ToxSci Advance Access originally published online on April 8, 2008
Toxicological Sciences 2008 104(1):155-162; doi:10.1093/toxsci/kfn072
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Nominal and Effective Dosimetry of Silica Nanoparticles in Cytotoxicity Assays
* Industrial Toxicology and Occupational Medicine unit, Université catholique de Louvain, Avenue E. Mounier, 53.02, 1200 Brussels, Belgium
Center for Surface Chemistry and Catalysis, Katholieke Universiteit Leuven, Kasteelpark Arenberg 23, 3001 Heverlee, Belgium
Laboratory of Cell Genetics, Vrije Universiteit Brussel, Pleinlaan, 2, 1050 Brussels, Belgium
Laboratory of Lung Toxicology, Katholieke Universiteit Leuven, Herestraat 49, 3000 Leuven, Belgium
¶ Department Metallurgy and Materials Engineering, Katholieke Universiteit Leuven, Kasteelpark Arenberg 44, bus 2450, 3001 Heverlee, Belgium
1 To whom correspondence should be addressed. Fax: +32-2-764-53-38. E-mail: dominique.lison{at}uclouvain.be.
Received December 6, 2007; accepted March 29, 2008
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ABSTRACT |
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Because of their small size and large specific surface area (SA), insoluble nanoparticles are almost not affected by the gravitational force and are generally formulated in stable suspensions or sols. This raises, however, a potential difficulty in in vitro assay systems in which cells adhering to the bottom of a culture vessel may not be exposed to the majority of nanoparticles in suspension. J. G. Teeguarden et al., 2007, Toxicol. Sci. 95, 300–312 have recently addressed this issue theoretically, emphasizing the need to characterize the effective dose (mass or number or SA dose of particles that affect the cells) which, according to their model based on sedimentation and gravitation forces, might only represent a very small fraction of the nominal dose. We hypothesized, in contrast, that because of convection forces that usually develop in sols, the majority of the particles may reach the target cells and exert their potential toxicity. To address this issue, we exposed three different cell lines (A549 epithelial cells, EAHY926 endothelial cells, and J774 monocyte-macrophages) to a monodisperse suspension of Stöber silica nanoparticles (SNP) in three different laboratories. Four different end points (lacticodehydrogenase [LDH] release, LDH cell content, tetrazolium salt (MTT), and crystal violet staining) were used to assess the cell response to nanoparticles. We found, in all cell lines and for all end points, that the cellular response was determined by the total mass/number/SA of particles as well as their concentration. Practically, for a given volume of dispersion, both parameters are of course intimately interdependent. We conclude that the nominal dose remains the most appropriate metric for in vitro toxicity testing of insoluble SNP dispersed in aqueous medium. This observation has important bearings on the experimental design and the interpretation of in vitro toxicological studies with nanoparticles.
Key Words: nanotoxicology; cytotoxicity; nanoparticles; monodisperse silica.
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INTRODUCTION |
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Nanomaterials are defined as particles, crystals, fibers, films, or composites with at least one dimension below 100 nm. Because of their nanoscale dimensions and hence large specific surface area (SA), nanomaterials exhibit remarkable properties. They represent a transition between bulk materials and atomic or molecular structures and, at this level, quantum effects lead to the occurrence of specific physicochemical properties (e.g., malleability, electrical conduction, magnetism). Because of their very specific nature, nanomaterials may also exhibit specific toxic properties. In recent years, the toxicology of nanomaterials has been the focus of a growing number of scientists (Nel et al., 2006
; Oberdorster et al., 2005). While the number of publications dealing with nanotoxicology and referenced in PubMed (keywords, "nanostructures/*toxicity") was limited to 12 in 2004, we counted 236 publications in January 2008. Assessing the toxic potential of nanomaterials represents, however, a serious challenge which has been addressed by several excellent reviews (Balshaw et al., 2005; Borm et al., 2006; Holsapple et al., 2005; Powers et al., 2006; Thomas and Sayre, 2005; Thomas et al., 2006; Tsuji et al., 2006; Warheit et al., 2007). In particular, defining the most appropriate dosimetry of nanomaterials remains a challenging issue, which has important impacts on both exposure assessment and experimental design (see e.g., Oberdorster et al., 2007; Wittmaack, 2007). A prevailing view is that nanoparticle SA is an important determinant of toxicity and could be proposed as a useful dose metric (Monteiller et al., 2007).
