Gold nanoparticles affect the antioxidant status in selected normal human cells

Figures & data

Table 1 Characterization of the AuNPs-PAA and AuNPs-PAA-Ctxb dispersed in different media.

	Nanoparticle type
Suspension medium	AuNPs-PAA	AuNPs-PAA-Ctxb
Modus size (nm) [5th and 95th percentile]
Class 1 water	4 [4–14]	26 [13–53]
DMEM	5 [4–11]	25 [13–66]
DMEM +10% FBS	5 [4–11]	19 [12–62]
Zeta potential (mV) + SD
Class 1 water	−30.53±0.25	−29.27±0.15
DMEM	−8.03±0.27	−7.59±0.45
DMEM +10% FBS	−8.37±0.25	−7.04±0.22

Notes: The size was measured with disc centrifugation. The values represent the modus particle size, the 5th and 95th percentile (nm). The zeta potential values were assessed with the Nanosizer Nano ZSP and are expressed as the mean nanoparticle surface charge ± standard deviation (n=3).

Abbreviations: AuNPs, gold nanoparticles; Ctxb, Cetuximab; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; mV, millivolts; PAA, polyallylamine; SD, standard deviation.

Table 2 Overview of the performed cell assays, exposure conditions and results

Cell type	Endpoint	Assay	Incubation time	Concentration	Results
A431, MDA-MB-453, HK-2, THLE-2, TIME	EGFR expression	Western blot	N.A.	N.A.	Relative EGFR expression compared to A431: HK-2 ±26%; THLE-2 ±3%; TIME ±2%; MDA-MB-453 ±0.14%.
	AuNPs internalization	TEM	3 h, 24 h	5.0 µg Au/mL (Only AuNPs-PAA-Ctxb)	AuNPs-PAA-Ctxb in intracellular vesicles. AuNPs-PAA-Ctxb clusters in TIME cells.
	ICP-MS	3 h, 6 h, 12 h, 24 h	5.0 µg Au/mL	Fast AuNPs-PAA-Ctxb interaction in A431 and HK-2 cells followed by a decrease. AuNPs-PAA(±Ctxb) interaction progressively increased in TIME, THLE-2 and MDA-MB-453 cells. Overall, AuNPs-PAA-Ctxb interaction was higher than AuNPs-PAA interaction.
	Cell viability	MTS assay	3 h, 24 h	0–50.0 µg Au/mL	Conjugation of Ctxb to AuNPs-PAA reduced cytotoxicity. TIME cells were the most sensitive cells followed by THLE-2, MDA-MB-453, HK-2 and A431 cells.
	Apoptosis	Live cell imaging	0–72 h	0–50.0 µg Au/mL	AuNPs-PAA(±Ctxb) caused apoptosis, which increased with an increasing concentration and exposure period.
	+2 nM NAC	0–72 h	0–50.0 µg Au/mL	Annexin V and caspase 3/7 activity reduced when cells were co-exposed to NAC.
HK-2, THLE-2, TIME	Mitochondrial membrane depolarization	TMRM	3 h, 6 h, 12 h, 24 h	3.0–5.0 µg Au/mL	Mitochondrial membrane potential decreased most profoundly in TIME cells followed by THLE-2 cells and HK-2 cells after 12 h. Mitochondrial membrane potential recovers after 24 h.
	+2 nM NAC	12 h	3.0–5.0 µg Au/mL	NAC prevented mitochondrial membrane depolarization.
	Enzyme activity	Glutathione and thioredoxin reductase	12 h	3.0–5.0 µg Au/mL	GR and TrxR enzyme activity decreased most profoundly in TIME cells followed by THLE-2 cells and HK-2 cells.

Abbreviations: A431, human epidermoid cancer cells; AuNPs-PAA, polyallylamine-coated gold nanoparticles; Ctxb, Cetuximab, EGFR, epidermal growth factor receptor; HK-2, human kidney cells; ICP-MS, inductively coupled plasma mass spectrometry; MDA-MB-453, human breast cancer cells; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); N.A., not applicable; NAC, N-acetyl L-cysteine; THLE-2, human liver cells; TIME, human telomerase immortalized microvascular endothelial cells; TMRM, tetramethylrhodamine methyl ester; GR, glutathione reductase; TrxR, thioredoxin reductase; TEM, transmission electron microscopy.

