G Model
ARTICLE IN PRESS
TOXLET 8701 1–8
Toxicology Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Cell uptake and oral absorption of titanium dioxide nanoparticles
1
2 3
Q1
G. Janer, E. Mas del Molino, E. Fernández-Rosas, A. Fernández, S. Vázquez-Campos ∗ LEITAT Technological Center, C/de la Innovació, 2, Terrassa, Barcelona 08225, Spain
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5 6 7 8 9 10
h i g h l i g h t s • • • •
TiO2 NPs were readily uptaken by A549 cells in vitro. Very low translocation of TiO2 NPs through a differentiated Caco-2 monolayer system. Titanium levels in tissues did not increase after a single oral dose of TiO2 NPs. Several NPs were observed in the cytoplasm of a cell from a Peyer’s Patch by TEM.
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Article history: Received 7 February 2014 Received in revised form 17 April 2014 Accepted 19 April 2014 Available online xxx
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Keywords: Oral bioavailability Caco-2 Biodistribution Clearance
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1. Introduction
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27 28 29 30 31 32 33 34 35 36 37
Large efforts are invested on the development of in vitro tests to evaluate nanomaterial (NM) toxicity. In order to assess the relevance of the adverse effects identified in in vitro toxicity tests a thorough understanding of the biokinetics of NMs is critical. We used different in vitro and in vivo test methods to evaluate cell uptake and oral absorption of titanium dioxide nanoparticles (TiO2 NPs). These NPs were readily uptaken by A549 cells (carcinomic human alveolar basal epithelial cells) in vitro. Such rapid uptake contrasted with a very low oral absorption in a differentiated Caco-2 monolayer system (human epithelial colorectal adenocarcinoma cells) and after oral gavage administration to rats. In this oral study, no significant increase in the levels of titanium was recorded by ICP-MS in any of the tissues evaluated (including among other: small intestine, Peyer’s patches, mesenteric lymph nodes, liver, and spleen). No NPs were observed by TEM in sections of the small intestine, except for several particles in the cytoplasm of a cell from a Peyer’s Patch area. The observation of NPs in Peyer’s Patch suggests that the Caco-2 monolayer system is likely to underestimate the potential for oral absorption of NPs and that the model could be improved by including M-cells in co-culture. © 2014 Published by Elsevier Ireland Ltd.
Due to ethical and economical considerations, large research efforts are dedicated to the development of toxicity test alternatives in vitro. When talking about NMs, the high number of variations that can exist among them (sizes, surface modifications, shape. . .) would lead to enormous economical and animal life costs if traditional in vivo testing methods had to be used. However, the use of in vitro data for toxicological risk assessment is not straight forward. It requires not only that the in vitro tests comprehensively cover the mechanisms of action leading to the relevant toxic effect, but also a very thorough understanding of the biokinetics of the substance/material.
∗ Corresponding author. Tel.: +34 93 788 23 00; fax: +34 93 789 19 06. E-mail addresses: [email protected], [email protected] (S. Vázquez-Campos).
