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Genetic toxicity assessment of engineered nanoparticles using a 3D in vitro skin model (EpiDerm ™ )

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ABSTRACT

Background: The rapid production and incorporation of engineered nanomaterials into consumer products alongside research suggesting nanomaterials can cause cell death and DNA damage (genotoxicity) makes in vitro assays desirable for nanosafety screening. However, conflicting outcomes are often observed when in vitro and in vivo study results are compared, suggesting more physiologically representative in vitro models are required to minimise reliance on animal testing.

Method: BASF Levasil® silica nanoparticles (16 and 85 nm) were used to adapt the 3D reconstructed skin micronucleus (RSMN) assay for nanomaterials administered topically or into the growth medium. 3D dose-responses were compared to a 2D micronucleus assay using monocultured human B cells (TK6) after standardising dose between 2D / 3D assays by total nanoparticle mass to cell number. Cryogenic vitrification, scanning electron microscopy and dynamic light scattering techniques were applied to characterise in-medium and air-liquid interface exposures. Advanced transmission electron microscopy imaging modes (high angle annular dark field) and X-ray spectrometry were used to define nanoparticle penetration / cellular uptake in the intact 3D models and 2D monocultured cells.

Results: For all 2D exposures, significant (p < 0.002) increases in genotoxicity were observed (≥100 μg/mL) alongside cell viability decreases (p < 0.015) at doses ≥200 μg/mL (16 nm-SiO2) and ≥100 μg/mL (85 nm-SiO2). In contrast, 2D-equivalent exposures to the 3D models (≤300 μg/mL) caused no significant DNA damage or impact on cell viability. Further increasing dose to the 3D models led to probable air-liquid interface suffocation. Nanoparticle penetration / cell uptake analysis revealed no exposure to the live cells of the 3D model occurred due to the protective nature of the skin model’s 3D cellular microarchitecture (topical exposures) and confounding barrier effects of the collagen cell attachment layer (in-medium exposures). 2D monocultured cells meanwhile showed extensive internalisation of both silica particles causing (geno)toxicity.

Conclusions: The results establish the importance of tissue microarchitecture in defining nanomaterial exposure, and suggest 3D in vitro models could play a role in bridging the gap between in vitro and in vivo outcomes in nanotoxicology. Robust exposure characterisation and uptake assessment methods (as demonstrated) are essential to interpret nano(geno)toxicity studies successfully.

Electronic supplementary material: The online version of this article (doi:10.1186/s12989-016-0161-5) contains supplementary material, which is available to authorized users.

No MeSH data available.


BASF Levasil® silicon dioxide nanoparticle primary characterisation: Bright field TEM micrographs of (a) 16 nm-SiO2, and (b) 85 nm-SiO2, allowed primary particle size, shape and morphology to be assessed. c Typical particle EDX spectrum relative to background confirming the presence of silicon and oxygen with no detectable contaminants (copper and carbon due to TEM grid and support film). d Schematic illustrating the negative surface charge of SiO2 particles, due to unbound surface oxygen groups
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Fig1: BASF Levasil® silicon dioxide nanoparticle primary characterisation: Bright field TEM micrographs of (a) 16 nm-SiO2, and (b) 85 nm-SiO2, allowed primary particle size, shape and morphology to be assessed. c Typical particle EDX spectrum relative to background confirming the presence of silicon and oxygen with no detectable contaminants (copper and carbon due to TEM grid and support film). d Schematic illustrating the negative surface charge of SiO2 particles, due to unbound surface oxygen groups

Mentions: This study used BASF Levasil® 200 and Levasil® 50 amorphous silica nanoparticles to optimise a 3D RSMN assay for nanomaterial test articles. Transmission electron microscopy (TEM) indicated both particles were spherical and had a relatively smooth surface morphology (Fig. 1a and b). Primary size (i.e., single particle) measurements from electron micrographs determined the average diameter of the Levasil® 200 to be 16.4 nm (manufacturer specified 15 nm) and Levasil® 50 to be 85.1 nm (manufacturer specified 55 nm) (Table 1). Therefore, text references hereafter refer to 16 nm-SiO2 or 85 nm-SiO2, respectively. No evidence of regular lattice planes was found at higher magnification confirming the expected amorphous structure. Nanoparticle composition and the presence of trace contaminants was investigated using energy dispersive X-ray (EDX) spectrometry. Comparing a blank area of the TEM grid to an area containing nanoparticles revealed a large shift in the ratio of the oxygen and silicon peaks, confirming the silicon dioxide particle composition (Fig. 1c). No evidence of unexpected elemental traces (e.g., impurities or unexpected suspension phase contaminants) was found by EDX. Dynamic Light Scattering (DLS) analysis at 300 μg/mL in ultra-pure water (MilliQ, 18MΩ·cm) showed number distribution peak maxima at 17 nm and 92 nm, respectively, for the 16 nm-SiO2 and 85 nm-SiO2. DLS size ranges in water were therefore concurrent to the primary sizes established by TEM, suggesting manufacturer-supplied suspensions were colloidally stable. Surface charge (zeta potential) measurements were strongly negative (< −40 mV) for both particles further indicative of colloidal stability in water and consistent with silicon dioxide’s surface chemistry of negative, unbound oxygen groups (Table 1 and Fig. 1d). Further TEM images, particle size distributions and EDX spectra are available in Additional files 1, 2 and 3.Fig. 1