Figure 1 EGFR expression in HK-2 cells, THLE-2 cells, TIME cells and MDA-MB-453 cells relative to EGFR expression in A431 cells, determined by Western blot. Results are expressed as mean ± SE and are obtained from at least 4 independent replicates. Protein bands were normalized against β-actin.

Abbreviations: A431, human epidermoid cancer cells; EGFR, epidermal growth factor receptor; HK-2, human kidney cells; MDA-MB-453, human breast cancer cells; SE, standard error; THLE-2, human liver cells; TIME, human telomerase immortalized microvascular endothelial cells.

Figure 2 Transmission electron microscopy images demonstrating the internalization of AuNPs-PAA-Ctxb in A431 cells (A and B), MDA-MB-453 cells (C and D), HK-2 cells (E and F), THLE-2 cells (G and H) and TIME cells (I and J). The cells were exposed to 5.0 µg Au/mL of AuNPs-PAA-Ctxb for 3 h (left column) or 24 h (right column). Arrows indicate AuNPs-PAA-Ctxb. Images were acquired at different magnifications. Size bar =1 µm, N = Nucleus.

Abbreviations: AuNPs-PAA-Ctxb, polyallylamine-coated gold nanoparticles conjugated to Cetuximab; A431, human epidermoid cancer cells; HK-2, human kidney cells; MDA-MB-453, human breast cancer cells, THLE-2, human liver cells; TIME, human telomerase immortalized microvascular endothelial cells.

Figure S2 Energy dispersive X-ray spectroscopy analysis on a representative THLE-2 cell (A), a TIME cell (G) and a HK-2 cell (M, N), which were treated with 5 µg Au/mL of AuNPs-PAA-Ctxb for 3 h or 24 h. Gold mapping was performed on the THLE-2 cell (B) and the TIME cell (H). Bright signal zones (red squares) were analyzed resulting in specific X-ray spectra (C, I, O) and quantification of gold, carbon and oxygen (E, K, Q). No-signal zones (blue squares) were analyzed resulting in specific X-ray spectra (D, J, P) and the detection of only carbon and oxygen (F, L, R).

Figure 3 Interaction of AuNPs-PAA(±Ctxb) with A431 cells (A), MDA-MB-453 cells (B), HK-2 cells (C), THLE-2 cells (D) and TIME cells (E) quantified with ICP-MS. The cells were exposed to 5.0 µg Au/mL of AuNPs-PAA(±Ctxb) for 3 h, 6 h, 12 h or 24 h. Results are expressed as mean pg gold per cell ± SD calculated from 3 replicates per condition obtained from one experiment. A significant difference compared to 3 h of exposure was calculated by a Kruskal-Wallis test and a Dunn’s post-hoc test (*p<0.05).

Abbreviations: A431, human epidermoid cancer cells; Ctxb, Cetuximab; Au, gold; AuNPs-PAA, polyallylamine-coated gold nanoparticles; HK-2, human kidney cells; ICP-MS, inductively coupled plasma mass spectrometry; MDA-MB-453, human breast cancer cells; SD, standard deviation; THLE-2, human liver cells; TIME, human telomerase immortalized microvascular endothelial cells.

Table 3 EC₅₀ values after 24 h of cell exposure and LOEC after 3 h and 24 h of exposure to AuNPs-PAA and AuNPs-PAA-Ctxb.

Cell type	AuNPs-PAA			AuNPs-PAA-Ctxb
	LOEC (3 h)	LOEC (24 h)	EC50+95% CI (24 h)	LOEC (3 h)	LOEC (24 h)	EC50+95% CI (24 h)
A431	25.0	12.5	13.7 [12.8–14.6]	50.0	25.0	26.9 [24.8–29.1]
MDA-MB-453	12.5	6.25	8.31 [7.67–8.96]	50.0	6.25	12.3 [11.6–13.0]
HK-2	>50	6.25	9.65 [8.60–10.7]	>50	12.5	16.7 [15.3–18.2]
THLE-2	12.5	3.12	5.08 [4.36–5.79]	3.12	6.25	9.75 [9.04–10.5]
TIME	12.5	6.25	5.47 [5.18–5.75]	12.5	6.25	5.67 [5.08–6.26]

Note: Values were calculated from the MTS viability assays and expressed as µg Au/mL (Figure 4).