It is anticipated that with inorganic NMs, the panel of mechanisms of toxicological action will comprise particle specific effects (e.g., impaired phagocytosis in particle-laden macrophages (Warheit et al., 1997)) and inorganic ion specific effects (e.g., after release of Ag+ ions from silver NPs). However, such panel of possible effects is expected to be considerably narrower than that for organic chemicals, which can specifically interact with binding sites of enzymes or receptors and result in an enormous number of toxicity pathways (Kavlock et al., 2012). There starts to be a considerable body of knowledge on the mechanisms of toxicity of NMs that have allowed the generation of hazard categorization trees on the basis of their physicochemical properties (RIP-oN2, 2001). On the other hand, the understanding and prediction of the biokinetic of NMs is still at a very early stage. In order to progress towards a future evaluation of NMs risks on the basis of in vitro tests, a better understanding of the critical biokinetic processes as well as the development of in vitro models to predict these processes is needed. The Caco-2 monolayer permeation test is a widely used in vitro test to predict oral absorption of organic compounds, particularly
http://dx.doi.org/10.1016/j.toxlet.2014.04.014 0378-4274/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Janer, G., et al., Cell uptake and oral absorption of titanium dioxide nanoparticles. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.04.014
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by the pharmaceutical industry (Artursson et al., 2001). A few reports also exist on the use of this test system for NMs (Koeneman et al., 2010; Al-Jubory and Handy, 2012; Antunes et al., 2013; Jin et al., 2013; Kenzaoui et al., 2012; Reix et al., 2012; Sonavane et al., 2008). These reports and the limited number of in vivo oral absorption or toxicity studies available suggest that NMs do cross the intestinal membrane, although at a very limited rate (Baek et al., 2012; Hillyer and Albrecht, 2001; Jani et al., 1994; Li et al., 2012; Schleh et al., 2012; Wang et al., 2007). The residence time of NMs in blood is short compared to most organic chemicals and dominated by the uptake by the mononuclear phagocyte system rather than elimination from the body (Landsiedel et al., 2012). Therefore, the evaluation of systemic absorption requires the quantification of NMs in tissues. This leads to several analytical challenges, both in case of organic and inorganic NMs. TiO2 NPs are one of the most commonly used NMs, due to its low costs and easiness to obtain at nano-sizes and its properties at this scale, such as reflection of UV sunrays and photocatalytic properties. Some reports exist on different aspects of the biokinetics of TiO2 NPs both in vitro (see below) and in vivo (Fabian et al., 2008; Patri et al., 2009; Shinohara et al., 2013; Wang et al., 2007). However, it is difficult to compare studies and to understand the predictive value of some of the in vitro studies because they used titania NMs of different crystal phase, size, shape and surface coatings. Most studies evaluating cell internalization of TiO2 NPs have found aggregates of these NMs in cytoplasmic vesicles (Allouni et al., 2012; Andersson-Willman et al., 2012; Halamoda Kenzaoui et al., 2012; Hussain et al., 2010; Jaeger et al., 2012; Kocbek et al., 2010; L’Azou et al., 2008; Singh et al., 2007; Wang et al., 2011; Zhu et al., 2012). This contrasts with the lack of internalization of TiO2 NPs reported by Fisichella et al. (2012) and the limited internalization reported by Koeneman et al. (2010) in Caco-2 cells. These differences are likely to be due to the cell type, but could also be related to the differences in the TiO2 NPs used in these studies. In the present report, we have used a single type of TiO2 NPs in different in vitro and in vivo systems in order to better understand how results from these studies can be extrapolated to other cell types or levels of organization (e.g., in vitro to in vivo). In particular, this manuscript describes the results of three studies: an in vitro cell uptake using A549 cells, an in vitro permeation test using differentiated Caco-2 cells, and an in vivo oral absorption test in rats. To overcome the analytical challenges associated to the tracking of unlabelled TiO2 NPs, both inductively coupled plasma mass spectrometry (ICP-MS) and transmission electron microscopy (TEM) were used.
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2. Methods
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2.1. NMs source and characterization
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ARTICLE IN PRESS
