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Uptake of different crystal structures of TiO2 nanoparticles by Caco-2 intestinal cells

Gitrowski, Constantinos, Al-Jubory, Aliaa R., Handy, Richard D.·2014·Toxicology Letters
DOI 10.1016/j.toxlet.2014.02.014
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Fig. 1 Transmission Electron microscope (TEM) images showing primary particles, and particle size distributions by nanoparticle tracking analysis (hydrodynamic diameter, Nanosight LM10) of; (A) bulk TiO2 particles, scale bar = 100 nm, (B) P25, scale bar = 50 nm, (C) anatase, scale bar = 50 nm and (D) rutile, scale bar = 50 nm at a concentration of 10 mg l -1 (dilutions of the 500 mg l -1 stocks) in Milliq water. Particle size distribution plots are individual examples from triplicate measurements.
Fig. 1 Transmission Electron microscope (TEM) images showing primary particles, and particle size distributions by nanoparticle tracking analysis (hydrodynamic diameter, Nanosight LM10) of; (A) bulk TiO2 particles, scale bar = 100 nm, (B) P25, scale bar = 50 nm, (C) anatase, scale bar = 50 nm and (D) rutile, scale bar = 50 nm at a concentration of 10 mg l -1 (dilutions of the 500 mg l -1 stocks) in Milliq water. Particle size distribution plots are individual examples from triplicate measurements.CC-BY-NC-ND · From the PDF
Fig. 2 (Experiment 1): Titanium accumulation in Caco-2 cells from exposures to 1 mg l -1 of different forms of TiO2 for 24 h. Data points are means ± SEM ( n = 3 replicates/treatment). Curves were fitted using Sigma plot version 12.0 to the raw data ( n = 3 observations/treatment) using the rectangular hyperbola function ( f = y 0 + a × (1 -exp( -b × x )). Equations for the curve fits were as follows: For bulk f = 2.64 + 11.54 × (1 -exp( -0.261 × x )), r 2 = 0.901; For P25, f = 1.16 + 12.54 × (1 -exp( -0.22 × x ), r 2 = 0.875; For anatase f = 1.71 + 7.01 × (1 -exp( -0.3017 × x ), r 2 = 0.952; For rutile f = 2.07 + 4.86 × (1 -exp( -0.67 × x )), r 2 = 0.731. The unexposed control showed no time-dependent changes and therefore a curve was not fitted. Accumulation of all titanium forms is significantly different to the controls (ANOVA P < 0.05 (*)) at 24 h. Accumulation of Bulk and P25 titanium forms at 24 h is significantly greater than the anatase and rutile forms (ANOVA P < 0.05 ( ∼ )).
Fig. 2 (Experiment 1): Titanium accumulation in Caco-2 cells from exposures to 1 mg l -1 of different forms of TiO2 for 24 h. Data points are means ± SEM ( n = 3 replicates/treatment). Curves were fitted using Sigma plot version 12.0 to the raw data ( n = 3 observations/treatment) using the rectangular hyperbola function ( f = y 0 + a × (1 -exp( -b × x )). Equations for the curve fits were as follows: For bulk f = 2.64 + 11.54 × (1 -exp( -0.261 × x )), r 2 = 0.901; For P25, f = 1.16 + 12.54 × (1 -exp( -0.22 × x ), r 2 = 0.875; For anatase f = 1.71 + 7.01 × (1 -exp( -0.3017 × x ), r 2 = 0.952; For rutile f = 2.07 + 4.86 × (1 -exp( -0.67 × x )), r 2 = 0.731. The unexposed control showed no time-dependent changes and therefore a curve was not fitted. Accumulation of all titanium forms is significantly different to the controls (ANOVA P < 0.05 (*)) at 24 h. Accumulation of Bulk and P25 titanium forms at 24 h is significantly greater than the anatase and rutile forms (ANOVA P < 0.05 ( ∼ )).CC-BY-NC-ND · From the PDF
Fig. 3 (Experiment 2): Effect of nystatin and vanadate on Ti accumulation in Caco2 cells incubated with 1 mg l -1 titanium forms at 24 h. The plots are means ± SEM ( n = 6) from the replicate data and are expressed as 'nmol [Ti] mg -1 protein'. Statistical analysis at 24 h time point showed that the levels of accumulation relative to the no drug controls were different (ANOVA P < 0.05 (*)). Symbol (#) indicates statistical differences between vanadate and nystatin (ANOVA P < 0.05). Letters represent differences between materials within drug treatment (materialtype effect) (ANOVA P < 0.05).