In addition, because of their small mass and large SA, nanoparticles are barely affected by the gravitational force, and Teeguarden et al. (2007) have recently addressed the difficulties to adequately characterize the dose of nanoparticles in suspension in in vitro systems. Assuming that particles have to sediment to reach their cellular target, they mainly considered gravitational settling and diffusion as the essential forces determining particle behavior in culture systems. They estimated that only a small fraction of nanoparticles in the lower part of the suspension would reach the cells by sedimentation. These investigators warned nanotoxicologists about the pitfalls of simply considering the nominal dose of nanomaterials in in vitro experiments and recommended to carefully consider what is actually delivered to the cells. In this modeling of nanoparticle migration, convection forces resulting from density fluctuations in suspensions were not considered.
We formulated the hypothesis that because of convection forces that are almost always present in suspensions, the majority of nanoparticles will collide with adherent cells and exert biological effects. To test this hypothesis, we produced a monodisperse suspension of Stöber amorphous silica nanoparticles (SNP) as a model and examined in vitro dose-effect relationships based on cytotoxicity assays.
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MATERIALS AND METHODS |
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Nanoparticle Preparation and Characterization
Monodisperse Stöber SNP were synthesized by mixing tetraethoxysilane (98%, Acros, Geel, Belgium) with a 25% ammonia solution (Fluka, Seelze, Germany) in the presence of absolute ethanol (VWR, Heverlee, Belgium) acting as a cosolvent (van-Blaaderen and Kentgens, 2007
). The molar concentrations of the reactants and characteristics of the dispersion are shown in Table 1. The dispersion was dialyzed during 24 h against heat-sterilized MilliQ water using Nadir dialysis membranes with average megawatt cut-off of 8000 Da (Spectrum, Breda, The Netherlands) in order to remove dissolved silicates, NH4OH, and ethanol.
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The hydrodynamic diameter of these particles was determined by dynamic light scattering (DLS) with an ALV High Performance Particle Sizer equipped with a He/Ne laser beam (wavelength 632.8 nm and power 3 mW). The scattered photons were detected under an angle of 173°. The autocorrelation functions were analyzed with inverse laplace transformation-maximum entropy method (Delsuc and Malliavin, 1998
) analysis and relative intensity distributions were calculated. A Gaussian fit on these monomodal distributions was used to calculate the mean and SD of these curves. Transmission electron microscopy images were also taken with a Philips CM200 FEG microscope operating at 200 kV (Table 1). Additional experiments were conducted with a stable monodisperse suspension of 15 nm nanoparticles obtained after extensive dialysis of a LudoxHS-40 preparation (Sigma Aldrich, Bornem, Belgium) against MilliQ water.
Cell Culture and Experimental Protocols
Different cell lines were used: J774, a mouse monocyte-macrophage American type culture collection (ATCC), at the Université catholique de Louvain; A549, a human type II lung epithelium (ATCC), at the Vrije Universiteit Brussel, and EAHY926, a human endothelium cell (Suggs et al., 1986), at the Katholieke Universiteit Leuven. The cells were seeded in 96-well plates (30,000, 10,000, and 66,000 cells/well, respectively) in Dulbecco's modified Eagle medium (DMEM, Invitrogen, Merelbeke, Belgium) supplemented with 10% fetal calf serum and 1% antibiotics (penicillin-streptomycin). After 24–48 h, preconfluent cells were washed twice with DMEM (without serum or antibiotics) and further incubated for 24 h in a given volume of DMEM containing the SNP dispersion at the desired concentration in the absence of serum (see below). The final SNP dispersions were prepared immediately before use from a common stock dispersion in DMEM diluted serially and vortexed before distribution in the culture wells. Control cells were incubated in a volume of DMEM equivalent to the corresponding test condition (see below). At the end of the exposure period, the cytotoxic response was evaluated with different assays. Lacticodehydrogenase (LDH) release and crystal violet staining were used to assess cytotoxicity in J774 cells. Briefly, at the end of the 24-h incubation period, the culture medium was harvested for the measurement of LDH activity (Lison and Lauwerys, 1990); the wells were rinsed once with phosphate-buffered saline (PBS) and stained with crystal violet (Flick and Gifford, 1984). Tetrazolium salt (MTT) reduction was used to assess cytotoxicity in A549 cells (Mosmann, 1983). Briefly, after 24 h of incubation with the particles, the medium was removed and the cells were incubated with MTT for 4 h before absorbance reading at 595 nm after complete solubilization of the formazan crystals in a 10% SDS/0.01M HCl solution. For EAHY926 cells, cell integrity was assessed by determining the percentage of LDH retained by the cellular layer (Roesems et al., 1997). The LDH activity of the medium (LDHmedium) and the cell lysate (LDHcells) was determined by monitoring spectrophotometrically the reduction of pyruvate. Cells viability was calculated according to the formula: % viability = (LDHcells/LDHcells + LDHmedium) x 100. The expression of LDH-based assays differed therefore between the laboratories using J774 and EAHY926 cells; the first one reported the fraction of extracellular enzyme activity (cytotoxicity was reflected by an increase of the measured parameter) whereas the second reported the remaining intracellular fraction (cytotoxicity was reflected by a decrease of the measured parameter). MTT assay was also used to monitor the cytotoxic response in EAHY926 cells. At the concentrations used, SNP did not influence LDH or MTT measurements.