TiO2 NPs, synthesized using the Flame Spray Pyrolisys, were obtained from L’Urederra (Spain) under the framework of the EU project Nanopolytox. Surface area and porosity of NMs were measured by a multi-point isotherm BET assay (equilibration time 60 s), using N2 as adsorbate, in a BET surface area analyzer (Nova 2200e series; Quantachrome). NMs were previously degassed overnight under vacuum at 105 ◦ C. Surface area was calculated using 12 points of relative pressure (r2 > 0.998), and porosity was obtained using the BJH calculation method in the desorption branch of the isotherm. NMs were dispersed with a tip sonicator (SONICS VCX 750; Braun) equipped with a standard ¼ (6,5 mm) × 5.6” (142 mm) tapered microtip (783630-0420; Izasa) at 1 mg/mL for 15 min without pauses, applying a power of 400 W. The dispersion was kept cold during the sonication process by using a water-ice bath around the solution flask. Samples were deposited on TEM
grids (formvar/carbon-coated 200 mesh Cu grids; Ted Pella Inc) just after sonication. Shape, size distribution and dispersibility of NMs were analyzed by TEM using an H-7000 operating at 120 KV (Hitachi). Finally, hydrodynamic diameters in relevant media were measured by dynamic light scattering (DLS) using a Zetasizer (nano series Nano-ZS; Malvern). 2.2. In vitro cell internalization A-549 cells were incubated with only cell media (controls) or with 100 g/mL TiO2 NPs for 72 h. Preliminary experiments had shown that this concentration did not affect cell viability (Alamar Blue® test) during this exposure period. Afterwards, the cells were washed with PBS and fixed with glutaraldehyde (2.5%) for 1 h. Cells were detached from the Petri dish by scraping and centrifuged (4 ◦ C, 1000 g, 5 min), to obtain a compact pellet. After washing with PB (0.1 M), cells were stained with OsO4 (1%) for 2 h and washed again with PB (0.1 M). Cells were dehydrated at 4 ◦ C through a series of acetone concentrations (50%, 70%, 90%, 96% and 100%), prior to being progressively (25%, 50%, 75% and 100%) embedded in Epon resin. After resin curing (60 ◦ C, 48 h), sections with a thickness of 50 nm were cut with an ultramicrotome and placed on Formvar carbon-coated Cu grids. Finally, these grids were further contrasted with uranyl acetate and lead citrate. All electron micrographs were obtained with a TEM (Jeol JEM 1010 MT) operating at 80 KV. Images were obtained with AnalySIS (SIS, Munster) on a Megaview III CCD camera. 2.3. In vitro oral absorption 2.3.1. Cell maintenance and exposure Caco-2 cells were kindly provided by Readycell SL for research purposes. These cells were grown in DMEM supplemented with 10% fetal calf serum (FCS) and antibiotics in an incubator at 37 ◦ C and 5% CO2 . For maintenance, cells were cultured in flasks and subcultured when they reached around 80% confluence. For the permeation experiments, Caco-2 cells were seeded in transwell inserts (PET membrane, 1 m pore diameter, BD Falcon) and grown for three weeks. The inserts selected had a pore size of 1 m, to minimize the retention of NP aggregates through this support membrane. At the end of the three weeks culture period, the transepithelial electric resistance (TEER) was evaluated to confirm the integrity of the cell membrane formed. TEER was measured using a Millicell® ERS reader (Millipore) coupled to electrodes from World Precision Instruments Inc. TEER values were considered acceptable when they were above 1000 Ohm/cm2 , and the permeation experiment was performed. TiO2 NPs were dispersed in the cell culture media (at 1 mg/mL) by horn sonication during 15 min using a Labsonic sonicator (BBraun) operated in continuous mode at 60% amplitude, and coupled to a 3 mm × 80 mm tip (BBraun). This stock solution was diluted in cell culture media to obtain a 100 g/mL stock that was applied in the apical side of the transwell inserts. Preliminary experiments had shown that exposure to 100 g/mL TiO2 NPs for 48 h did not affect Caco-2 membrane integrity (evaluated by TEER and Lucifer Yellow permeability). After 24 and 48 h of exposure, the basal compartment was collected to determine the permeability of the NP across the cell membrane. In order to test whether NMs could cross the polymeric membrane of the inserts, some of the transwell inserts were not seeded. Preliminary experiments had shown that the salt content on the cell media used in the assays considerably decreased the sensitivity of the analysis by ICP-MS. Due to this reason we introduced a dialysis step (24 h dialysis in a volume ratio of 1:1000 in a MWCO 3500 Da membrane from Spectrum Labs) prior to analysis. The
Please cite this article in press as: Janer, G., et al., Cell uptake and oral absorption of titanium dioxide nanoparticles. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.04.014
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calibration standards were prepared using the same NP dispersions and were also dialyzed prior to analyses.
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2.3.2. ICP-MS analyses Acidic digestion (hydrofluoric acid:nitric acid; 1:20) with an analytical microwave (CEM, model: Mars) was performed to completely dissolve the NPs into metal ions. The digested samples were analyzed by ICP-MS (Agilent, model: 7500ce). The temperature ramp used was 15 min to 190 ◦ C and hold 10 min, and the RF power: 400, 800 or 1000 W depending on the number of samples. Other ICP-MS parameters for the determination of metals are presented in the Supplementary Material (Table S1).