Genetic toxicity assessment of engineered nanoparticles using a 3D in vitro skin model (EpiDerm ™ )
BASF Levasil® silicon dioxide nanoparticle primary characterisation: Bright field TEM micrographs of (a) 16 nm-SiO2, and (b) 85 nm-SiO2, allowed primary particle size, shape and morphology to be assessed. c Typical particle EDX spectrum relative to background confirming the presence of silicon and oxygen with no detectable contaminants (copper and carbon due to TEM grid and support film). d Schematic illustrating the negative surface charge of SiO2 particles, due to unbound surface oxygen groups
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Related In: Results  -  Collection

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Fig1: BASF Levasil® silicon dioxide nanoparticle primary characterisation: Bright field TEM micrographs of (a) 16 nm-SiO2, and (b) 85 nm-SiO2, allowed primary particle size, shape and morphology to be assessed. c Typical particle EDX spectrum relative to background confirming the presence of silicon and oxygen with no detectable contaminants (copper and carbon due to TEM grid and support film). d Schematic illustrating the negative surface charge of SiO2 particles, due to unbound surface oxygen groups
Mentions: This study used BASF Levasil® 200 and Levasil® 50 amorphous silica nanoparticles to optimise a 3D RSMN assay for nanomaterial test articles. Transmission electron microscopy (TEM) indicated both particles were spherical and had a relatively smooth surface morphology (Fig. 1a and b). Primary size (i.e., single particle) measurements from electron micrographs determined the average diameter of the Levasil® 200 to be 16.4 nm (manufacturer specified 15 nm) and Levasil® 50 to be 85.1 nm (manufacturer specified 55 nm) (Table 1). Therefore, text references hereafter refer to 16 nm-SiO2 or 85 nm-SiO2, respectively. No evidence of regular lattice planes was found at higher magnification confirming the expected amorphous structure. Nanoparticle composition and the presence of trace contaminants was investigated using energy dispersive X-ray (EDX) spectrometry. Comparing a blank area of the TEM grid to an area containing nanoparticles revealed a large shift in the ratio of the oxygen and silicon peaks, confirming the silicon dioxide particle composition (Fig. 1c). No evidence of unexpected elemental traces (e.g., impurities or unexpected suspension phase contaminants) was found by EDX. Dynamic Light Scattering (DLS) analysis at 300 μg/mL in ultra-pure water (MilliQ, 18MΩ·cm) showed number distribution peak maxima at 17 nm and 92 nm, respectively, for the 16 nm-SiO2 and 85 nm-SiO2. DLS size ranges in water were therefore concurrent to the primary sizes established by TEM, suggesting manufacturer-supplied suspensions were colloidally stable. Surface charge (zeta potential) measurements were strongly negative (< −40 mV) for both particles further indicative of colloidal stability in water and consistent with silicon dioxide’s surface chemistry of negative, unbound oxygen groups (Table 1 and Fig. 1d). Further TEM images, particle size distributions and EDX spectra are available in Additional files 1, 2 and 3.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: The rapid production and incorporation of engineered nanomaterials into consumer products alongside research suggesting nanomaterials can cause cell death and DNA damage (genotoxicity) makes in vitro assays desirable for nanosafety screening. However, conflicting outcomes are often observed when in vitro and in vivo study results are compared, suggesting more physiologically representative in vitro models are required to minimise reliance on animal testing.

Method: BASF Levasil&reg; silica nanoparticles (16 and 85&nbsp;nm) were used to adapt the 3D reconstructed skin micronucleus (RSMN) assay for nanomaterials administered topically or into the growth medium. 3D dose-responses were compared to a 2D micronucleus assay using monocultured human B cells (TK6) after standardising dose between 2D / 3D assays by total nanoparticle mass to cell number. Cryogenic vitrification, scanning electron microscopy and dynamic light scattering techniques were applied to characterise in-medium and air-liquid interface exposures. Advanced transmission electron microscopy imaging modes (high angle annular dark field) and X-ray spectrometry were used to define nanoparticle penetration / cellular uptake in the intact 3D models and 2D monocultured cells.

Results: For all 2D exposures, significant (p&thinsp;&lt;&thinsp;0.002) increases in genotoxicity were observed (&ge;100&nbsp;&mu;g/mL) alongside cell viability decreases (p&thinsp;&lt;&thinsp;0.015) at doses &ge;200&nbsp;&mu;g/mL (16&nbsp;nm-SiO2) and &ge;100&nbsp;&mu;g/mL (85&nbsp;nm-SiO2). In contrast, 2D-equivalent exposures to the 3D models (&le;300&nbsp;&mu;g/mL) caused no significant DNA damage or impact on cell viability. Further increasing dose to the 3D models led to probable air-liquid interface suffocation. Nanoparticle penetration / cell uptake analysis revealed no exposure to the live cells of the 3D model occurred due to the protective nature of the skin model&rsquo;s 3D cellular microarchitecture (topical exposures) and confounding barrier effects of the collagen cell attachment layer (in-medium exposures). 2D monocultured cells meanwhile showed extensive internalisation of both silica particles causing (geno)toxicity.

Conclusions: The results establish the importance of tissue microarchitecture in defining nanomaterial exposure, and suggest 3D in vitro models could play a role in bridging the gap between in vitro and in vivo outcomes in nanotoxicology. Robust exposure characterisation and uptake assessment methods (as demonstrated) are essential to interpret nano(geno)toxicity studies successfully.

Electronic supplementary material: The online version of this article (doi:10.1186/s12989-016-0161-5) contains supplementary material, which is available to authorized users.

No MeSH data available.