Abbreviations: A431, human epidermoid cancer cells; AuNPs, gold nanoparticles; CI, confidence interval; EC₅₀, Half maximal effect concentration; HK-2, human kidney cells; LOEC, Lowest observed effect concentration; MDA-MB-453, human breast cancer cells, THLE-2, human liver cells; TIME, human telomerase immortalized microvascular endothelial cells; Ctxb, cetuximab.

Figure 4 Cell viability after exposure to AuNPs-PAA (black curves) and AuNPs-PAA-Ctxb (gray curves) of A431 cells (A and B), MDA-MB-453 cells (C and D), HK-2 cells (E and F), THLE-2 cells (G and H) and TIME cells (I and J). Cells were exposed to increasing concentrations of AuNPs-PAA or AuNPs-PAA-Ctxb for 3 h or 24 h. The number of viable cells was assessed by MTS assay. The results are expressed as the mean percentage of viable cells relative to the unexposed cells ± SE and are obtained from at least three independent experiments with a minimum of three replicates per condition. A significantly reduced viability compared to the unexposed control was calculated by a one-way ANOVA and a Dunnett post-hoc test (*p<0.05, **p<0.01, ***p<0.001 ****p<0.0001).

Abbreviations: A431, human epidermoid cancer cells; ANOVA, analysis of variance; AuNPs-PAA, polyallylamine-coated gold nanoparticles; Ctxb, Cetuximab; HK-2, human kidney cells; MDA-MB-453, human breast cancer cells; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); SE, standard error; THLE-2, human liver cells; TIME, human telomerase immortalized microvascular endothelial cells.

Figure 5 Live cell imaging for caspase 3/7 activation (left lanes) and annexin V labeling (right lanes) in A431 cells (A and B, K and L), MDA-MB-453 cells (C and D, M and N), HK-2 cells (E and F, O and P), THLE-2 cells (G and H, Q and R) and TIME cells (I and J, S and T). The cell lines were exposed to increasing concentrations of AuNPs-PAA or AuNPs-PAA-Ctxb. Time-lapse pictures were taken every 2 h for 72 h. The results are expressed as the mean area of caspase 3/7 or annexin V per well ± SE and are obtained from at least three replicates. A significant increase in caspase 3/7 activity and annexin V labeling compared to unexposed control cells was calculated by a two-way ANOVA and a Dunnett post-hoc test. The start of significance is visualized by vertical bars (*p<0.05, **p<0.01, ***p<0.001 ****p<0.0001).

Abbreviations: A431, human epidermoid cancer cells; ANOVA, analysis of variance; AuNPs-PAA, polyallylamine-coated gold nanoparticles; Ctxb, Cetuximab; HK-2, human kidney cells; MDA-MB-453, human breast cancer cells; SE, standard error; THLE-2, human liver cells; TIME, human telomerase immortalized microvascular endothelial cells.

Figure 6 Mitochondrial membrane potential measurements of the HK-2 cells (A), THLE-2 cells (B) and TIME cells (C) after exposure to AuNPs-PAA or AuNPs-PAA-Ctxb. TIME cells were exposed to 3.0 µg Au/mL, THLE-2 and HK-2 cells were exposed to 5.0 µg Au/mL of AuNPs-PAA or AuNPs-PAA-Ctxb. During the 12 h exposure, 2 nM NAC was added to assess oxidative stress. The mitochondrial membrane potential was measured with TMRM fluorescence, detected by flow cytometry. The results are expressed as mean TMRM fluorescent signal relative to the unexposed control cells (red line) ± SE and are obtained from at least three independent experiments with a minimum of three replicates per condition. Significant difference in TMRM signal compared to the unexposed control was calculated by a one-way ANOVA and a Dunnett post-hoc test (bullets). Significant difference in TMRM fluorescent signal between AuNPs-PAA and AuNPs-PAA-Ctxb exposed cells, or between nanoparticle-exposed cells and NAC co-exposed cells, was calculated by a Student’s t-test (asterisks) (*p<0.05, **p<0.01, ***p<0.001 ****p<0.0001).