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2.4. In vivo oral absorption
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2.4.4. ICP-MS tissue analyses Tissue samples were acid digested and analyzed by ICP-MS (Agilent 7500ce) as described above. 2.4.5. Electron microscopy analyses Peyer’s patch and smooth small intestine samples preserved in buffered glutaraldehyde-paraformaldehyde were fixed with OsO4 . This was followed by serial dehydration through a series of acetone increasing concentrations, and embedding in Epon resin. After resin curing, 50 nm ultrafine cuts were obtained and processed as described above for the cell samples. 2.5. Statistical analyses
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2.4.1. Animals and housing conditions Sprague-Dawley male rats (180–230 g at the beginning of the experiment) were allocated to the vehicle or the TiO2 NPs groups (4 animals per group). Animals were housed in the Animal Facilities of the Barcelona Scientific Park, under a 12:12 h light:dark cycle at 20 ± 2 ◦ C and 55 ± 30% humidity. The rats had ad libitum access to Protein Rodent Maintenance Diet 2914C (Harlan) and autoclaved water. Animal work was approved by the Barcelona Scientific Park Animal Care Committee. 2.4.2. NM dispersions, dose selection, and administration procedure TiO2 NPs were dispersed at a concentration of 10 mg/mL in MQ water containing sodium citrate at 2 mM (as dispersant). Dispersion was achieved by horn sonication using the same conditions described for the transwell experiments. The dose (100 mg/Kg by oral gavage in a volume of 10 mL/Kg) was selected on the basis of previous toxicological information from literature, so that it could be tolerated while high enough to enable analytical procedures. 2.4.3. Experimental procedures Administered rats were weighted and observed to assess clinical signs of toxicity on the day of administration and on the following day, before sacrifice. At termination, spleen, liver, small and large intestines, and mesenteric lymph nodes were removed. The intestines were carefully washed with phosphate buffer (pH 7.4) to remove their content. Peyer’s patches were excised and separated from the rest of the small intestine. The caecum was separated from the rest of the large intestine. One of the Peyer’s patches and a piece of smooth small intestine were immediately preserved in buffered (pH 7.4) glutaraldehyde (2.5%) paraformaldehyde (2%) for later TEM analyses. The remaining samples were kept at −20 ◦ C for chemical analyses.
T-test analyses (p < 0.05) were performed to evaluate statistical significance of differences between the control group and the treated group.
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3. Results
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3.1. NM properties
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The TiO2 NPs used in this study were spherical and had a size range of 18 ± 8 nm. Their surface area was 89.8 m2 /g and the porosity was 0.0372 cm3 /g with a porus diameter of 12.9 nm. The size of the NP aggregates depended on the medium used for dispersion. The hydrodynamic diameter (Z-average) in DMEM, as used in the in vitro studies, was 270 nm for a dispersion at 0.1 mg/mL and 367 nm for a dispersion at 1 mg/mL. In sodium citrate as used for the oral administrations, the hydrodynamic diameter was 202 nm at 1 mg/mL and 117 at 10 mg/mL. 3.2. Cell internalization TiO2 NPs were present in form of agglomerates of different sizes within cytoplasmic vesicles. In none of the cells evaluated, particles were found freely in the cytoplasm or in the nucleus. In some cases, the NPs agglomerates were bound to the cell membranes. Although most had sizes around 20–30 nm, some NPs were considerably larger (200–300 nm, see upper left corner of Fig. 1). The size of the agglomerates ranged between a few NPs (total size less than 100 nm) to hundreds of NPs, with total size above 1 m. 3.3. In vitro oral absorption TiO2 NPs could freely move through the insert membrane in the absence of cells, but a very small proportion (below or close to the detection limit, i.e., 0.1 ppm or 0.4% of the applied concentration)
Fig. 1. TEM micrographs of A-549 cells exposed to 100 g/mL TiO2 NP for 72 h. Nucleus (n).