Experiment 1.
Preconfluent cells were incubated during 24 h with increasing concentrations of SNP in a fixed volume of 200 µl per well. Effective concentrations of SNP (EC, defined as the concentration causing about 50% reduction of cell viability or 50% of the maximal cell response) were selected for each cell line and for each end point.
Experiment 2.
The cells were incubated for 24 h with a fixed concentration of SNP dispersions (equal to EC values obtained in experiment 1, expressed in µg/ml) but in increasing volumes (40, 80, 120, 160, 180, 200, and 240 µl per well), i.e., with increasing total mass/number/SA of particles.
Experiment 3.
The cells were incubated for 24 h with a fixed mass of SNP (equal to EC values obtained in experiment 1, expressed in µg per well) but in increasing volumes (40, 80, 120, 160, 180, 200, and 240 µl per well), i.e., with decreasing particle concentrations.
Silicon Content Measurements
Experiments were conducted with preconfluent J774 cells to determine the amount of SNP in contact with the cell layer (within or on the cells) after 6 h of exposure. In order to obtain a sufficient amount of material for reliable elemental determinations, the experiment was conducted in 48-well plates, and the volumes of culture medium were adapted to keep the same ratio of nanoparticle mass per cell. Since the SA of a 48-well (0.81 cm2) is 2.25-fold larger than that of a 96 well (0.36 cm2), the volumes were multiplied by 2.25. After 6 h of contact with the suspensions, the cells were washed with PBS and solubilized in MilliQ water containing 0.1% Triton X100 before direct Si measurement by inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7500) using a certified Si solution as standard.
Statistics
Results of cytotoxicity assays are expressed as percent of the response measured in control cells exposed in an equivalent volume of medium (arithmetic means ± SD). Dose-effect relationships were assessed by ANOVA and subsequent trend tests. p values <0.05 were considered significant.
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RESULTS |
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We first verified that the SNP dispersion remained stable during the experimental procedure. DLS measurements (Fig. 1) confirmed that the hydrodynamic diameter of the particles remained practically unchanged after dialysis and dilution in DMEM. The particles remained monodisperse for at least 25 and 4 days in MilliQ water and in DMEM, respectively.
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We then selected effective concentrations of nanoparticles (EC) in the different cytotoxicity assays in order to define a range of dose where slight variations would have a maximal impact on the measured end point. The cells were exposed to different concentrations of SNP in a fixed volume of medium. Figures 2–4
(experiment 1) show the dose-effect relationships for the three cell lines. EC values selected for the three cell lines and for the respective cytotoxicity assays are summarized in Table 2.
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We then reasoned that if the majority of SNP present in a given volume of culture medium would contribute to the cytotoxic activity, the amplitude of the effect would vary with the total volume of nanoparticle dispersion. We therefore incubated cells with these EC but in volumes below and above 200 µl (same concentration, increasing volume, increasing particle mass, total number, or total SA). The results depicted in Figures 2–4 (experiment 2) clearly indicate that, in all cell lines and for all cytotoxicity tests, the response increased with the volume of dispersion which is linked with the total mass, number, or SA of particles present in the culture medium (linear trend test, p < 0.0001 in all cases).
We also assessed the amount of SNP in contact with the cells by measuring the silicon content in the cellular layer. This measurement was performed after 6 h of incubation to avoid the possible impact of cell loss which might have followed cytotoxicity if it was assessed at 24 h. The results shown in Figure 5A clearly indicate that the target dose of SNP, i.e. in contact with or within the cells (about 10% of the total mass of Si in suspension), increased with the total mass of particles in suspension. These results further supported our hypothesis that the majority of suspended particles determine the cell response.
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We then incubated the cells with a fixed mass of particles suspended in decreasing volumes (same mass/number/SA, decreasing volume, increasing concentration) to assess the influence of nanoparticle concentration. The results depicted in Figures 2–4 (experiment 3) clearly indicate that the cytotoxic responses increased with particle concentration (linear trend test, p < 0.0001 in all cases). The cellular silicon content (Figure 5B) although less influenced by the concentration than by particle mass increased with particle concentration. The cellular dose measured in cells incubated with the lowest volume (90 µl) did, however, not fit the general trend of increasing dose with increasing concentration.