Abbreviations: ANOVA, analysis of variance; AuNPs-PAA, polyallylamine-coated gold nanoparticles; Ctxb, Cetuximab; HK-2, human kidney cells; NAC, N-acetyl L-cysteine; SE, standard error; THLE-2, human liver cells; TIME, human telomerase immortalized microvascular endothelial cells; TMRM, tetramethylrhodamine methyl ester.

Figure 7 Thioredoxin reductase and glutathione reductase activity in the HK-2 cells (A), THLE-2 cells (B) and TIME cells (C) after exposure to AuNPs-PAA or AuNPs-PAA-Ctxb. TIME cells were exposed to 3.0 µg Au/mL, THLE-2 and HK-2 cells were exposed to 5.0 µg Au/mL of AuNPs-PAA or AuNPs-PAA-Ctxb for 12 h. The enzyme activity rate per minute was measured by the increase in absorbance at 405 nm for GR and at 412 nm for TrxR. The results are expressed as mean enzyme activity rate per minute per 100 µg of protein ± SE and are obtained from at least three independent experiments with a minimum of two replicates per condition. Significant difference in enzyme activity rate compared to the unexposed control was calculated by a Student’s t-test (*p<0.05, **p<0.01, ***p<0.001 ****p<0.0001).

Abbreviations: AuNPs-PAA, polyallylamine-coated gold nanoparticles; Ctxb, Cetuximab; GR, Glutathione reductase; HK-2, human kidney cells; SE, standard error; THLE-2, human liver cells; TIME, human telomerase immortalized microvascular endothelial cells; TrxR, Thioredoxin reductase.

Figure 8 Live cell imaging for caspase 3/7 activation (left panels) and annexin V labeling (right panels) in A431 cells (A and B, K and L), MDA-MB-453 cells (C and D, M and N), HK-2 cells (E and F, O and P), THLE-2 cells (G and H, Q and R) and TIME cells (I and J, S and T). The cell lines were co-exposed to increasing concentrations of AuNPs-PAA or AuNPs-PAA-Ctxb and 2 nM of NAC. Time-lapse pictures were taken every 2 h for 72 h. The results are expressed as the mean area of caspase 3/7 or annexin V per well ± SE and are obtained from at least three replicates. A significant increase in caspase 3/7 activity and annexin V labeling compared to unexposed control cells was calculated by a two-way ANOVA and a Dunnett post-hoc test. The start of significance is visualized by vertical bars (*p<0.05, **p<0.01, ***p<0.001 ****p<0.0001).

Abbreviations: A431, human epidermoid cancer cells; ANOVA, analysis of variance; AuNPs-PAA, polyallylamine-coated gold nanoparticles; Ctxb, Cetuximab; HK-2, human kidney cells; MDA-MB-453, human breast cancer cells; SE, standard error; NAC, N-acetyl L-cysteine; THLE-2, human liver cells; TIME, human telomerase immortalized microvascular endothelial cells.

Figure 9 Correlation analyses showing the relation between the residual mitochondrial membrane potential and cell viability (A); the basal TrxR activity and cell viability (B); the extent of TrxR activity and cell viability (C); and the extent of TrxR inhibition and the residual mitochondrial potential (D). Data are presented as mean ± SD (for mitochondrial membrane potential and TrxR activity) or 95% CI (for EC₅₀). 1= HK-2 cells, 2= THLE-2 cells and 3= TIME cells.

Abbreviations: CI, confidence interval; HK-2, human kidney cells; SD, standard deviation; THLE-2, human liver cells; TIME, human telomerase immortalized microvascular endothelial cells; TrxR, thioredoxin reductase; AuNPs-PAA, polyallylamine-coated gold nanoparticles; Ctxb, cetuximab; EC₅₀, half maximal effect concentration.

Figure S1 UV-Vis spectrum of AuNPs-PAA and AuNPs-PAA-Ctxb (A). Gel electrophoresis of Ctxb, AuNPs-PAA + Ctxb and AuNPs-PAA-Ctxb (B). Size distribution curves (relative number) of AuNPs-PAA and AuNPs-PAA-Ctxb in Class 1 water, DMEM and DMEM +10% FBS obtained from CPS Disk Centrifugation (Benelux Scientific, Eke, Belgium) (C).

Abbreviations: AuNPs-PAA, polyallylamine-coated gold nanoparticles; Ctxb, Cetuximab; FBS, Fetal bovine serum; DMEM, Dulbecco’s Modified Eagle Medium.