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Oxide Nanoparticle Uptake in Human Lung Fibroblasts: Effects of Particle Size, Agglomeration, and Diffusion at Low Concentrations LUDWIG K. LIMBACH,† YUCHUN LI,‡ ROBERT N. GRASS,† TOBIAS J. BRUNNER,† MARCEL A. HINTERMANN,† MARTIN MULLER,§ DETLEF GUNTHER,‡ AND W E N D E L I N J . S T A R K * ,† Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland, Laboratory of Inorganic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland, and Electron Microscopy Facility, Department of Physics, ETH Zurich, CH-8093 Zurich, Switzerland
Quantitative studies on the uptake of nanoparticles into biological systems should consider simultaneous agglomeration, sedimentation, and diffusion at physiologically relevant concentrations to assess the corresponding risks of nanomaterials to human health. In this paper, the transport and uptake of industrially important cerium oxide nanoparticles, into human lung fibroblasts is measured in vitro after exposing thoroughly characterized particle suspensions to a fibroblast cell culture for particles of four separate size fractions and concentrations ranging from 100 ng g-1 to 100 µg g-1 of fluid (100 ppb to 100 ppm). The unexpected findings at such low but physiologically relevant concentrations reveal a strong dependence of the amount of incorporated ceria on particle size, while nanoparticle number density or total particle surface area are of minor importance. These findings can be explained on the basis of a purely physical model. The rapid formation of agglomerates in the liquid is strongly favored for small particles due to a high number density while larger ones stay mainly unagglomerated. Diffusion (size fraction 25-50 nm) or sedimentation (size fraction 250-500 nm) limits the transport of nanoparticles to the fibroblast cells. The biological uptake processes on the surface of the cell are faster than the physical transport to the cell at such low concentrations. Comparison of the colloid stability of a series of oxide nanoparticles reveals that untreated oxide suspensions rapidly agglomerate in biological fluids and allows the conclusion that the presented transport and uptake kinetics at low concentrations may be extended to other industrially relevant materials.
* Corresponding author phone: +41 44 632 09 80; fax:+41 44 633 10 83; e-mail:
[email protected]. † Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences. ‡ Laboratory of Inorganic Chemistry, Department of Chemistry and Applied Biosciences. § Department of Physics. 9370
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Introduction Nanoparticles have become a polarizing issue beyond the scientific community, and widespread fear of possible negative impacts of anthropogenic particle emissions has even triggered calls to ban nanotechnology per se (1, 2). Rapidly growing reports on novel applications of nanoparticles fail to alleviate the often cited public unease toward the invisible technology (3). Spectacular findings of nanomaterials in the bodies of rats after exposure to carbon nanotubes or titania nanoparticles (4, 5) are in sharp contrast to a series of successful market introductions of consumer goods and slow legislation. Among numerous nanosized materials being currently produced, a different level of risk to human exposure arises for varying areas of applications. While semiconductor or metallic nanoparticles are mainly used in the fabrication of components and the risk of exposure to humans is limited, oxide nanoparticles have applications that reach far into our everyday life. Polymer additives in packaging or tires or nanoparticles for catalysts (6) or in sunscreen formulations undoubtedly pose a source of particle uptake. During production or nanoparticle handling, dust uptake through the lung has raised concerns of asbestos-like toxic effects (3). Studies on degradable polymer nanoparticles (7), surface-modified particles (8, 9), and quantum dots as fluorescence labels (10) indicate that other small particles may also exhibit high mobility within human cells. Despite the prominence of oxide nanoparticles in the current research, exploration of their interactions with living cells remains at a very early stage. This reflects, in part, the inherent difficulties of detecting untreated nanoparticles at relevantly low concentrations. The aim of this in vitro cell culture study was therefore to measure the uptake of an industrially important representative, cerium oxide nanoparticles (11, 12), into human lung fibroblasts. The latter offer a suitable cell type both for comparison to earlier studies and for their involvement in the formation of silicosis and asbestosis (13-15). Since such untreated oxides are not detectable by fluorescence microscopy at concentrations of 100 ng g-1, whole cell element analysis at nanogram per gram concentrations was applied to quantify the nanoparticle uptake into cells. The local enrichment of nanoparticles within the cells is shown by transmission electron microscopy of thin film sections. A quantitative physical model is developed on the basis of these findings and allows a more in-depth understanding of the behavior of nanoparticles in biological systems. These in vitro findings can now be applied to in vivo studies for further investigations on quantitative assessment of health effects.