Please cite this article in press as: Janer, G., et al., Cell uptake and oral absorption of titanium dioxide nanoparticles. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.04.014
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Table 1 Levels of titanium present in the basal compartment after exposure to 100 g/mL TiO2 NPs. Results are average from at least duplicated experiments. The detection limit (d.l.) was 0.1 ppm (equivalent to 0.4% migration to basal compartment). Control blanks % titanium in the basal compartment
ARTICLE IN PRESS
TOXLET 8701 1–8
Toxicology Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Cell uptake and oral absorption of titanium dioxide nanoparticles
1
2 3
Q1
G. Janer, E. Mas del Molino, E. Fernández-Rosas, A. Fernández, S. Vázquez-Campos ∗ LEITAT Technological Center, C/de la Innovació, 2, Terrassa, Barcelona 08225, Spain
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5 6 7 8 9 10
h i g h l i g h t s • • • •
TiO2 NPs were readily uptaken by A549 cells in vitro. Very low translocation of TiO2 NPs through a differentiated Caco-2 monolayer system. Titanium levels in tissues did not increase after a single oral dose of TiO2 NPs. Several NPs were observed in the cytoplasm of a cell from a Peyer’s Patch by TEM.
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Article history: Received 7 February 2014 Received in revised form 17 April 2014 Accepted 19 April 2014 Available online xxx
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Keywords: Oral bioavailability Caco-2 Biodistribution Clearance
26
1. Introduction
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27 28 29 30 31 32 33 34 35 36 37
Large efforts are invested on the development of in vitro tests to evaluate nanomaterial (NM) toxicity. In order to assess the relevance of the adverse effects identified in in vitro toxicity tests a thorough understanding of the biokinetics of NMs is critical. We used different in vitro and in vivo test methods to evaluate cell uptake and oral absorption of titanium dioxide nanoparticles (TiO2 NPs). These NPs were readily uptaken by A549 cells (carcinomic human alveolar basal epithelial cells) in vitro. Such rapid uptake contrasted with a very low oral absorption in a differentiated Caco-2 monolayer system (human epithelial colorectal adenocarcinoma cells) and after oral gavage administration to rats. In this oral study, no significant increase in the levels of titanium was recorded by ICP-MS in any of the tissues evaluated (including among other: small intestine, Peyer’s patches, mesenteric lymph nodes, liver, and spleen). No NPs were observed by TEM in sections of the small intestine, except for several particles in the cytoplasm of a cell from a Peyer’s Patch area. The observation of NPs in Peyer’s Patch suggests that the Caco-2 monolayer system is likely to underestimate the potential for oral absorption of NPs and that the model could be improved by including M-cells in co-culture. © 2014 Published by Elsevier Ireland Ltd.
Due to ethical and economical considerations, large research efforts are dedicated to the development of toxicity test alternatives in vitro. When talking about NMs, the high number of variations that can exist among them (sizes, surface modifications, shape. . .) would lead to enormous economical and animal life costs if traditional in vivo testing methods had to be used. However, the use of in vitro data for toxicological risk assessment is not straight forward. It requires not only that the in vitro tests comprehensively cover the mechanisms of action leading to the relevant toxic effect, but also a very thorough understanding of the biokinetics of the substance/material.
∗ Corresponding author. Tel.: +34 93 788 23 00; fax: +34 93 789 19 06. E-mail addresses: [email protected], [email protected] (S. Vázquez-Campos).
It is anticipated that with inorganic NMs, the panel of mechanisms of toxicological action will comprise particle specific effects (e.g., impaired phagocytosis in particle-laden macrophages (Warheit et al., 1997)) and inorganic ion specific effects (e.g., after release of Ag+ ions from silver NPs). However, such panel of possible effects is expected to be considerably narrower than that for organic chemicals, which can specifically interact with binding sites of enzymes or receptors and result in an enormous number of toxicity pathways (Kavlock et al., 2012). There starts to be a considerable body of knowledge on the mechanisms of toxicity of NMs that have allowed the generation of hazard categorization trees on the basis of their physicochemical properties (RIP-oN2, 2001). On the other hand, the understanding and prediction of the biokinetic of NMs is still at a very early stage. In order to progress towards a future evaluation of NMs risks on the basis of in vitro tests, a better understanding of the critical biokinetic processes as well as the development of in vitro models to predict these processes is needed. The Caco-2 monolayer permeation test is a widely used in vitro test to predict oral absorption of organic compounds, particularly
