Letters to the Editor pubs.acs.org/crt
Letter to the Editor Regarding the Article by Wittmaack o the Editor: In a perspective paper in Chemical Research in Toxicology this year, Dr. K. Wittmaack reanalyzed our data on the cytotoxicity of silica nanoparticles (SNP) in different cell lines.1 The aim of this reassessment was to derive a new dose metric that would better account for the cytotoxic activity of these particles in vitro. To perform this analysis, Dr. Wittmaack assumed that “gravitational settling must have contributed significantly [to the cytotoxic effect], presumably as the result of the formation of large agglomerates”. On the basis of this assumption, Dr. Wittmaack hypothesized that the cells were covered with several closely packed layers of SNP, “the dose required for complete cell death ranged between 4 and about 20 layers of NPs”. This reasoning led Dr. Wittmaack to conclude that the cytotoxicity observed in our experiments resulted from an aspecific overload phenomenon, “the toxic potential of individual silicate NPs [being] very low”. With the present letter, we would like to draw the attention of your readers to the fact that these conclusions should be, at least, questioned. In our original study2 as well as in a companion publication,3 it was already explained that the cytotoxicity experiments were conducted with monodisperse SNP (29.3 nm), taking great care to minimize effects of aggregation or agglomeration. We provided dynamic light scattering (DLS) measurements (Figure 1 in ref 2) clearly demonstrating that SNP remained monodisperse during 24 h in Dulbecco's modified Eagle's medium (DMEM) without fetal calf serum. At that time, we did not consider, however, the possible effects of cellular constituents that might induce aggregation, through either depletion forces or bridging flocculation.4 To verify the hypothesis of Dr. Wittmaack, we performed additional experiments to check whether SNP did aggregate to a sufficient extent, such that sedimentation may occur on the cultured cell layer. We performed turbidity and backscattering measurements (Turbiscan 2000 MA, Formulaction, France), which allow a direct statistical characterization of positional aggregation since the local scattering is expected to increase upon aggregation. Simultaneously, new DLS measurements (Brookhaven 90 Plus, BIC, United States), measuring light scattering at a fixed angle of 90°, were performed. DLS is exquisitely sensitive to detect the formation of aggregates because the signal intensity recorded by the instrument is expected to be proportional to the sixth power of the particle size, in the particle size regime considered in our study, so that the relative proportions of aggregates versus primary particles can be calculated. In this letter, we focus on monodisperse Stöber SNP with primary particle diameter (TEM) of 19 nm to reproduce the experimental conditions of the original paper.2 The synthesis procedure and the physicochemical characteristics of these 19 nm SNP were very similar to the SNP used in this original paper. We first incubated SNP during 24 h in DMEM without serum. No aggregation of the SNP could be detected by DLS (Figure 1A), and no sedimentation was evidenced by turbidity measurements (Figure 1B).
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© 2011 American Chemical Society
To verify that sufficient contrast existed for the studied SNP to detect large aggregates and particle sedimentation, we included a positive control condition (Figures 1C,D) using the same concentration of the SNP suspensions, incubated in the presence of 2.6 mM chloroquine, an agent previously reported to induce SNP aggregation.5 Clear changes in particle size and light transmission were observed, consistent with a rapid sedimentation of the aggregated SNP. We then examined whether the cells or their products potentially released during a cytotoxicity experiment could influence the behavior of the SNP. We cultured endothelial cells (EA.hy926, ref 2) at the bottom of the measurement tubes and monitored the aggregation status and sedimentation of cytotoxic SNP concentrations incubated with cells in DMEM without serum. For the EA.hy926 cells exposed to the highest particle concentration, we noted some slight aggregation of the SNP after 6 h (Figure 1E) but not to a sufficient extent (in size and/or in quantity) as to induce detectable sedimentation in a turbidity experiment (Figure 1F). However, the shift to smaller aggregate sizes for long incubation times (20 h in Figure 1E) and the decrease in the intensity generated by primary particles (see the trend in Figure 1E) might indicate slight sedimentation. The latter might, however, also result from the uptake of primary particles by the cells. Hence, we estimated the number of particles that may sediment and eventually end up covering the cells. We calculated the relative abundance of aggregates and primary particles from the intensity generated by both particle populations, taking the average particle diameter and the particle scattering efficiency under the 90° detector angle into account (see the Supporting Information). The number ratio of aggregates to primary particles was 1/5.7 × 106. Hence, we calculated, considering a density of 2 g/cm3 for solid SNP, that if all of the aggregates with a minimal diameter of 400 nm would sediment in 24 h, they would cover only 8% of the bottom surface area (see the Supporting Information). This estimation is largely an upper limit because aggregates were not expected to be extremely dense but rather to have a low to medium fractal dimension, and they would sediment more slowly. The value of 8% coverage is thus definitely a maximum estimate. It is also important to realize that after 6 h, and certainly after 20 h of incubation, cells were dying. We, therefore, verified the possibility that cellular components released by dying cells might be the cause of the slight aggregation observed, and we incubated SNP with a cellular lysate (Figure 1G,H). As in the system containing cell cultures at the bottom, slight aggregation and possible sedimentation were found from DLS measurements. Turbidity data, however, did not indicate sedimentation. These results suggest that cellular components, possibly released by death or dying cells, slightly modify the organization and stability of the SNP suspension. This aggregation is probably due to weak protein bridging between particles. Published: December 15, 2011 4