Materials and Methods General Experimental Design. The investigation of size and concentration effects on the quantitative uptake of oxide nanoparticles into human lung fibroblasts was investigated by exposing corresponding cell cultures in vitro to a thoroughly characterized ceria dispersion for a specific time. After removal of the suspension and repeated washing, the nanoparticles remaining within the cells were measured by element analysis using inductively coupled plasma mass spectroscopy. The time-dependent uptake was determined for four different size fractions previously prepared by fractionating centrifugation (fractions ranging from 25-50 up to 250-500 nm in hydrodynamic particle diameter) at exposure concentrations of 100 ng g-1 and 1 µg g-1 of fluid, e.g., 100 ppb and 1 ppm. The concentration-dependent uptake of the above size fractions was measured after 10 min 10.1021/es051043o CCC: $30.25
2005 American Chemical Society Published on Web 10/12/2005
of exposure to nanoparticle dispersions containing 100 ng to 100 µg of nanoparticles per gram of fluid (100 ppb to 100 ppm). Nanoparticle Size Classification. Ceria nanoparticles (16) made by flame spray synthesis using chlorine-free carboxylate precursors (17) were dispersed in ultrapure, filtrated water (Millipore, S185, resistance >18.2 MΩ) and deagglomerated by sonication (Hielscher GmbH UP-400S). To study the effect of different particle sizes, four size ranges were separated by centrifugation (Sigma 3K30 and Sorvall RC5C Plus, centrifugation time for all runs 5 min). Particle sizes were determined by X-ray disk centrifugation (XDC, Brookhaven Instruments, BI-XDC), N2 adsorption (Brunauer-Emmett-Teller, Micromeritics Tristar 3000), transmission electron microscopy (CM30 ST, Philips, LaB6 cathode, operated at 300kV, point resolution ∼2 Å), and X-ray diffraction (Siemens powder X-ray diffractometer using Ni-filtered Cu KR radiation in step mode with a step size of 0.3 Å). Size fraction I (20-50 nm) was the supernatant after consecutive centrifugation for 5 min each at 45gn, 173gn, 375gn, 1110gn, 3500gn, 12 800gn, 12 800 gn, and 10 000gn (Sigma 3K30). (The Sorvall RC5C Plus centrifuge was used unless otherwise stated.) Size fractions II (40-80 nm) and III (80-150 nm) were made from CeO2 nanoparticles after sintering at 700 °C for 16 h and dispersion and sonication as above. Centrifugation for 5 min at 4000gn (Sigma 3K30) resulted in a supernatant, size fraction II. The residue of consecutive centrifugation steps at 3000gn and 2500gn (Sigma 3K30) was redispersed, centrifuged at 500gn, and yielded the supernatant as size fraction III. Size fraction IV was made from CeO2 nanoparticles previously sintered at 1000 °C for 16 h. First deagglomerated in a ball mill (W. Wirth Multiflex) for 2 h, the dispersion was stabilized with citric acid for sonication and washed with 0.02 M citric acid solution to remove abrasive impurities. The resulting suspension in the supernatant of centrifugation at 50gn (Sigma 3K30) was size fraction IV. Surface Charge Measurements. To analyze the stability of nanoparticle suspensions against agglomeration within biological systems, zeta-potential and X-ray disk centrifuge (XDC) measurements were preformed. The zeta-potential was measured from 2 wt % metal oxide suspensions in water by the colloidal vibration current method (Dispersion Technologie DT 1200). The stock solutions were diluted 1:1 either with water, RPMI 1680, or cell culture medium containing 10% fetal calf serum (FCS). Cell Culture. Human lung fibroblasts (ATCC, MRC-9) were maintained with RPMI 1640 medium + Glutamax (Gibco, NY) with 10% fetal calf serum (PAA Laboratories GmbH, Linz, Austria) and 100 units g-1 penicillin and 100 µg g-1 streptomycin (Gibco, NY), referred to as cell culture medium. The fibroblasts were passaged according to ATCC Technical Bulletin No. 3, and experiments were carried out using cells between passages 11 and 19, incubated at 37 °C under a humidified atmosphere of 5% CO2 and 95% air, and subcultivated using trypsin-EDTA (Gibco, NY) after reaching confluence. Exposure of Nanoparticles to Cells. The dependence of nanoparticle uptake has been analyzed for different sizes, concentrations, and exposure times. The fibroblasts were seeded in six-well plates (TPP AG, Trasadingen, Switzerland) as nearly confluent cell layers, counting about 500 000 cells per well (Figure 3D). Corresponding freshly diluted nanoparticle-water suspensions (size fractions I-IV) were mixed with the medium (1:9), shortly sonicated, and added to the well (2 mL each). For analysis cells were washed three times with potassium- and calcium-free phosphate-buffered solution (PBS, 8200 ppm NaCl, 600 ppm Na2HPO4, 200 ppm NaH2PO4, pH 7.4) and dried in air at ambient temperature. Element Analysis. Dried cells were transferred into precleaned digestion vials by consecutive dissolution in 1