http://dx.doi.org/10.1016/j.toxlet.2014.04.014 0378-4274/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Janer, G., et al., Cell uptake and oral absorption of titanium dioxide nanoparticles. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.04.014
38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56
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G. Janer et al. / Toxicology Letters xxx (2014) xxx–xxx
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by the pharmaceutical industry (Artursson et al., 2001). A few reports also exist on the use of this test system for NMs (Koeneman et al., 2010; Al-Jubory and Handy, 2012; Antunes et al., 2013; Jin et al., 2013; Kenzaoui et al., 2012; Reix et al., 2012; Sonavane et al., 2008). These reports and the limited number of in vivo oral absorption or toxicity studies available suggest that NMs do cross the intestinal membrane, although at a very limited rate (Baek et al., 2012; Hillyer and Albrecht, 2001; Jani et al., 1994; Li et al., 2012; Schleh et al., 2012; Wang et al., 2007). The residence time of NMs in blood is short compared to most organic chemicals and dominated by the uptake by the mononuclear phagocyte system rather than elimination from the body (Landsiedel et al., 2012). Therefore, the evaluation of systemic absorption requires the quantification of NMs in tissues. This leads to several analytical challenges, both in case of organic and inorganic NMs. TiO2 NPs are one of the most commonly used NMs, due to its low costs and easiness to obtain at nano-sizes and its properties at this scale, such as reflection of UV sunrays and photocatalytic properties. Some reports exist on different aspects of the biokinetics of TiO2 NPs both in vitro (see below) and in vivo (Fabian et al., 2008; Patri et al., 2009; Shinohara et al., 2013; Wang et al., 2007). However, it is difficult to compare studies and to understand the predictive value of some of the in vitro studies because they used titania NMs of different crystal phase, size, shape and surface coatings. Most studies evaluating cell internalization of TiO2 NPs have found aggregates of these NMs in cytoplasmic vesicles (Allouni et al., 2012; Andersson-Willman et al., 2012; Halamoda Kenzaoui et al., 2012; Hussain et al., 2010; Jaeger et al., 2012; Kocbek et al., 2010; L’Azou et al., 2008; Singh et al., 2007; Wang et al., 2011; Zhu et al., 2012). This contrasts with the lack of internalization of TiO2 NPs reported by Fisichella et al. (2012) and the limited internalization reported by Koeneman et al. (2010) in Caco-2 cells. These differences are likely to be due to the cell type, but could also be related to the differences in the TiO2 NPs used in these studies. In the present report, we have used a single type of TiO2 NPs in different in vitro and in vivo systems in order to better understand how results from these studies can be extrapolated to other cell types or levels of organization (e.g., in vitro to in vivo). In particular, this manuscript describes the results of three studies: an in vitro cell uptake using A549 cells, an in vitro permeation test using differentiated Caco-2 cells, and an in vivo oral absorption test in rats. To overcome the analytical challenges associated to the tracking of unlabelled TiO2 NPs, both inductively coupled plasma mass spectrometry (ICP-MS) and transmission electron microscopy (TEM) were used.
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2. Methods
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2.1. NMs source and characterization
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TiO2 NPs, synthesized using the Flame Spray Pyrolisys, were obtained from L’Urederra (Spain) under the framework of the EU project Nanopolytox. Surface area and porosity of NMs were measured by a multi-point isotherm BET assay (equilibration time 60 s), using N2 as adsorbate, in a BET surface area analyzer (Nova 2200e series; Quantachrome). NMs were previously degassed overnight under vacuum at 105 ◦ C. Surface area was calculated using 12 points of relative pressure (r2 > 0.998), and porosity was obtained using the BJH calculation method in the desorption branch of the isotherm. NMs were dispersed with a tip sonicator (SONICS VCX 750; Braun) equipped with a standard ¼ (6,5 mm) × 5.6” (142 mm) tapered microtip (783630-0420; Izasa) at 1 mg/mL for 15 min without pauses, applying a power of 400 W. The dispersion was kept cold during the sonication process by using a water-ice bath around the solution flask. Samples were deposited on TEM
