Letters to the Editor pubs.acs.org/crt
Reply to the Letter to the Editor Regarding My Article on Dose Metrics in Nanotoxicity Studies (Wittmaack, 2011)
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manufacturer's homepage (http://www.formulaction.com/ technology_mls.html)). The notation turbidity commonly serves to specify the opacity or muddiness of water (http:// www.waterontheweb.org/under/waterquality/turbidity.html). Here, the correct terminology for the data shown in the righthand column of Figure 1 in the letter of Lison et al. is
o the Editor: The letter sent by Dr. D. Lison and coworkers to the editor of Chemical Research in Toxicology questions the findings of my recent article in this journal in which I presented a reanalysis of previously published data. Lison et al.1 had performed meaningful experiments involving in vitro measurements of cell viability after 24 h of exposure to dispersed silica nanoparticles (NPs). Extending the approach employed by other groups, they varied not only the nominal mass concentration in cell culture media of constant volume or height but also the height at constant mass and mass concentration. Dose−response data were shown in separate diagrams, for each of the three sets of experiments. The results showed very clearly thatin contrast to common consensus the mass concentration must be excluded as the prime dose metric in such investigations. The inevitable conclusion had to be that in vitro nanotoxicity studies are impossible to interpret in a quantitative manner, unless one could identify an alternative dose parameter that might allow integration of all data in a universal diagram. My remarkable finding2 was that the wide spectrum of data1 did in fact fall on a common dose− response curve for each cell line, “provided the dose was quoted in terms of the areal density of NP mass delivered to the cells.” I did not hypothesize “that the cells were covered with several closely packed layers of silica NP.” Instead, I concluded that “loss of viability became observable only if cells were exposed to the equivalent of 1 to 5 closely packed layers of NPs.” It was made clear2 that the quoted number of NP layers served only as a secondary dose parameter, aimed at assisting the reader in trying “to appreciate the meaning” or the magnitude of the respective areal densities. In my paper,2 I further concluded “that cell-death phenomena frequently reported in the literature for in vitro exposure to NPs are most likely not due to some unknown toxic potential of the NPs but rather a consequence of cell overload with nanostructured matter...The ability to derive this very important result rested on the availability of a data basis containing not only information on cell viability but also on the uptake of NPs by the cells.” The very important point here is that the data which I used to quantify NP uptake were contained in the same paper of Lison et al.1 from which I extracted the cell viability data. Now, in their letter, Lison et al. present additional data aimed at showing that it is impossible to deliver NPs to cells to the extent that they previously measured and reported themselves. To make their point, Lison et al. used dynamic light scattering (DLS) to measure size distributions of silica NPs in Dulbecco's modified Eagle's medium (DMEM) without serum. Whereas in their earlier work1 DLS spectra were determined at a wavelength λ of 632.8 nm and a scattering angle θ of 173°, the new data were obtained using a different instrument operating at λ = 659 nm and θ = 90°. Attempts were also made to characterize the agglomeration state of the silica particles in DMEM by performing “turbidity and backscattering measurements” at λ = 880 nm (wavelength derived from the © 2011 American Chemical Society
Figure 1. Light optical image illustrating the growth of Aerosil NP matter at the bottom of a well. The Aerosil powder was dispersed in water containing BSA (height 6.2 nm) at 35 μg/mL and was allowed to settle for 19.5 h.
