Label-free in vitro toxicity and uptake assessment of citrate stabilised gold nanoparticles in three cell lines

Abstract

Background: Reliable in vitro toxicity testing is needed prior to the commencement of in vivo testing necessary for hazard identification and risk assessment of nanoparticles. In this study, the cytotoxicity and uptake of 14 nm and 20 nm citrate stabilised gold nanoparticles (AuNPs) in the bronchial epithelial cell line BEAS-2B, the Chinese hamster ovary cell line CHO, and the human embryonic kidney cell line HEK 293 were investigated.

Methods: Cytotoxicity of the AuNPs was assessed via traditional XTT-, LDH-, and ATP-based assays, followed by cell impedance studies. Dark-field imaging and hyperspectral imaging were used to confirm the uptake of AuNPs into the cells.

Results: Interference of the AuNPs with the XTT- and ATP-based assays was overcome through the use of cell impedance technology. AuNPs were shown to be relatively non-toxic using this methodology; nevertheless CHO cells were the most sensitive cell type with 20 nm AuNPs having the highest toxicity. Uptake of both 14 nm and 20 nm AuNPs was observed in all cell lines in a time- and cell type-dependent manner.

Conclusions: Using the cell impedance and dark-field hyperspectral imaging technologies, it was possible to study the toxicity of AuNPs in different cell lines and show that these cells could internalize AuNPs with their subsequent intracellular aggregation. It was also possible to show that this toxicity would not correlate with the level of uptake but it would correlate with cell-type and the size of the AuNPs. Therefore, these two label-free methodologies used in this study are suitable for in vitro studies on the effects of AuNPs, and could present themselves as appropriate and valuable methodologies for future nanoparticle toxicity and uptake studies.

Figure 1

Absorbance spectrum of the gold nanoparticles. The UV–vis spectrum of 14 nm and 20 nm AuNPs at 1 nM in A) milli-Q water, B) in RPMI culture medium, and C) in Ham’s F12 culture medium.

Figure 2

Characterisation of the gold nanoparticles in milli-Q water and in culture medium using Transmission Electron Microscopy. TEM images of 14 nm AuNPs in (A) water, (B) RPMI culture medium, and (C) Ham’s F12 culture medium; and 20 nm AuNPs in (D) water, (E) RPMI culture medium, and (F) Ham’s F-12 culture medium.

Figure 3

Toxicity studies using conventional assays: XTT, LDH, and ATP assays. (A) BEAS-2B cells were treated with either 1 nM or 5 nM of 14 nm AuNPs for 1 hour prior to toxicity testing using the In Vitro Toxicology Assay Kit (XTT assay). Absorbance measured at 450 nm and a reference wavelength of 600 nm. (B) BEAS-2B cells were treated with either 1 nM or 5 nM of 14 nm AuNPs for 1 hour prior to toxicity testing using the CytoTox-ONE™ assay (LDH assay). Lysis solution was used as a positive control for maximum LDH release and sample LDH is expressed as a percentage of this maximum release. Fluorescence measured at 560 nm excitation, 590 nm emission. (C) BEAS-2B cells were treated with either 1 nM or 5 nM of 14 nm AuNPs for 1 hour prior to toxicity testing using the CellTiter Glo assay (ATP assay). Luminescent signal was measured and viability is expressed as a percentage of the untreated control cells. (D) The absorbance of 1 nM and 5 nM AuNPs in culture medium and with unreduced XTT at 450 nm. (E) Absorbance values obtained when data from (D) was subtracted from (A). (F) Luciferin substrate from the CellTiter Glo assay was incubated with ATP and 14 nm AuNPs to produce the luminescent product oxyluciferin prior to measurement of the luminescent signal.

Figure 4

Normalised cell index of cells treated with AuNPs. BEAS-2B cells (A and B), HEK 293 cells (C and D), and CHO cells (E and F) were treated with 14 nm AuNPs (left panel) and 20 nm AuNPs (right panel). BEAS-2B, HEK 293, and CHO cells were seeded and allowed to recover for 24 hours prior to treatment for 72 hours, 36 hours, or 42 hours, respectively. The slope of the curves was calculated and t-test conducted to determine differences between treated and untreated control cells. In (A) and (C), all treatments were statistically different from untreated control (p < 0.05) whilst no significant differences were observed in (B) and (D). In (E), 2 nM (p < 0.05) and 5 nM (p < 0.01) were statistically different from untreated control; whilst in (F) both 2 nM and 5nM treatments were statistically different from the untreated control at p < 0.01; n ≥ 3.

Figure 5

Dark-field image at 100x magnification of BEAS-2B cells incubated with 14 nm AuNPs for 1 hr. Cells were washed, fixed, and immobilised onto slides prior to image capture using the CytoViva system. Four focal planes, (A) through to (D), are observed as the focus of the microscope moves through the cell.

Figure 6

Dark-field images of BEAS-2B cells incubated with AuNPs. Dark-field images were captured at 60x magnification of BEAS-2B cells incubated with either (A) 14 nm or (B) 20 nm AuNPs for 1 hr, 4 hrs, or 6 hrs.

Figure 7

Dark-field images of HEK 293 cells incubated with AuNPs. Dark-field images were captured at 60x magnification of HEK 293 cells incubated with either (A) 14 nm or (B) 20 nm AuNPs for 1 hr, 4 hrs, or 6 hrs.

Figure 8

Dark-field images of CHO cells incubated with AuNPs. Dark-field images were captured at 60x magnification of CHO cells incubated with either (A) 14 nm or (B) 20 nm AuNPs for 1 hr, 4 hrs, or 6 hrs.

Figure 9

Spectral profiles of 14 nm and 20 nm AuNPs. Ten spectral profiles of randomly selected AuNPs that appeared visually to be either (A and C) in single suspension, or (B and D) aggregated nanoparticles. Each coloured line represents the spectrum from a single pixel.

Figure 10

Representative images of the SAM analyses. (A) HSI scan of BEAS-2B cells treated with 1 nM 14 nm AuNPs for 4 hours; (B) SAM image indicating pixels of (A) that matched the spectral library of 14 nm singularly dispersed nanoparticles; (C) SAM image indicating pixels of (A) that matched the spectral library of the 14 nm aggregated nanoparticles.