grids (formvar/carbon-coated 200 mesh Cu grids; Ted Pella Inc) just after sonication. Shape, size distribution and dispersibility of NMs were analyzed by TEM using an H-7000 operating at 120 KV (Hitachi). Finally, hydrodynamic diameters in relevant media were measured by dynamic light scattering (DLS) using a Zetasizer (nano series Nano-ZS; Malvern). 2.2. In vitro cell internalization A-549 cells were incubated with only cell media (controls) or with 100 g/mL TiO2 NPs for 72 h. Preliminary experiments had shown that this concentration did not affect cell viability (Alamar Blue® test) during this exposure period. Afterwards, the cells were washed with PBS and fixed with glutaraldehyde (2.5%) for 1 h. Cells were detached from the Petri dish by scraping and centrifuged (4 ◦ C, 1000 g, 5 min), to obtain a compact pellet. After washing with PB (0.1 M), cells were stained with OsO4 (1%) for 2 h and washed again with PB (0.1 M). Cells were dehydrated at 4 ◦ C through a series of acetone concentrations (50%, 70%, 90%, 96% and 100%), prior to being progressively (25%, 50%, 75% and 100%) embedded in Epon resin. After resin curing (60 ◦ C, 48 h), sections with a thickness of 50 nm were cut with an ultramicrotome and placed on Formvar carbon-coated Cu grids. Finally, these grids were further contrasted with uranyl acetate and lead citrate. All electron micrographs were obtained with a TEM (Jeol JEM 1010 MT) operating at 80 KV. Images were obtained with AnalySIS (SIS, Munster) on a Megaview III CCD camera. 2.3. In vitro oral absorption 2.3.1. Cell maintenance and exposure Caco-2 cells were kindly provided by Readycell SL for research purposes. These cells were grown in DMEM supplemented with 10% fetal calf serum (FCS) and antibiotics in an incubator at 37 ◦ C and 5% CO2 . For maintenance, cells were cultured in flasks and subcultured when they reached around 80% confluence. For the permeation experiments, Caco-2 cells were seeded in transwell inserts (PET membrane, 1 m pore diameter, BD Falcon) and grown for three weeks. The inserts selected had a pore size of 1 m, to minimize the retention of NP aggregates through this support membrane. At the end of the three weeks culture period, the transepithelial electric resistance (TEER) was evaluated to confirm the integrity of the cell membrane formed. TEER was measured using a Millicell® ERS reader (Millipore) coupled to electrodes from World Precision Instruments Inc. TEER values were considered acceptable when they were above 1000 Ohm/cm2 , and the permeation experiment was performed. TiO2 NPs were dispersed in the cell culture media (at 1 mg/mL) by horn sonication during 15 min using a Labsonic sonicator (BBraun) operated in continuous mode at 60% amplitude, and coupled to a 3 mm × 80 mm tip (BBraun). This stock solution was diluted in cell culture media to obtain a 100 g/mL stock that was applied in the apical side of the transwell inserts. Preliminary experiments had shown that exposure to 100 g/mL TiO2 NPs for 48 h did not affect Caco-2 membrane integrity (evaluated by TEER and Lucifer Yellow permeability). After 24 and 48 h of exposure, the basal compartment was collected to determine the permeability of the NP across the cell membrane. In order to test whether NMs could cross the polymeric membrane of the inserts, some of the transwell inserts were not seeded. Preliminary experiments had shown that the salt content on the cell media used in the assays considerably decreased the sensitivity of the analysis by ICP-MS. Due to this reason we introduced a dialysis step (24 h dialysis in a volume ratio of 1:1000 in a MWCO 3500 Da membrane from Spectrum Labs) prior to analysis. The
Please cite this article in press as: Janer, G., et al., Cell uptake and oral absorption of titanium dioxide nanoparticles. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.04.014
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calibration standards were prepared using the same NP dispersions and were also dialyzed prior to analyses.
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2.3.2. ICP-MS analyses Acidic digestion (hydrofluoric acid:nitric acid; 1:20) with an analytical microwave (CEM, model: Mars) was performed to completely dissolve the NPs into metal ions. The digested samples were analyzed by ICP-MS (Agilent, model: 7500ce). The temperature ramp used was 15 min to 190 ◦ C and hold 10 min, and the RF power: 400, 800 or 1000 W depending on the number of samples. Other ICP-MS parameters for the determination of metals are presented in the Supplementary Material (Table S1).
