Supplementary information
Impact of serum as a dispersion agent for in vitro and in vivo toxicological
assessments of TiO2 nanoparticles
Sandra Vranic1, 2, *, §, Ilse Gosens3, *, Nicklas Raun Jacobson4, Keld A. Jensen4, Bas Bokkers5, Ali Kermanizadeh6, 7,
Vicki Stone6, Armelle Baeza-Squiban1, Flemming R. Cassee3, Lang Tran8, Sonja Boland1
1
Univ Paris Diderot, Sorbonne Paris Cité, Unit of Functional and Adaptive Biology (BFA), UMR 8251 CNRS,
Laboratory of Molecular and Cellular Responses to Xenobiotics 5 rue Thomas Mann, 75 013 Paris, France
2
Nanomedicine Lab, Institute of Inflammation and Repair, Faculty of Medical and Human Sciences & National
Graphene Institute, AV Hill Building, University of Manchester, Upper Brook Street, Manchester M13 9PT, United
Kingdom
3
Centre for Sustainability, Environment and Health, National Institute for Public Health and the Environment,
Bilthoven, the Netherlands
4
Danish Centre for Nanosafety, National Research Centre for the Working Environment, Copenhagen, Denmark
5
Centre for Safety of Substances and Products, National Institute for Public Health and the Environment, Bilthoven,
the Netherlands
6
Heriot-Watt University, School of Life Sciences, John Muir building, Edinburgh, UK
7
University of Copenhagen, Department of Public Health, Section of Environmental Health, Copenhagen, Denmark
8
Institute of Occupational Medicine, Edinburgh, UK
* equal contributions
§
Corresponding author:
Sandra Vranic
[email protected]
1. Physicochemical properties of primary TiO2 NPs
Physicochemical characteristics of primary TiO2 NPs have been previously studied in detail (Kermanizadeh et al.
2013) and are summarized in Supplementary Table 1 and Figure 1.
Composition
XRD size
Crystalline
phase
BET surface
area
Elemental
impurities
Coating
TiO2
10 nm
Rutile
84 m2/g
<1%
Triethoxypropyl
aminosilane
Supplementary Table 1: Physicochemical properties of primary TiO2 NPs (previously published data (Kermanizadeh
et al. 2013)).
Transmission electron microscopy was carried out on diluted samples dispersed in Nanopure-filtered water added to
200 mesh holey carbon-coated Cu-grids which were air-dried in a HEPA-filtered biosafety Class II cabinet (Holten,
DK) before analysis on a 200 kV Tecnai G20 Transmission Electron Microscope (FEI Company, Oregon, USA).
TEM-grids were stored in individual 1 ml Eppendorf tubes until analysis to reduce the risk of contamination.
5
0
n
m
A)
Supplementary Figure 1: Transmission electron microscopy image of TiO2 NPs.
Analysis by TEM in the current study showed that the sample by number was dominated by <15 nm-size individual
crystallites and dense aggregates of 20 to 50 nm-size primary particles. The small crystallite size is in good agreement
with the specific surface area of 84 m2/g.
2. Size distributions of TiO2 NPs diluted in culture media
DLS measurements were performed as described in materials and methods. Representative size distribution of NPs
dispersed in water or in the presence of 2% serum and then diluted to 1/10 in RPMI cell culture medium, measured by
DLS.
Supplementary Figure 2. Size distributions of TiO2 NPs dispersed in water (A) or 2% serum (B) after a 1/10 dilution
in RPMI cell culture media measured by DLS.
3. WST-1 assay
In order to assess the cytotoxicity of TiO2 NPs, a viability assay (WST-1 assay) based on the measurement of cellular
metabolic activity was performed (Supplementary Figure 3). Cells were seeded in 96-well plates at 10,000 cells per
well in complete culture medium and incubated for 48 h before treatment with 100 µl of NPs. Metabolic activity was
assessed using the WST-1 cell proliferation reagent (Roche, Meylan, France) according to the manufacturer’s
recommendations. After 2 h of incubation with cells, supernatants were transferred to a fresh plate in order to decrease
the potential interference of NPs during the absorbance measurement at 450 and 630 nm using a microplate reader
(ELx800, BioTek, USA).
