Keywords:
Biological effects, Experimental investigations, Experimental, Contrast agents
Authors:
C. Strobel1, R. Herrmann2, A. A. Torrano3, C. Bräuchle3, W. A. Kaiser1, I. Hilger1; 1Jena/DE, 2 Augsburg/DE, 3Munich/DE
DOI:
10.1594/ecr2013/C-2054
Methods and Materials
TiO2 nanoparticle samples #1,
#3 and #4 (Table 1) were obtained from Merck KGaA (Germany; Eusolex® T [sample #1]; Eusolex® T-2000 [sample #3] and Eusolex® T-ECO [sample #4]).
Samples #2,
#5 and #6 were isolated from sun protection agents (sample #2: Babysmile Sonnenmilch (Win Cosmetic,
Germany); sample #5: Babylove Sonnencreme (dm-drogerie markt,
Germany) and sample #6: Ladival® Sonnenschutz Milch (Stada,
Germany)).
Fourier transform infrared measurement was used to determine the composition of the coating and secondary shell of the TiO2 nanoparticles.
The size and shape of the TiO2 nanoparticles were determined by transmission electron microscopy (TEM),
whereas the hydrodynamic diameters deduced by dynamic light scattering (DLS) and the ζ-potential were measured with a Zetasizer (Nano ZS Malvern Instruments,
UK).
To elucidate the possible effect of nanoparticles on model targets after intravenous application,
the permanent human microvascular endothelial cell line HMEC-1 was used.
The relative dehydrogenase activity (rcDH activity [%]) and relative ATP content (rATP content [%]) of cells following exposure to nanoparticles were determined by MTS (CellTiter 96® AQueous One Solution Cell Proliferation Assay Kit,
Promega GmbH,
Germany) and ATP assay (CellTiter-Glo® Luminescent Cell Viability Assay,
Promega GmbH,
Germany),
respectively.
To interpret the impact of the nanoparticles on endothelial cells,
the threshold for cytotoxicity according to DIN EN ISO 10993-5:2009-10 was considered.
A commercial ELISA kit (RayBiotech,
USA) was used to determine the presence of the monocyte chemo-attractant protein-1 (MCP-1) which was released from cells after nanoparticle treatment.
This readout was used as marker for pro-inflammatory impact.
TiO2 nanoparticles with a fluorescence label (N-(2,5-bis(dimethylethyl)phenyl)-N’-(3-(triethoxysilyl)-propyl-perylene-3,4,9,10-tetracarboxylic acid diimide) were used to visualize the localization of the nanoparticles within the cells via laser scanning microscopy.
For statistical analysis IBM SPSS Statistics,
version 19.0 (©2010 SPSS Statistics 19 Inc,
an IBM Company,
USA) was used,
employing ANOVA.
To determine differences between different groups the post hoc Bonferroni test was used.
Results were considered as statistically different at P ≤ 0.05.