Fig. 3 (Experiment 2): Effect of nystatin and vanadate on Ti accumulation in Caco2 cells incubated with 1 mg l -1 titanium forms at 24 h. The plots are means ± SEM ( n = 6) from the replicate data and are expressed as 'nmol [Ti] mg -1 protein'. Statistical analysis at 24 h time point showed that the levels of accumulation relative to the no drug controls were different (ANOVA P < 0.05 (*)). Symbol (#) indicates statistical differences between vanadate and nystatin (ANOVA P < 0.05). Letters represent differences between materials within drug treatment (materialtype effect) (ANOVA P < 0.05).CC-BY-NC-ND · From the PDF
Fig. 4 (Experiment 3): Effects of chloropromazine, genistein and amiloride on Ti accumulation in Caco-2 cells incubated with 1 mg l -1 titanium forms at 24 h. The plots are means ± SEM ( n = 6) from the replicate data and are expressed as 'nmol [Ti] mg -1 cell protein'. Statistical analysis at 24 h showed that the levels of accumulation relative to the no drug controls were different (ANOVA P < 0.05 (*)). Letters represent differences between materials within drug treatment (materialtype effect) (ANOVA P < 0.05).
Fig. 4 (Experiment 3): Effects of chloropromazine, genistein and amiloride on Ti accumulation in Caco-2 cells incubated with 1 mg l -1 titanium forms at 24 h. The plots are means ± SEM ( n = 6) from the replicate data and are expressed as 'nmol [Ti] mg -1 cell protein'. Statistical analysis at 24 h showed that the levels of accumulation relative to the no drug controls were different (ANOVA P < 0.05 (*)). Letters represent differences between materials within drug treatment (materialtype effect) (ANOVA P < 0.05).CC-BY-NC-ND · From the PDF
Fig. 5 (A-D) SEM images of Caco-2 cells 96 h after seeding having been exposed to 1 mg l -1 TiO2 forms. (A) Backscatter image (BSI) of Caco-2 cells exposed to 1 mgl -1 'anatase' TiO2'A' showing subsurface Ti with EDS spectra below; (B) Secondary electron image (SEI) of surface bound anatase TiO2 (scale bar represents 2 𝛍 m); (C) BSI of 'D' showing subsurface Ti; (D) SEI image of cell surface exposed to P25 TiO2. The cell membrane appears to have covered the TiO2 (projections are microvilli, scale bar represents 100 nm); Spectra are labelled according to which image they belong to. The spectrum labelled Image D is the spectra associated with an EDS map of the entire Image 6 D.
Fig. 5 (A-D) SEM images of Caco-2 cells 96 h after seeding having been exposed to 1 mg l -1 TiO2 forms. (A) Backscatter image (BSI) of Caco-2 cells exposed to 1 mgl -1 'anatase' TiO2'A' showing subsurface Ti with EDS spectra below; (B) Secondary electron image (SEI) of surface bound anatase TiO2 (scale bar represents 2 𝛍 m); (C) BSI of 'D' showing subsurface Ti; (D) SEI image of cell surface exposed to P25 TiO2. The cell membrane appears to have covered the TiO2 (projections are microvilli, scale bar represents 100 nm); Spectra are labelled according to which image they belong to. The spectrum labelled Image D is the spectra associated with an EDS map of the entire Image 6 D.CC-BY-NC-ND · From the PDF
Fig. 6 (A-D) TEM images of cells grown for 21 days in culture inserts; (A) TEM of control cells (scale bar represents 5 𝛍 m); (B) TEM image of cells following exposure to 10 mg l -1 P25 TiO2 NPs (Note the invagination in progress (measuring 271 nm across)) (scale bar represents 1 𝛍 m; (C) TEM image of intracellular matrix comprising of two vesicles containing NPs (Vesicles are of differing sizes 300 nm and 800 nm diameter, Scale bar represents 1 𝛍 m); (D) SEM blockface image in backscatter mode with EDS showing intracellular Ti underneath the apical membrane (scale bar represents 10 𝛍 m) The thickness of the monolayer is between 30 and 40 𝛍 m.
Fig. 6 (A-D) TEM images of cells grown for 21 days in culture inserts; (A) TEM of control cells (scale bar represents 5 𝛍 m); (B) TEM image of cells following exposure to 10 mg l -1 P25 TiO2 NPs (Note the invagination in progress (measuring 271 nm across)) (scale bar represents 1 𝛍 m; (C) TEM image of intracellular matrix comprising of two vesicles containing NPs (Vesicles are of differing sizes 300 nm and 800 nm diameter, Scale bar represents 1 𝛍 m); (D) SEM blockface image in backscatter mode with EDS showing intracellular Ti underneath the apical membrane (scale bar represents 10 𝛍 m) The thickness of the monolayer is between 30 and 40 𝛍 m.CC-BY-NC-ND · From the PDF

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Authors
Gitrowski, C., Al-Jubory, A. R., & Handy, R. D.
Venue
Toxicology Letters
Publisher
Elsevier BV
Published
2014-05
Volume
226
Issue
3
Pages
264-276
ISSN
0378-4274
DOI
10.1016/j.toxlet.2014.02.014
Cited by
72
References
1
Language
en
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