To verify whether our observations would also apply to other SNPs, we tested Stöber nanoparticles with other sizes (15 to >100 nm, not shown) or 15 nm Ludox nanoparticles (Fig. 6) and obtained similar responses.
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DISCUSSION |
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Monodisperse particles diffuse, settle, and agglumerate in culture medium as a function of medium density and viscosity, and depending on particle size, shape, charge, and/or density. To examine whether these phenomena could significantly affect the accurate estimation of the dose actually delivered to the cells tested, Teeguarden et al. 2007
developed a model based on the assumption that particles have to sediment to reach their target. They mainly considered gravitational settling and diffusion as the essential forces determining particle behavior in culture systems. Considering these mechanisms of particle migration, it was estimated that the active fraction of particles (i.e., reaching adherent cells at the bottom of the culture well, delivered dose) might be extremely low or even negligible for particles with nanoscale dimensions. It was calculated that it would take about 14 h for a 50-nm diameter particle to move over a distance of 1 mm by diffusion, and, when considering gravitational force, it would take about 17 days for a 50-nm SNP to sink over the same distance. Consequently, these investigators warned nanotoxicologists about the pitfalls of simply considering the nominal dose of nanomaterials in in vitro experiments and recommended to carefully consider what is actually delivered to the cells.
To examine this issue experimentally, we choose amorphous SNP as a model because they were previously shown to be cytotoxic (Chang et al., 2007; Jin et al., 2007; Lin et al., 2006). Stöber nanoparticles can be produced under sterile conditions, formulated in stable monodisperse suspensions, and allow the specific and sensitive measurement of silicon in target cells to monitor the delivered dose. Moreover, contrary to other nanomaterials (Wörle-Knirsch et al., 2006), we found that Stöber nanoparticles did not interfere with the cytotoxicity assays that were used in this study. We needed, however, to consider the possibility that Stöber nanoparticles were solubilized partly or totally in the culture medium, which would have impacted on the interpretation of the experimental results. This hypothesis could, however, be excluded both theoretically and experimentally. The kinetics of silica dissolution in water is, indeed, relatively slow with a dissolution equilibrium that is reached after about 15 days in aqueous suspensions (Iler, 1979). In cell culture medium, amorphous silica is considered as insoluble (Brunner et al., 2006). The DLS measurements conducted on the suspensions in water or in culture medium confirmed that we used monodisperse suspensions of insoluble nanoparticles. The results of cytotoxicity experiments showed that although the Stöber nanoparticles would not significantly sediment according to the diffusion/gravitational settling model of Teeguarden et al. (2007), the cytotoxic effects were related to the total mass (or total number or total SA) and concentration of nanoparticles in suspension of the test system. If, on the contrary, the diffusion/gravitation model (Teeguarden et al., 2007) was at stake, only a very small fraction of nanoparticles suspended in a volume about 3 mm above the adherent layer (about 100 µl based on a SA of 0.360 cm2 for a 96-well plate) would have reached the cells over an incubation period of 24 h (about 8 h to diffuse over 1 mm according to equation 3 in Teeguarden et al., 2007). This fraction would have been minimal and, in any case, independent on the total dispersion volume, certainly when it exceeded 100 µl.
The measurement of Si in or on the J774 cells, indicating that the cellular dose increased with the nominal dose and nanoparticle concentration, further supported our finding. Why the cellular dose was less influenced by particle concentration than by mass, number, or SA is not immediately clear and deserves further investigations. The lower Si cellular content observed at the highest concentration (Fig. 5B, 90 µl or 185 µg/ml) might reflect early cytotoxicity and early loss of adherent cells.
The consistent findings in our three laboratories using monodisperse suspensions of SNP, three different cell lines, and different cytotoxicity assays strongly support the robustness of our observation which has important bearing on the experimental design and the interpretation of in vitro toxicological studies with nanoparticles. Extrapolation of these findings to nanoparticles other than silica remains an important issue, but no data are available.
The fact that some engineered nanomaterials tend to aggregate and agglomerate rapidly to form larger particles, with dimensions often exceeding the nanometric range, may introduce an additional level of complexity to characterize particle behavior in in vitro systems. However, these larger aggregates/agglomerates will generally settle rapidly and be effectively delivered to target cells.
Summing up, we found that the in vitro cytotoxic activity of monodisperse suspended SNP on target cells is essentially determined by their total mass (or total number or total SA) and their concentration. Practically, both parameters are of course intimately interdependent for a given volume of dispersion. We conclude that the nominal dose of insoluble monodisperse nanoparticles remains the most appropriate metric for in vitro toxicity testing.
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FUNDING |
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Federal Science Policy (Belgium) program "Science for a Sustainable Development" (Convention SD/HE/02A).
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