dx.doi.org/10.1021/tx2003382 | Chem. Res. Toxicol. 2012, 25, 4−6
Chemical Research in Toxicology
Letters to the Editor
Figure 1. Stability of a 19 nm amorphous SNP suspension under several conditions. Left panel: DLS measurements probing the particle size in the suspension at 1.5 cm above the bottom of the measurement tube. Middle panel: Schematic representation of the corresponding experimental setup and dispersion status of the nanoparticles. Right panel: light transmission through the measurement tube every 40 μm over the whole height of the aqueous suspension (17 mm). (A) DLS measurement of 700 μg SNP/mL in DMEM without serum showing a stable particle dispersion over 24 h. (B) Turbidity measurement of 700 μg SNP/mL in DMEM without serum showing no changes in sample turbidity for the duration of the experiment, confirming the stability of the sample in DMEM. (C) DLS measurement of a 700 μg/mL SNP aqueous suspension with 2.6 mM chloroquine. Large aggregates were detected immediately after introducing chloroquine. After 5 h, no more particles or aggregates were detected by DLS measurements, indicating fast sedimentation. (D) Turbidity measurement of 700 μg/mL SNP in an aqueous suspension with 2.6 mM chloroquine showing a turbid suspension at 0 h. Sedimentation of the large aggregates occurred rapidly, decreasing the light transmission through the lower part of the measurement tube where the particles sedimented (height 0−3 mm) and increasing light transmission through the upper part of the liquid from which the aggregates settled. The fast disappearance of aggregates from the suspension was confirmed since no changes in sample turbidity were observed after 4 h. (E) DLS measurement of 70 μg SNP/mL in DMEM without serum in contact with EA.hy926 cells grown at the bottom of the measurement tube (200 × 103 cells/cm2). This concentration corresponds to the EC50 of the EA.hy926 cells (not shown). At the beginning of the experiment, all particles were monodisperse. After 6 h, some larger entities, presumably small particle aggregates, were detected. (F) Turbidity of the 70 μg SNP/mL in DMEM without serum in contact with EA.hy926 cells grown at the bottom of the measurement tube. The turbidity of the sample did not change during the time course of the experiment, indicating that no massive sedimentation occurred. (G) DLS measurement of 700 μg SNP/mL in presence of a cell lysate, obtained from EA.hy926 cells disrupted in pure water (300 × 103cells/mL). At all time points, aggregates were observed next to primary particles. (H) Turbidity of the 700 μg SNP/mL in DMEM without serum in contact with EA.hy926 cells grown at the bottom of the measurement tube. The turbidity of the sample did not change during the time course of the experiment, indicating that no significant aggregation or sedimentation occurred.
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dx.doi.org/10.1021/tx2003382 | Chem. Res. Toxicol. 2012, 25, 4−6
Chemical Research in Toxicology
Letters to the Editor
To conclude, we thank Dr. Wittmaack for giving us the opportunity to further examine aggregation and sedimentation that may occur under the actual conditions of cytotoxicity experiments. The hypothesis of cytotoxicity induced by the formation of a dense layer of SNP covering the cells appears, however, very unlikely. Leen C. J. Thomassen† Dorota Napierska‡ Kasper Masschaele† Laetitia Gonzalez§ Virginie Rabolli∥ Micheline Kirsch-Volders§ Jan Vermant⊥ Peter H. Hoet‡ Johan A. Martens† Dominique Lison*,∥ † Center for Surface Chemistry & Catalysis, Katholieke Universiteit Leuven, Kasteelpark Arenberg 23, 3001 Heverlee, Belgium ‡
Laboratory of Lung Toxicology, Katholieke Universiteit, Herestraat 49, 3000 Leuven, Belgium §
Laboratory of Cell Genetics, Vrije Universiteit Brussel, Pleinlaan, 2, 1050 Brussels, Belgium ∥ Louvain Centre for Toxicology and Applied Pharmacology, Université Catholique de Louvain, Avenue E. Mounier, 53.02, 1200 Brussels, Belgium ⊥
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Department of Chemical Engineering, K. U. Leuven, W. de Croylaan 46, B-3001 Leuven, Belgium
ASSOCIATED CONTENT
S Supporting Information *
Numerical estimation of the number of aggregates that might have sedimented during the experiment. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
(1) Wittmaack, K. (2011) Novel dose metric for apparent cytotoxicity effects generated by in vitro cell exposure to silica nanoparticles. Chem. Res. Toxicol. 24 (2), 150−158. (2) Lison, D., Thomassen, L. C., Rabolli, V., Gonzalez, L., Napierska, D., Seo, J. W., Kirsch-Volders, M., Hoet, P., Kirschhock, C. E., and Martens, J. A. (2008) Nominal and effective dosimetry of silica nanoparticles in cytotoxicity assays. Toxicol. Sci. 104 (1), 155−162. (3) Thomassen, L. C., Aerts, A., Rabolli, V., Lison, D, Kirsch-Volders, M., Gonzalez, L., Napierska, D., Hoet, P. H., Kirschhock, C. E., and Martens, J. A. (2010) Synthesis and Characterization of Stable Monodisperse Silica Nanoparticle Sols for In Vitro Cytotoxicity Testing. Langmuir 26 (1), 328−335. (4) Nel, A. E., Madler, L., Velegol, D., Xia, T., Hoek, E. M., Somasundaran, P., Klaessig, F., Castranova, V., and Thompson, M. (2009) Understanding biophysicochemical interactions at the nanobio interface. Nat. Mater. 8 (7), 543−557. (5) Rabolli, V., Thomassen, L. C., Princen, C., Napierska, D., Gonzalez, L., Kirsch-Volders, M., Hoet, P. H., Huaux, F., Kirschhock, C. E., Martens, J. A., and Lison, D. (2010) Influence of size, surface area and microporosity on the in vitro cytotoxic activity of amorphous silica nanoparticles in different cell types. Nanotoxicology 4 (3), 307− 318.
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dx.doi.org/10.1021/tx2003382 | Chem. Res. Toxicol. 2012, 25, 4−6
