Physicochemical Properties and Cellular Toxicity of Nanocrystal Quantum

Transcription

1 Physicochemical Properties and Cellular Toxicity of Nanocrystal Quantum Dots Depend on their Surface Modification. Akiyoshi Hoshino 1,2,3, Kouki Fujioka 1,3, Taisuke Oku 1, Masakazu Suga 1, Yu F. Sasaki 4, Toshihiro Ohta 5, Masato Yasuhara 2, Kazuo Suzuki 3 and Kenji Yamamoto 1,2 1 Department of Medical Ecology and Informatics, Research Institute, International Medical Center of Japan, Toyama , Shinjuku, Tokyo , Japan 2 Department of Pharmacokinetics and Pharmacodynamics, Hospital Pharmacy, Tokyo Medical and Dental University, Yushima , Bunkyo-ku, Tokyo , Japan 3 Department of Bioactive Molecules, National Institute of Infectious Diseases, Toyama , Shinjuku, Tokyo , Japan 4 Faculty of Chemical and Biological Engineering, Hachinohe National College of Technology, Tamonoki Uwanotai 16-1, Hachinohe, Aomori , Japan 5 School of Life Science, Tokyo University of Pharmacy and Life Science,Horinouchi , Hachioji, Tokyo , Japan.

2 Contents: Experimental Procedures: Preparation of CdSe/ZnS fluorescent nanocrystal Qdots, Preparation of surface-modified nanocrystal QDs, and Protocols for Comet assay and cell viability assays Experimental Procedures Preparation of surface-modified nanocrystal QDs ZnS-coated CdSe nanocrystal QDs (fluorescence wavelength: approximately 518 nm, emitted green) were synthesized as previously described The synthesized QDs, which were enfolded into the micelle of n-trioctylphosphine oxide (TOPO), were dissolved into tetrahydrofuran in 4ml-volume flask. Then 11-mercaptoundecanioc acid (MUA), 2-aminoethanethiol (cysteamine hydrochloride), and 3-mercapto 1,2-propanediol (thioglycerol) were added in order to replace surface TOPO in these molecules. After moderately heating at 65 C for 24 hours under reflux conditions, distilled water was added to the flask and mixed. The turbid solution was collected and centrifuged to remove the water-insoluble residue. Next, supernatant fraction containing soluble QDs was applied to a Sephadex G-25 column (Amersham Biosciences, Piscataway, NJ) to remove aggregated nanocrystal complexes and

3 impurities such as TOPO and ZnS, and then flow-through was purified using an ultra-filtration membrane (Millipore) to remove low molecular ingredients such as MUA, cysteamine and thioglycerol. Finally, purified QD solution was powderized by vacuum distillation. QDs were reconstituted in distilled water and filtrated with a 0.1-µm filters (Millipore) immediately before use. The physiochemical properties of obtained QDs were investigated; the fluorescence intensity and peak emission wavelength were measured using a fluorescence spectrometer FP-6500 (JASCO corp., Japan), and the effective particle size and zeta-potential in aqueous solution were concurrently measured using particle characterizer ZetaSizer-Nano (Sysmex corp., Kobe, Japan). Comet assay MUA (Aldrich Chemical, Milwaukee, NJ) and TOPO (Wako Pure Chemical Industries, Japan) were dissolved in DMSO. Cysteamine and thioglycerol (Wako Pure Chemical) were dissolved in sterile water. For the comet assay, QDs were mixed with sterile sheep serum albumin solution at a final concentration of 10 µm. WTK1 human lymphoma cells 33 were maintained in RPMI-1640 medium supplemented with 10% FBS, 200 µg/ml sodium pyruvate, and 200 µg/ml streptomycin at 37 C under a 5% CO 2 atmosphere. Each 2 ml of cell suspension was placed in 5-cm dishes, and treated with

4 the test compound for 2 or 12 h. Immediately after treatment, cells were harvested, embedded in 1% GP42 agarose (Nakalai Tesque, Japan) dissolved in saline, and layered on a slide glass 27. Slides were placed in a chilled lysing solution (2.5 M NaCl, 100 mm Na 4 -EDTA, 10 mm Tris-HCl, 1% sarkosyl, 10% DMSO, and 1% Triton X-100, ph 10) and kept at 0 C in the dark for more than 60 min, then in chilled alkaline solution (300 mm NaOH and 1 mm Na 2 -EDTA, ph 13) for 20 min in the dark at 0 C. Electrophoresis was performed at 0 C in the dark for 20 min at 25 V (0.96 V/cm) and approximately 250 ma. The slides were then neutralized and stained with ethidium bromide. The length of the whole comet was measured for 50 nuclei for each dose using a fluorescence microscope (200 magnification) with a green filter. The difference between the means in the treated and control plates was compared with the Dunnett test after one-way ANOVA. A p-value less than 0.05 was considered statistically significant. Cell culture and viability assays Vero cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM/F12 culture media (Invitrogen, Carlsbad, CA) supplemented with 5% heat-inactivated FBS (Gibco, Grand Island, NY) at 37 C. Cell viability was assessed by

5 MTT assay and Flow cytometry. In MTT assay, cells were plated at cells on 96-well plates. Then QD solution was directly added to culture media 14,25. Then Cell Proliferation Kit (Roche Diagnostics) was added into wells. After incubation for 4h, cells were lysed on the plate and absorbance was measured by microplate reader (BioRad). In flow cytometry, cells were plated at cells on 24-well plate and incubate for 12 h with various QDs. After incubation, cells were detached without trypsin from culture plates using modified Puck s EDTA solution (140 mm NaCl, 5 mm KCl, 5.5 mm glucose, 4 mm NaHCO 3, 0.8 mm EDTA, and 9 mm HEPES buffer; ph 7.3). Then Cells were wshed, stained with propidium iodide, fixed with 1% paraformaldehyde-contained PBS, and the fluorescence of QDs and PI was analyzed by flow cytometry (BD biosciences).