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2.4.4. ICP-MS tissue analyses Tissue samples were acid digested and analyzed by ICP-MS (Agilent 7500ce) as described above. 2.4.5. Electron microscopy analyses Peyer’s patch and smooth small intestine samples preserved in buffered glutaraldehyde-paraformaldehyde were fixed with OsO4 . This was followed by serial dehydration through a series of acetone increasing concentrations, and embedding in Epon resin. After resin curing, 50 nm ultrafine cuts were obtained and processed as described above for the cell samples. 2.5. Statistical analyses
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2.4.1. Animals and housing conditions Sprague-Dawley male rats (180–230 g at the beginning of the experiment) were allocated to the vehicle or the TiO2 NPs groups (4 animals per group). Animals were housed in the Animal Facilities of the Barcelona Scientific Park, under a 12:12 h light:dark cycle at 20 ± 2 ◦ C and 55 ± 30% humidity. The rats had ad libitum access to Protein Rodent Maintenance Diet 2914C (Harlan) and autoclaved water. Animal work was approved by the Barcelona Scientific Park Animal Care Committee. 2.4.2. NM dispersions, dose selection, and administration procedure TiO2 NPs were dispersed at a concentration of 10 mg/mL in MQ water containing sodium citrate at 2 mM (as dispersant). Dispersion was achieved by horn sonication using the same conditions described for the transwell experiments. The dose (100 mg/Kg by oral gavage in a volume of 10 mL/Kg) was selected on the basis of previous toxicological information from literature, so that it could be tolerated while high enough to enable analytical procedures. 2.4.3. Experimental procedures Administered rats were weighted and observed to assess clinical signs of toxicity on the day of administration and on the following day, before sacrifice. At termination, spleen, liver, small and large intestines, and mesenteric lymph nodes were removed. The intestines were carefully washed with phosphate buffer (pH 7.4) to remove their content. Peyer’s patches were excised and separated from the rest of the small intestine. The caecum was separated from the rest of the large intestine. One of the Peyer’s patches and a piece of smooth small intestine were immediately preserved in buffered (pH 7.4) glutaraldehyde (2.5%) paraformaldehyde (2%) for later TEM analyses. The remaining samples were kept at −20 ◦ C for chemical analyses.
T-test analyses (p < 0.05) were performed to evaluate statistical significance of differences between the control group and the treated group.
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3. Results
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3.1. NM properties
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The TiO2 NPs used in this study were spherical and had a size range of 18 ± 8 nm. Their surface area was 89.8 m2 /g and the porosity was 0.0372 cm3 /g with a porus diameter of 12.9 nm. The size of the NP aggregates depended on the medium used for dispersion. The hydrodynamic diameter (Z-average) in DMEM, as used in the in vitro studies, was 270 nm for a dispersion at 0.1 mg/mL and 367 nm for a dispersion at 1 mg/mL. In sodium citrate as used for the oral administrations, the hydrodynamic diameter was 202 nm at 1 mg/mL and 117 at 10 mg/mL. 3.2. Cell internalization TiO2 NPs were present in form of agglomerates of different sizes within cytoplasmic vesicles. In none of the cells evaluated, particles were found freely in the cytoplasm or in the nucleus. In some cases, the NPs agglomerates were bound to the cell membranes. Although most had sizes around 20–30 nm, some NPs were considerably larger (200–300 nm, see upper left corner of Fig. 1). The size of the agglomerates ranged between a few NPs (total size less than 100 nm) to hundreds of NPs, with total size above 1 m. 3.3. In vitro oral absorption TiO2 NPs could freely move through the insert membrane in the absence of cells, but a very small proportion (below or close to the detection limit, i.e., 0.1 ppm or 0.4% of the applied concentration)
Fig. 1. TEM micrographs of A-549 cells exposed to 100 g/mL TiO2 NP for 72 h. Nucleus (n).
Please cite this article in press as: Janer, G., et al., Cell uptake and oral absorption of titanium dioxide nanoparticles. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.04.014
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ARTICLE IN PRESS G. Janer et al. / Toxicology Letters xxx (2014) xxx–xxx
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Table 1 Levels of titanium present in the basal compartment after exposure to 100 g/mL TiO2 NPs. Results are average from at least duplicated experiments. The detection limit (d.l.) was 0.1 ppm (equivalent to 0.4% migration to basal compartment). Control blanks % titanium in the basal compartment