Only a slight cytotoxicity was observed after 24h of treatment with TiO2 NPs dispersed in water whereas TiO2 NPs
dispersed in the presence of serum were found to be non-cytotoxic.
Supplementary Figure 3. Cytotoxicity of TiO2 NPs. Metabolic activity of cells after 24h of exposure to TiO2 NPs
dispersed in the presence or absence of serum, assessed by WST-1 assay. * significantly reduced compared to the
control, ≠ significant difference between two conditions, (p<0.05 ANOVA followed by Dunnet’s t test).
4. Induction of IL-8mRNA by TiO2 NPs in NCI-H292 cells
RT-qPCR analysis was performed after seeding cells in 6 well plates at 10,000 cells/cm2 in complete cell culture
medium and cultured for 48 h before treatment with NPs for 24 h. mRNA extraction, purification and analysis were
performed as previously described (Ramgolam et al. 2012). Briefly, RNA was extracted by TRI REAGENT®
(Euromedex, France) quantified by Nanodrop 2000 (Thermo Scientific, France). Reverse transcription was done by
M-MLV Reverse Transcriptase kit (Promega, Charbonnieres-les-bains, France). Quantitative PCR was performed
with a Roche LC480 using LightCyclerR480 SYBR Green I Master (Roche Diagnostics, Mannheim, Germany) with 5
µL of cDNA diluted to 1/20 using specific primers (Sense : 5’-CTC TCT TGG CAG CCT TCC T-3’, Anti-sense: 5’AAT TTC TGT GTT GGC GCA GT-3’). The Ct values and the normalized ratio were determined with
LightCyclerR480 software 1.5. Levels of gene expression were normalized using Ribosomal Protein L19 (RPL19,
Sense: 5GGC TCG CCT CTA GTG TCC TC3 5C, Anti sense: 5CAA GGT GTT TTT CCG GCA TC3), as its
expression remained unaltered with our treatment conditions.
Supplementary Figure 4. Expression of IL-8 mRNA in NCI-H292 cells after 24 hours of exposure to TiO2 NPs
dispersed in the presence or absence of serum measured by RT-qPCR and normalized to the control. Error bars
indicate SEM of 6-15 samples. * significant difference compared to the control, ≠ significant difference between two
conditions, (p<0.05 ANOVA followed by Dunnet’s t test).
Induction of a pro-inflammatory response was confirmed by quantifying the mRNA expression of Il-8. A high
induction of IL-8 mRNA expression was only observed when NPs were dispersed in the absence of serum
(Supplementary Figure 4).
5. Characterization of TiO2 agglomerate size for intratracheal instillation
For in vivo experiments, Nanosight analysis (LM20, NanoSight Ltd, UK) was performed immediately before
intratracheal instillation on stock suspensions diluted in ultrapure water (0.1 µg/ml TiO2). The data were compiled
from three separate measurements and the median and mean particle size was determined. Similar particle size
distributions were obtained for TiO2 dispersions in ultrapure water with and without serum.
NP dispersion
TiO2 NPs diluted in ultrapure water
TiO2 NPs diluted in ultrapure water + 2% mouse serum
Mean particle
size (nm)
Median particle size
(nm)
159 +/-5
137 +/- 8
148 +/- 23
103 +/- 19
Supplementary Table 2. Hydrodynamic diameter of TiO2 NP dispersions.
.
Kermanizadeh A, Vranic S, Boland S, et al. (2013) An in vitro assessment of panel of engineered nanomaterials using
a human renal cell line: cytotoxicity, pro-inflammatory response, oxidative stress and genotoxicity. BMC
Nephrol 14:96 doi:10.1186/1471-2369-14-96
Ramgolam K, Hamel R, Rumelhard M, Marano F, Baeza-Squiban A (2012) Autocrine effect of EGFR ligands on the
pro-inflammatory response induced by PM(2.5) exposure in human bronchial epithelial cells. Arch Toxicol
86(10):1537-46 doi:10.1007/s00204-012-0863-x