| [Objective]Nanomaterials have been applied to a wide range of products, ranging from cosmetics, printer toners, clothing, electronics, and to drug delivery formulations. With this explosion in applications, concerns about the potential toxicity of nanomaterials are raised from both the scientific community and the public (for review see Colvin (12) and Nel (13)). Nanomaterials have unique biological effects due to increased surface-to-mass ratio, small size, and increased reactivity.Qdots, a type of semiconductor nanocrystals, are widely used in cellular studies due to their physicochemical properties such as bright fluorescence, narrow emission band, and high photostability (23-25), especially the CdSe/ZnS core/shell Qdots. Researchers have also explored in vivo labeling of Qdots in live animals to obtain optical imaging of cancer (26,27). For Qdots, studies so far have showed that after exposed to Qdots for 48 h, cells exhibited decreased survival rate (9,14,28). A dose-and size-dependent cytotoxic effect of Qdots was also observed (14,29). The hypothesis for the mechanism of the cytotoxicity of Qdots is that Cd2+ released from CdSe might trigger oxidative stress in cells (21).Previous studies have indicated that surface plays an important role in nanotoxicity (9,15,16,30,31). It has been demonstrated that the toxicity of nanoparticle could be influenced by surface functionalization (9,28). For instance, CdSe/ZnS solubilized by a simple ligand exchange with a mercaptoacid are less soluble and are toxic to breast cancer cells; in contrast, the toxicity is noticeably reduced if Qdots are embedded in a cross-linked shell (9,28,31). We have also shown that polyethylene glycol silanized Qdots (PEG-Qdots) did not induce any statistically significant cell cycle changes and minimal apoptosis/necrosis in human cells (17). In addition, a high throughput transcriptome-wide analysis revealed less than 50 genes (equivalent to-0.2% of total genes) out of more than 22,000 probed showed significant changes by PEG-Qdots treatment (17). Therefore, it is suggested that at least the biological properties of Qdots are attributable to the QD-capping material rather than to the core metalloid complex itself (15).Thus, in the present study, we evaluated the cytotoxic and genotoxic effects of QDs on human skin fibroblasts, in particular, PEG coating versus non-coated QDs.[Methods]1. Cell culture and Qdots treatment:Human skin fibroblasts (HSF) were routinely subcultured in a-modified Minimum Essential Medium (α-MEM) (Invitrogen, Carlsbad, CA), containing 10% newborn calf serum,100 U/ml penicillin,125μg/ml streptomycin, and 0.03% glutamine.80% confluence HSF were-exposed to 8 nM PEG-Qdot440,8 nM PEG-Qdot680,80 nM PEG-Qdot440, or 80 nM PEG-Qdot680, respectively, for various time spots. PBS was used as negative control.2. Cytotoxicity experiments1) MTS:Cells were seeded into 96-well culture plates at a density of 1 X 104 cells /well. After various QDs treatment,20 ul of MTS(5 mg/ml in PBS) was added to each well. Plates were further incubated in 37℃for another 45 mins. Absorbance at 490 nm was recorded on a microplate reader.2) Trypan Blue assay Suspend cells with PBS to final density at around 1 X 103/mL.75μl cell suspension was mixed with 75μl 0.4% Trypan blue solution.50μl was applied to the hemacytometer and the unstained (viable) and stained (nonviable) cells were counted separately in the hemacytometer to obtain the total number of viable cells3. Detection of yH2AX:1) Immunofluorescent microscopy:to observe the distributaion and formation of yH2AX foci in nucleus after treatment with Qdots.2) Western blotting:Extract nuclear protein from HSF cells after drug treatment and further subject to Western blot to detect yH2AX expression level.4. Comet assay:After treatment with Qdots, cells were resuspended to a density of 6×1010L-1. "Sandwich" gel was prepared followed by lysis, electrophoresis, DAPI stain procedure and finally analyzed using immunofluorescent microscope.5. ROS measurement:10 mM DCFH-DA stock solution (in methanol) was diluted 500-fold in PBS to yield a 20μM working solution. After QDs treatment, the cells in the 96-well plate were washed twice with PBS and then incubated in 100μl working solution of DCFH-DA at 37℃for 30 min. Fluorescence was then determined at 485 nm excitation and 520 nm emission wavelength using a microplate reader (Infinite M200).6. Dimensional (2D) protein electrophoresis:HSF were treated with PEG coated Qdots at 8 nM or 80 nM for 24 hours, then whole protein was extracted for 2D electrophoresis. [Results]1. PEG coating attenuates cytotoxicity.Without the PEG coating, at 8 nM, neither U-Qdot440 nor U-Qdot680 showed significant cytotoxicity, judging by relative cell survival, and cell proliferation. However, at 80 nM, U-Qdot680 exhibited cytotoxicity, leading to a decrease of cell survival. On the other hand, no significant cytotoxic effects were observed for both PEG-Qdots at either low (8 nM) or high (80 nM) concentrations during the 24 h period.2. Exposure to U-Qdots could induce DNA damage by measuringγH2AX foci formation, a sensitive indicator for DNA double strand breaks (DSBs).It was found that exposure to 80 nM PEG-Qdot440 or PEG-Qdot680 did not induceγH2AX foci formation during a 24 hr period, while a positive control, MNNG induced strongγH2AX foci formation. In contrast, exposure to 80 nM U-Qdot440 or U-Qdot680 increasedγH2AX foci formation, both in the percentage of cells containing foci and the number of foci per cell, in a time-dependent manner.3. Neutral comet assay confirmed U-Qdots indeed induced DNA DSBs.4. ROS might be responsible for the induction ofγH2AX foci by U-Qdots exposure.5. Proteomic analysis showed that at concentrations of 8 and 80 nM, both PEG-Qdot-440 and PEG-Qdot-680 induced only minor protein expression perturbations. [Conclusions]1. PEG as a coating material can reduce the cytotoxicity of Qdots.2. U-Qdots could induce DNA damage by measuringγH2AX foci formation, a sensitive indicator for DNA double strand break。3. ROS might be responsible for the induction of yH2AX foci by U-Qdots exposure.
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