SUPPORTING INFORMATION

Transcription

1 SUPPORTING INFORMATION SUPPLEMENTARY FIGURE LEGENDS Fig. S1. Separation of non-dissolved nanoparticles. Tests were conducted on the separation of non-dissolved nanoparticles added in excess to BEGM (A) and complete DMEM (B) cell culture media. The maximum dissolved zinc concentrations that can be attained by dissolution in the cell culture media were measured. The nanoparticles cannot dissolve more than permitted by this effective saturation concentration. Centrifugation at 22, g for 5 minutes successfully removes solid phase from BEGM, but is unable to remove all excess from DMEM solutions. Fig. S2. Assessment of cellular viability using MTS and PI uptake assays. (A) After exposure to 25 µg/ml of the NP suspension for 1-16 hrs, cells were incubated with the MTS reagent for 3 min and the absorbance was measured at 49 nm. All the MTS values for different doses were normalized according to the control values (no particle exposure), which were regarded as 1% cell viability. (B) After exposure to metal oxide nanoparticles at doses of 6-1 μg/ml for 16 hrs, cells were stained with 47.5 μg/ml PI and immediately analyzed by flow cytometry. The % PIpositive (M1-gated) cells were scored by Cellquest. Data are presented as mean ± standard deviation. p<.1. Data are representative of 3 separate experiments with at least 3 wells per treatment. 1

2 Fig. S3. Calibration of CNT-NADH peroxidase (Npx)-bioelectrode using H 2 O 2. The bioelectrodes were equilibrated in the reaction buffer (acetate buffer, ph 6) or water, then scanned to obtain initial cyclic voltammograms (CV) of the enzyme assembly prior to sample measurements. The data points represent the readings generated by the nanobiosensor (electric current in µa) at different H 2 O 2 concentrations. Calibration curves were generated with linear regression based on the data gathered using standard H 2 O 2 solutions in (A) low (< 1 nm) and (B) high (1-4 nm) range. Fig. S4. Relative mrna levels in cells after 6 hr exposure to 25 μg/ml metal oxide NP. After NP exposure, semi-quantitative Nrf2 and NQO-1 mrna levels in RAW and BEAS-2B cells were determined using realtime PCR analysis as described in Materials and Methods. Results represent the fold change (means ± SD) compared with the control group. p <.5. Fig. S5. Electron microscopy to determine ultrastructural changes in BEAS-2B cells after exposure to 25 μg/ml metal oxide NP for 16 hr. Panels on the left depict 4,8 magnification, while panels on right depict 15, magnification to show the organellar details. Please notice disappearance of the mitochondria (mito), nuclear condensation, and vacuolar appearance of the cell after exposure. Fig. S6. Electron microscopy to determine the ultrastructural changes in RAW cells after exposure to 25 μg/ml metal oxide nanoparticles for 16 hr. Panels on the left depict 4,8 magnification, while panels on right depict magnification at 15, to show the mitochondrial details. Please notice the mitochondrial disappearance and nuclear fragmentation after treatment. 2

3 Fig. S7. Surface modification and fluorescent labeling of metal oxides. The surfaces of the metaloxide (,, ) nanoparticles were functionalized with primary amines by reacting the surface hydroxyl groups with the alkoxy silane groups of aminopropyltriethoxysilane (APTS). The amines introduced on the particle surface and then reacts with fluorescein isothiocyanate (FITC) dye molecules. The details of these labeling procedures were described in Materials and Methods. Fig. S8. Confocal microscopy to study the cellular distribution of Zn 2+ in RAW cells. RAW cells were treated with 25 μg/ml NP for 6 hr. Cells were stained with 5 µm Newport Green DCF for 3 min. After fixation cells were visualized in a confocal microscope. 3

4 Figure S1 A BEGM B CDMEM Zn concentration (µm) Total Zn concentration Max Zn concentration Zn concentration (µm) Total Zn concentration Max Zn concentration Centrifugation time (min) Centrifugation time (min)

5 Figure S2 A % Cell Viability (MTS) B % PI + Cells RAW Time (h) RAW Dose (μg/ml) % Cell Viability (MTS) % PI + Cells BEAS-2B BEAS-2B Time (h) Dose (μg/ml)

6 Figure S3 A 2.5 B 6 Current /µa Current /µa [H 2 O 2 ]/ nm [H 2 O 2 ]/ nm

7 Figure S4 Relative mrna levels Relative mrna levels RAW Nrf2 BEAS-2B Nrf2 Relative mrna levels Relative mrna levels Φ Φ NQO-1 Φ NQO-1 Φ

8 Figure S5 BEAS-2B Φ 4 µm 2 µm

9 Figure S6 RAW Φ Ap Ap Ap Ap 4 µm 2 µm

10 Figure S7 Surface Modification and Fluorescent Labeling of Metal Oxides H 2 N H 2 N H 2 N NH 2 Si O Si O Si O Si Si O Si O Si O ( Si O O O) ( O O O) O O O O = FITC Metal oxides coated with NH 2 -silane FITC-labeled metal oxides

11 Figure S8 A ZnSO 4 25 µg/ml LAMP-1 Newport Green DCF Overlay RAW µm B Caveolin-1 Newport Green DCF BEAS-2B Overlay 1 µm