and 0.5 mL subboiled nitric acid (Merck, p.a., 65%; subboiled, MLS GmbH, Duopor acid purification system) per well for 30 min. After addition of 200 µL of H2O2 (Fluka, trace select, 30%) the vials were sonicated for 10 min (Bandelin Elecronic, RK510) and fully digested (digestion unit, MLS GmbH, Ultra Clave II) by increasing temperature and pressure to 230 °C at 90 bar for 75 min. After digestion the samples were diluted with ultrapure water (resistance >18.2 MΩ) to a total weight of 10 g. In and Y (10 ng g-1) were added to the sample digests as internal standards. In all samples the Ce and K concentrations were measured using an inductively coupled plasma sector field mass spectrometer (ICP-SF-MS, Element 2, ThermoFinnigan, Bremen, Germany). Ce and K standard solutions (0.1-500 ng g-1, prepared from 1000 mg L-1 stock solutions, Merck, Germany) including 10 ng g-1 In and Y as internal standards were used for calibration. Particle Agglomeration, Diffusion, and Sedimentation. To investigate the influence of mass transport on the uptake of nanoparticles into cells, a simple model describing particle sedimentation, aggregation, and particle diffusion was derived. Fractal structures as observed by aggregation of nanoparticles can be described by the following scaling relation
N)
() Rg r
Df
(1)
where N is the number of primary particles with radius r (m) per agglomerate, Df is the fractal dimension, and Rg (m) is the characteristic radius of gyration. The sedimentation rate of a fractal aggregate with N primary particles in a liquid medium can be calculated using a force balance assuming the aggregate hydrodynamic radius equal to the radius of gyration (Rg)
vsed )
2(Fs - Fm)gNr3 9µRg
(2)
where Fs and Fm (kg m-3) are the density of the solid and medium, respectively, g (m s-2) is the gravitational force, and µ (Pa s) is the dynamic viscosity of the medium. For the calculation of the rate of diffusion, particles were assumed to diffuse from the liquid toward the cells in one dimension (top to bottom of the culture well). If particles are taken up by the cells at a rate much larger than the rate of diffusion (diffusion is the rate-limiting step), then the transport process can be represented by Fick’s second law (18)
∂c ∂2c )D 2 ∂t ∂z
(3)
where c (kg m-3) is the particle concentration, D (m2 s-1) is the diffusion coefficient, t (s) is time, and z (m) is the spatial coordinate (from bottom to top of the culture well) with the following initial and boundary conditions where cinit (kg m-3) is the initial ceria concentration (1 × 10-3 kg m-3)
t ) 0, all z, c ) cinit t > 0, z ) 0, c ) 0 t > 0, z ) ∞, cinit
(4)
After solution and in combination with Fick’s first law, the uptake flux j (kg m-3 s-1) through diffusion for a cell of volume V (m3) and the exposed cell surface area A (m2) can be derived
j)
xπtD (0 - c )
A V
(5)
∞
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FIGURE 1. Classification of oxide nanoparticles (ceria) in four size fractions (I-IV) by a series of centrifugation steps results in particle fractions with identical chemical compositions. (A) X-ray disk centrifugation reveals clearly separated size fractions of nanoparticles ranging from around 20 to 500 nm. (B) Transmission electron microscopy images of the corresponding size classes reveals crystalline particles of ceria, bar size 50 nm. Larger particle fractions (II-IV) were obtained after sintering the nanoparticles of size fraction I. The diffusion coefficient D of small particles can be derived using the Stokes-Einstein equation
D)
kBT 6πµRg
(6)
where kB is the Bolzmann constant and T (K) is the temperature of the medium. The aggregation of nanoparticles in a medium can be calculated by solving the Smoluchowski equation (19). In a simplified form the particle population can be given as monodisperse with a total concentration nt (m-3) and an aggregation rate constant β (m3 s-1) as well as the stability ratio W relating to the steric and electronic hindrance toward aggregation and giving the ratio of the aggregation constant of diffusion-limited cluster aggregation and the observed aggregation constant
dnt 1 β 2 )n dt 2W t
(7)
resulting in a characteristic time of doublet formation of
τ)
2W 3µW ) β 4kBTn0
(8)
where n0 (m-3) represents the initial particle concentration. 9372