transmittance.3 It is worth noting that the employed instrument was designed to characterize concentrated liquid dispersions under conditions of multiple scattering (rather than single scattering). A problem associated with the characterization of engineered silica NPs is that they are highly or fully transparent, depending on cleanliness and compactness. Therefore, transmitted light loses intensity only by scattering, not by absorption. Nevertheless, it is possible to identify transparent matter that may pile up at the transparent bottom of vials containing NPs dispersed in liquid media. In a recent study,4 I used optical microscopy to explore settling phenomena for a variety of commercially available NP powders. For lack of space, detailed results for amorphous silicon dioxide could not be included in the previous paper. Of interest in the present context is Aerosil (manufactured by Evonik, formerly Degussa). Scanning electron microscopy (SEM) showed that the primary particle size in the analyzed samples was 30 ± 5 nm. DLS measurements of the same material, sonicated in high-purity water [plus bovine serum albumin (BSA) at 0.17 mg/mL], revealed prominent peaks centered at hydrodynamic diameters between 100 and 160 nm and, in one of three spectra, a small peak located at about 700 nm. The progress of settling was studied with a sample containing 35 μg/mL of Aerosil (200 μL in one well of a 96-well plate, mean height of the liquid 6.2 mm). With a mass density of 2.3 g/cm3 for silica, the areal density of Aerosil contained in the liquid sample (21.7 μg/cm2) was equivalent to a compact layer with a thickness of 94 nm. An image taken after a storage time of 19.5 h is presented in Figure 1. Settled matter in the form of flat, mostly circular disks, Published: December 15, 2011 7
dx.doi.org/10.1021/tx200381e | Chem. Res. Toxicol. 2012, 25, 7−10
Chemical Research in Toxicology
Letters to the Editor
630 nm “as the average diameter of the aggregate population exceeding 400 nm in diameter.” Differential cross-sections for scattering angles away from 0° depend strongly on particle size and (not shown) on the refractive index, to the end that any quantitative estimate makes sense only if these particle parameters are known exactly. In the size range 330 ≤ D ≤ 630 nm, differences in size by 100 nm can change σ′(90°) by up to a factor of 10; see Figure 2. General lack of knowledge on particle size, agglomeration state, compactness, refraction index, etc., is one reason for the fact that manufacturers of DLS instruments implement software that delivers spectra only in normalized form, the largest peak intensity set to unity. To proceed, we assume that the particle parameters of interest are known. In analogy to the approach used by Lison et al., the dissolved NP matter is considered to be contained in compact spherical particles of diameter D, with a refractive index n = 1.54. The number density ND of these particles is ND = 6mc/ρπD3, with mc and ρ denoting the mass concentration and the mass density, respectively. The DLS signal I may be written I(90°) ∝ NDwσ′(90°), where w is the width of the medium viewed by the DLS detector. Calculated DLS signals thus derived are presented in Figure 3. The data are normalized
is clearly visible. The disks cover about 10% of the inspected area. Let us assume that, to produce the observed contrast and diffraction at the edges, the disks must have had a thickness of 500 nm. Spread out over the whole area, the mean thickness would be 50 nm. This implies that 53% of the dispersed matter had settled after 19.5 h. The corresponding mean settling velocity would be 3.1 mm/19.5 h = 0.16 mm/h. Making use of Stoke's law, as before,2 it turns out that this settling velocity will be achieved by compact (!) Aerosil agglomerates with a diameter of 310 nm. Whereas the results of Figure 1 provide clear evidence that agglomerates made of rather small primary particles of silica can settle rapidly, discussion of the new data presented in the letter of Lison et al. requires extended calculations on the basis of established theories. Adopting the approach taken by Lison et al., I used the so-called Mie Scattering Calculator by Scott Prahl (http://omlc.ogi.edu/calc/mie_calc.html) to perform more than 200 calculations of total and differential scattering cross sections, σ and σ′ = dσ/dΩ, respectively, for spherical particles with diameters D between 13 nm and 1.8 μm, λ = 659 and 880 nm, with real indices of refraction n of 1.33 for the medium (water) and 1.54 (silica), 1.50, and 1.44 for the particles, the latter two cases serving to explore the sensitivity of Mie scattering features to variations of n (Ω denotes the solid angle into which photons are scattered). It is well-known that for particle sizes D < λ/2π the cross-section for light scattering off spherical particles can be described by the Rayleigh theory, σ ∝ D6 (for example, http://en.wikipedia.org/wiki/Rayleigh_ scattering). For larger particles, the much more complex Mie scattering theory applies. To appreciate the size dependence of scattering cross-sections, Figure 2 shows a summary of results
Figure 3. Normalized signal of light scattered from spherical silica particles in water at 90°. The number of particles was normalized to a constant mass concentration.
