic and toxicology research is still in its infancy, the literature is mainly observational reports, such as quantum dots observed after intravenous injection in vivo distribution (7,8 ) as well as different package was modified quantum dots metabolism. However, at the molecular level whether the quantum dot impact physiological function of cells, especially cells of the body to even more worthy of attention. A compound of the cell at the molecular level of the cells from its two most important substances that affect the protein and DNA to study. The interference of DNA is that many compounds cause functional changes in cell physiology major pathways, such as the modification of base pairs, resulting in DNA single-strand breaks (single strand breaks, SSBs), double-strand breaks (double strand breaks, DSBs), chain or between chain crosslinking within the (cross-link) and so on. DNA damage may be due to genetic traits affecting change, so that the characteristic function of DNA damage is called \In various DNA damage, DSBs are regarded as the most serious kind. Study found that cells subjected to DNA damage, especially DNA double-strand breaks after DNA double-strand breaks sites will occur one kind H2AX protein phosphorylation. As a eukaryotic cell DNA with histones binding component, H2AX containing the C-terminal domain of a Ser residue occurs when the DSBs, the phosphorylated residue (called γH2AX). γH2AX further raise other DNA repair proteins to damage sites, the formation of \As γyH2AX closely related to the appearance and DSBs, γH2AX become detect DNA damage, a new specific targets. Some studies have shown that cadmium telluride or cadmium selenide quantum dot toxicity as the core part in the solution free from cadmium or cadmium into the cells and release elements (9,10). On the other hand, cadmium telluride or cadmium selenide quantum dots as the material having a light-induced oxidation and air-induced oxidation, the resulting free radicals may be generated by the quantum dot another major mechanism of toxicity. In order to increase the stability of quantum dots, to broaden its scope of application and to reduce the side effects of the core material, the researchers cadmium selenide or cadmium telluride quantum dots coupled with different outer wrapping materials in order to reduce the possible release of cadmium and increasing bio-compatible sex. ZnS is a more universal application materials. It can improve elements such as tellurium, cadmium selenide shells under the instability caused by the track, thus enhancing the fluorescence efficiency (11). Synthesis at the beginning, the quantum dot hydrophilic poor, researchers using mercaptopropionic acid (mercaptopropionic acid) and poly-ethylene glycol (polyethylene glycol) (PEG) to increase the water solubility of quantum dots and prevent aggregation of the nanoparticles ( 6). For these reasons, we intend on quantum dot cytotoxicity and genotoxicity in-depth study. The topic chosen human skin fibroblasts (HSF) as target cells were detected CdSe QDs and a PEG-coated CdSe QDs on the HSF cell cytotoxicity; selection γH2AX to detect targets, using immunofluorescence studies of these two materials HSF cells against DNA damage; through the comet assay to further verify the genetic material toxicity. Detection using fluorescent probes DCF ROS levels to explore its role in DNA damage. As quantum dots on human skin fibroblasts of toxicological basis to provide a mechanism and explore its corresponding. [Method] 1, cell culture and quantum dot processing: HSF cells were cultured in 10% fetal calf serum, 0.03% L-glutamine, 104 U · L-1 penicillin, 125 mg · L-1 streptomycin α- MEM medium. Cells were subcultured to logarithmic phase, the quantum dots were added to 440,680, making quantum dots each culture medium at a final concentration reached 8 nM and 80 nM, at 37 ℃, 5? 2 continue to foster 2,8 and 24 h. The control group with the same volume of PBS in treated cells. 2, cytotoxicity assays 1) MTS assay: a 96-well plate, respectively, with quantum dots 440 and 680 for 2,8 and 24 h treatment, each plate is divided into blank group (without quantum point processing), quantum dots 8 nM, 80 nM treatment group, reagent control. Cells were cultured to the corresponding time points after treatment, plus 5g · L-1 MTS 20μl, 37 ℃ incubated for 45 minutes under the conditions, termination of culture. 490 wavelength selection in each well was measured on a microplate absorbance (A 490). 2) Trypan blue counting experiment: Take 75μl mixed with cell culture medium with 75μl diluted concentration of 0.4% trypan blue with mix, take 50μl mixture drops in the counting board, observed under the microscope counting. 3, γH2AX detection '1) Immunofluorescence: Using quantum dots at different time points after treatment of cells by immunofluorescence detection yH2AX means of distribution in the nucleus. 2) Western blot: quantum dots treated cells using western blotting assay γH2AX protein expression. 4, comet assay: Cells treated with quantum dots After centrifugation, the cell concentration adjusted to 6 × 1010L-1. Preparation of \Sequentially cleaved, 25V electrophoresis, DAPI staining, observed under fluorescence microscope. 5, ROS detection mechanism: in the cell after treatment with 0.25% trypsin digestion, PBS resuspended by adding DCFH-DA concentration was adjusted to work (20μM), incubated in the dark for 30 min, using a microplate reader at excitation waves were detected at 485 nm. 6, two-dimensional electrophoresis: HSF cells were treated with PEG coated quantum dots 8 nM or 80 nM for 24 hours after the extraction of two-dimensional electrophoresis protein. [Results] 1, no PEG coated quantum dots 680 in 80 nM concentration effect 24 hours significantly affect cell survival. MTS assay shows, wrapped with a PEG 680 and 440 of quantum dots 8 nM and 80 nM treatment of cells under conditions for 24 hours, cells in the control group had no significant difference. But not both of these PEG wrapped QD effect at high concentration 8 hours after the cell begins to show a negative effect on cell survival. Trypan blue experiments further show that 8 nM concentration without PEG-modified quantum dots 680 and 440 at the 24-hour observation period of HSF cells almost no effect; the quantum dots 680 in high doses case show the cytotoxic, number of living cells was significantly reduced. 2, without the PEG-modified quantum dots doses at high concentrations can cause γH2AX foci formation. γH2AX focus is to detect DNA damage sensitive indicators. Immunofluorescence experiments show QDs with PEG 440 and 680 in the case of parcels within 24 hours γH2AX focus formation compared with the negative control and no significant difference. While exposed to high doses of quantum dots appear γH2AX focus HSF under the number showing time-dependent manner. Westernblot and to verify this trend. 3, the neutral comet assay showed no PEG-modified DNA double quantum dot can cause breakage. 4, ROS induced γH2AX quantum dot is one of the reasons the focus. 5, there is PEG-modified quantum dots 440 and 680 changes in protein expression levels for almost no effect. [Conclusion] 1, PEG is better to reduce the cytotoxicity of quantum dot material 2, no PEG coated quantum dots can cause DNA damage, in particular in γH2AX foci formation and DNA double bond breakage. ; 3, oxidative stress is caused by non-toxic materials, PEG-modified quantum one of the reasons.

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