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FIGURE 2. (A) Number of primary particles per aggregate versus time for 25 nm nanoparticles with agglomeration. Values are calculated for ceria in cell culture medium at a concentration of 1 µg g-1. The surface charge of particles may slow agglomeration by changing the stability ratio (W), which results in much slower agglomeration. For all conditions nanoparticles are agglomerating during the time scale of biological experiments. (B) Characteristic doublet formation time for diffusion-limited agglomeration (W ) 1) versus particle concentration for three different ceria particle sizes (25, 100, and 320 nm). For all calculations, body temperature (310 K) and the literature properties for ceria were assumed. The viscosity of the cell culture medium was measured as 1.4 × 10-3 Pa s using a viscosimeter (Ares, Rheometric Scientific). The cell volume (V ) 1.8 × 10-15 m3) and the cell surface area (A ) 6 × 10-10 m2) were estimated by cell counting using a Neubauer chamber and light microscopy. The resulting cell dimensions are comparable to literature data (20). Figure 2A shows the strong influence of the stability ratio W on the evolution of the aggregate mass for 25 nm particles at 1 µg g-1. Small changes on the surface of the particle can have a very large impact on the stability ratio and can therefore significantly slow agglomeration. Figure 2B shows the influence of particle concentration and particle size on the characteristic doublet formation time for ceria nanoparticles. Larger particles have mean doublet formation times (collision of two particles forming an agglomerate of the two primary particles) up to several hours for low concentrations. Nanoparticles (25 nm) are agglomerating within seconds even at concentrations as low as 1 µg g-1.
Results and Discussion The application of low exposure concentrations of 100 ng g-1 or 1 µg g-1 ceria in the culture medium allows avoidance of particle overload effects and resulting unspecific changes
FIGURE 3. Nanoparticle uptake for four different size classes. (A) Time-dependent nanoparticle uptake expressed as ceria concentration within cells after exposure to 100 ng ceria g-1 culture medium. (B) Time-dependent ceria concentration after exposure to 1 µg ceria g-1 medium. (C) Variation of ceria exposure concentration over 4 orders of magnitude reveals consistent differences between size classes. The ceria concentration in cells was measured after 10 min of exposure. (D) Human fetal lung fibroblasts (MRC-9). (E) Fibroblasts after 1 h of exposure to 1 ppm ceria nanoparticles, bar size 250 µm. in cell proliferation, morphology, and metabolism. Both timeand concentration-dependent uptake of ceria (Figure 1) into cells were measured over 4 orders of magnitude. Experiments were conducted at similar cell densities and at least 3 days after subcultivation. The fibroblast cells were absorbing ceria particles linearly with exposure time (Figure 3). At both 100 ng g-1 and 1 µg g-1 ceria exposure concentrations, size fraction I showed much less ceria mass uptake than dispersions with progressively increasing ceria particle sizes (size fractions II-IV). After 2 h, ceria concentrations within cells reached up to 70 µg g-1 in the case of size fraction IV (1 µg g-1 exposure concentration) and corroborated an active accumulation of nanoparticles within the cells. Studies at much higher concentrations on the uptake of degradable polymer particles (21) or carbon nanotubes in macrophages (22) showed similar linear uptake kinetics but inverse size dependence (23). The concentration dependence of all size fractions was investigated at exposure concentrations ranging from 100 ng g-1 to 100 µg g-1. High exposure concentrations resulted in a proportional increase in ceria uptake, which is consistent with studies on polymer nanoparticles (21). The size dependence of the amount of incorporated ceria was consistent over 4 orders of magnitude, and larger size fractions resulted in a higher ceria content in the cells than smaller nanoparticle dispersions. For the low concentrations, no saturation of the ceria concentration was observed within 4 h. To compare different mechanisms for particle transport, the ceria uptake by diffusion-limited transport for size fraction I (numberaveraged diameter of 25 nm) and the sedimentation of particles on the cell surface followed by instantaneous uptake as suggested by Gorelik et al. (24) for size fraction IV (numberaveraged diameter of 320 nm) were calculated only using measurable parameters (Figures 6A and 6B, respectively).