to the signal calculated for D = 30 nm, which corresponds to the low end of the hydrodynamic diameter assigned in panels E and G of Lison et al. to scattering from primary silica NPs. Lison et al. calculated the scattering signal due to primary particles by assigning to them not the measured hydrodynamic diameter quoted above but rather a diameter of 19 nm, as derived by transmission electron microscopy (TEM). By contrast, a hydrodynamic diameter was assigned to the agglomerates (D = 630 nm). Using the TEM diameter, Lison et al. constructed a factor of (30/19)6 = 15.5 in favor of their arguments. Furthermore, with measured agglomerate sizes between 330 and 630 nm, Figure 2 shows clearly that selecting 630 nm as a pseudorepresentative case is not justified because, for a given DLS signal, a high scattering cross-section for agglomerates implies a low number density (as Lison et al. like to show). Lison et al. specified the ratio of the integral intensities of aggregates to primary particles as 18.5. Given the similarity of peak shapes, one can assess the intensity ratio also from the products of the heights and the widths of the peaks. On that basis, the ratio in panel E of Figure 1 of Lison et al. turns out to
Figure 2. Normalized cross-sections for scattering of light from spherical silica particles (diameter D) dispersed in water, at two different wavelengths in vacuum (for details, see the text). For convenience, the calculated data were divided by D6 and normalized to the results for a wavelength of 659 nm.
calculated for the two relevant wavelengths. The calculated data were divided by D6 and normalized by appropriate factors so that, for λ = 659 nm, the scaled cross-sections are unity in the Rayleigh regime, that is, at small particle sizes. Of particular interest are the differential cross-sections σ′(90°), the case reflecting the DLS measurements. In Figure 1 of Lison et al., the examples referred to as showing detectable but “not significant aggregation”, the most prominent DLS peaks appear at hydrodynamic diameters of about 500 and 630 nm (panel E) and between 330 and 500 nm (panel G). Lison et al. quoted 8
dx.doi.org/10.1021/tx200381e | Chem. Res. Toxicol. 2012, 25, 7−10
Chemical Research in Toxicology
Letters to the Editor
range between 45 and 50. Let us settle on an average ratio of 35, represented by the dash-dotted line in Figure 3. The message is clear: If all NP mass were contained in compact agglomerates with a hydrodynamic diameter of 500 nm (denoted by a cross in Figure 3), the expected integral DLS signal would be 35 times larger than the signal for the case that all mass were contained in primary particles with a hydrodynamic diameter of 30 nm; that is, the measured (average) ratio and the calculated ratio agree perfectly. Hence, we may consider the NP laden medium as having half of the mass contained in primary particles, the remainder in “agglomerates”. From Stoke's law, one finds the settling velocity of 500 nm silica spheres to be 0.42 mm/h, so that even in a medium with a height as large as 1.7 cm (Lison et al.), about 50% of the agglomerates should have settled after 20 h. For 70 μg/mL, the areal density of the settled NP matter would be 0.5 × 0.5 × 1.7 × 70 = 30 μg/cm2 or 115 nm. The above estimates are based on the assumption that the agglomerates are compact spheres of exactly known size so that Mie scattering theory can be applied. In reality, the situation is much more complex; that is, the agglomerates may at best be partially compact and fluffy elsewhere, so that the DLS signal generated by an entity executing Brownian motion like a 500 nm sphere will be due to light scattering from a geometrically complex arrangement to which the laws of Mie scattering cannot be applied directly. Researchers active in the field of nanotoxicity must accept that DLS signals contain qualitative, not quantitative, information on particle concentrations. If one likes to assess the amount of settled NP matter, one should not rely on DLS but rather monitor the arrival of material directly by optical microscopy.4 Finally, the transmittance data of Lison et al. deserve a few comments. In panels B, F, and H of their Figure 1, the transmittance varies between 0.945 and 0.97, as indicated by horizontal lines in Figure 4. Given the fact that the NP mass
commonly added to DMEM. The signal fluctuations measured as a function of height suggest that the transmittance of the vials was not constant along the walls. Calculations of transmittance τ in the single scattering regime are meaningful only at τ values between 1.0 and 0.9, at most down to τ = 0.8. Multiple scattering applies for τ < 0.6 (see Figure 4). Size-dependent changes in transmittance due to the presence of compact silica spheres in water were calculated under the assumption of single scattering for three mass concentrations, ranging from 7 to 700 μg/mL. The results suggest that at a mass concentration of 7 μg/mL, the change in transmittance produced by spheres with diameters