The measurements showed that 20-50 nm ceria uptake at low concentrations is a diffusion-limited transport process. For size fraction IV, a mean sedimentation velocity of 260 nm s-1 in the exposure wells made the sedimentation of particles onto the cell the dominant transport process for 250-400 nm ceria particles. After uptake, electron microscopy revealed ceria nanoparticles in vesicles within the cytoplasm (Figure 4). Transmission electron micrographs of 50 nm thin slides of fibroblast cells (after 2 h of exposure to 10 µg g-1) showed ceria nanoparticle agglomerates confined by single or multiple lipid bilayers. The ceria nanoparticles stayed clearly visible, accounting for their high electron density (Ce atomic number 58). No particles were found outside of vesicles or flowing freely in the cytoplasm. Particles were exclusively present in the form of agglomerates (Figure 4) as predicted by the agglomeration model. In contrast to these oxide nanoparticles, polymer micelles were found to move further into other organelles such as mitochondria (25). No evidence of particles entering mitochondria or the cell nucleus could be found from electron microscopy images. Since a variety of different oxide nanoparticles are used industrially, a selection of different oxide nanoparticles of 20-70 nm in size was compared in terms of their colloid stability in water and cell culture medium (Figure 5). In pure water, the zeta-potential measurements of oxide dispersions cover a wide range from -25 to 55 mV. Once these dispersions were mixed with cell culture medium, protein adsorption strongly affected the surface charge distribution of oxides (26) and shifted the zetapotential to around -18 mV (27). The comparable charge of seven different oxides in the medium suggested that protein adsorption dominated the surface charge distribution for oxide nanoparticles. Small surface charges allowed primary VOL. 39, NO. 23, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Exposure of human lung fibroblast cells to ceria nanoparticles of 20-50 nm in diameter results in the uptake of agglomerates. (A) Vesicles inside a fibroblast cell with ceria agglomerates. The high atomic mass of ceria and resulting contrast make the particles visible as dark spots. (B) A series of nanoparticle agglomerates close to the cell membrane. (C) Nanoparticles both inside the cell (vesicle) and outside are exclusively found in the form of agglomerates, confirming the dominant role of agglomeration. All bar sizes are 1.5 µm. The sensitivity of the model is illustrated by computing rates of particle transport to the cells for different mean agglomerate sizes containing 10-40 particles each. No assumptions beyond measurable parameters were necessary for this calculation and corroborate the validity of the model. Sedimentation of larger particles is strongly affected by the particle density as shown in Figure 6A. The large particles behave as if they have a slightly smaller apparent density.
FIGURE 5. Stability of different oxide nanoparticles against agglomeration displayed by the zeta-potential. In ultrapure water (empty columns), the colloid stability is characteristic for different materials. Suspensions in cell culture medium undergo protein adsorption, which results in comparable low surface charge density (below -25 mV) and favors rapid agglomeration as observed for ceria. particles to overcome the electrostatic repulsion, and nanoparticles rapidly agglomerated. The resulting grown clusters of loosely connected nanoparticles are shown in Figure 4. Under this perspective, the current findings for diffusionlimited agglomeration and size-dependent mean mass uptake determined for ceria may be also valid for most oxide nanoparticles. Discussions on nanoparticle-related toxicity have suggested particle size, degree of agglomeration, specific surface area, and number concentration as parameters to correlate to nanoparticle uptake (3, 5, 9, 28, 29). At a given size class, e.g., size fraction I, the mass uptake increases linearly with the primary particle number concentration of nanoparticles, which is proportional to the mass concentration (Figure 3). Both transport to the cell by sedimentation or diffusion linearly depend on mass concentration. Assuming fast uptake at the cell membrane, the calculated rates of transport using eqs 2 and 5 reveal the dominance of diffusion for small particles while larger particles are transported by sedimentation (Figure 6). 9374
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The influence of particle size was furthermore investigated at a constant mass concentration to compare the relative particle uptake to the relative number concentration, the relative specific surface area, and the relative size of the primary particles (Figure 7) calculated according to ref 30. The mass uptake of different size classes did not correlate to the primary particle number concentration and also did not correlate to the specific surface area. At the same mass concentration, larger particles are significantly better taken up than smaller nanoparticles. These results may be discussed in terms of agglomeration. The rapid in situ agglomeration of dispersions of size fractions I and II disqualifies the concept of primary particle size. Their mean agglomerate doubling time is in the range of seconds even at concentrations as low as 1 µg g-1. This is supported by electron micrographs depicting agglomerates of several hundred nanometers in diameter, consisting of a large number of primary particles. The low density of such impaction-derived agglomerates (28) (fractal dimensions around 2) reduces the ceria mass uptake per absorbed agglomerate, the diffusion coefficient, and the speed of sedimentation, which all contribute to a lower ceria uptake. As a consequence, the uptake of single, unagglomerated oxide nanoparticles of 20-50 nm in size was not observed. Calculations for even lower concentrations can be found earlier (Figure 2), and it can be concluded that single oxide nanoparticle uptake may only occur if particles are surface-modified to restrict or change protein adsorption (31-33) or at concentrations below nanograms per gram concentrations. This study shows that human lung fibroblast cells rapidly absorb ceria nanoparticles from culture medium even at concentrations as low as 100 ng g-1. Small oxide nanoparticles undergo fast agglomeration upon contact to cell culture
FIGURE 6. Physical models for ceria nanoparticle transport. (A) Calculated ceria transport after sedimentation (dotted lines) of 320 nm ceria spheres for different particle densities and ceria transport to the cell by diffusion (red solid line) together with experimental data (black squares). (B) Calculated ceria transport by sedimentation (dotted lines) or diffusion (solid lines) for ceria aggregates of 10, 20, and 40 primary particles per agglomerate. Calculations for 25 nm particles and experimental data (black squares) show a good agreement.
FIGURE 7. Uptake of ceria nanoparticles of different size classes best correlates to the particle diameter. The low surface charge and high relative number density of small particles (20-50 nm) induces rapid agglomeration, and the cell effectively only absorbs agglomerates. Larger particles with resulting low number densities slowly agglomerate and can penetrate into the cells much more efficiently. medium unless they are surface-modified (8, 34, 35). The uptake of single, unagglomerated nanoparticles therefore becomes very improbable for particles of less than 50 nm in size. In contrast to this, larger particles around 200 nm have very low number densities at concentrations around 1 µg g-1. Their mean agglomeration time is high, and cells mainly take up single particles presumably by vesiculation after sedimentation of such particles onto the cell. Comparison of the mass uptake of four different size classes of ceria nanoparticles shows that particle size is indirectly the dominant factor determining the rate of uptake, while primary particle number concentration and total surface area are of minor importance.
Acknowledgments We thank L. Diener and F. Krumeich for the preparation of microtome slides and electron microscopy images of cells and ceria nanoparticles, P. Wick, A. Bruinink, and H. Plattner for discussions on vesicle transport, and K. Rezwan for particle
size analysis. Financial support by the ResOrtho, the Gebert Ru ¨f Foundation, and the ETH Zurich is kindly acknowledged.
Supporting Information Available Crystallinity of nanoparticles and infrared spectroscopy and surface properties. This material is available free of charge via the Internet at http://pubs.acs.org.
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Received for review June 3, 2005. Revised manuscript received August 25, 2005. Accepted September 8, 2005. ES051043O
