Gold nanoparticles affect the antioxidant status in selected normal human cells

Abstract

Purpose:

This study evaluates the cytotoxicity of AuNPs coated with polyallylamine (AuNPs-PAA) and conjugated or not to the epidermal growth factor receptor (EGFR)-targeting antibody Cetuximab (AuNPs-PAA-Ctxb) in normal human kidney (HK-2), liver (THLE-2) and microvascular endothelial (TIME) cells, and compares it with two cancer cell lines that are EGFR-overexpressing (A431) or EGFR-negative (MDA-MB-453).

Results:

Conjugation of Cetuximab to AuNPs-PAA increased the AuNPs-PAA-Ctxb interactions with cells, but reduced their cytotoxicity. TIME cells exhibited the strongest reduction in viability after exposure to AuNPs-PAA(±Ctxb), followed by THLE-2, MDA-MB-453, HK-2 and A431 cells. This cell type-dependent sensitivity was strongly correlated to the inhibition of thioredoxin reductase (TrxR) and glutathione reductase (GR), and to the depolarization of the mitochondrial membrane potential. Both are suggested to initiate apoptosis, which was indeed detected in a concentration- and time-dependent manner. The role of oxidative stress in AuNPs-PAA(±Ctxb)-induced cytotoxicity was demonstrated by co-incubation of the cells with N-acetyl L-cysteine (NAC), which significantly decreased apoptosis and mitochondrial membrane depolarization.

Conclusion:

This study helps to identify the cells and tissues that could be sensitive to AuNPs and deepens the understanding of the risks associated with the use of AuNPs in vivo.

Keywords:

• cytotoxicity
• EGFR
• Cetuximab
• oxidative stress

Acknowledgments

We would like to thank Ornella Fichera (University of Namur [UNamur]) for the production of the AuNPs-PAA and size distribution analyses; Corry Charlier and Caroline De Bona (UNamur) for processing the TEM samples and the support during TEM imaging; Ziekenhuis Sint-Dimpna and CHR de Namur for the delivery of Cetuximab; Sandra Bouchy (UNamur) for her guidance on the MTS experiments and TEM sample preparation; Diane Van Houtven (Flemish Institute For Technological Research) for her guidance on the zeta potential measurements; Prisca Verheyen, Karolien Van Rompaey and Luc Gelens for the ICP-MS measurements; Andrew Dobney for English proofreading of this paper. We are thankful to the technological platforms SIAM and Morph-Im (UNamur).

Disclosure

Noami Daems is the recipient of a FRIA grant from the Fonds de la Recherche Scientific (F.R.S-FNRS). Sébastien Penninckx is funded by the F.R.S-FNRS. The authors report no other conflicts of interest in this work.

Supplementary materials

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.

$\mathrm{y}=\frac{\mathrm{k}}{1+{\left(\frac{\mathrm{x}}{{\mathrm{x}}_{50}}\right)}^{\mathrm{s}}}$

Equation S1 Log-logistic function for curve fitting of the MTS viability data.

$\mathrm{y}=\frac{\mathrm{k}\left(1+\mathrm{g}\mathrm{x}\right)}{1+\left(1+2\mathrm{g}{\mathrm{x}}_{50}\right){\left(\frac{\mathrm{x}}{{\mathrm{x}}_{50}}\right)}^{\mathrm{s}}}$

Equation S2 Modified log-logistic function for curve fitting of the MTS viability data.

In both equations, y represents the experimental cell viability, x is the gold concentration and ^Sdefines the slope parameter. Moreover, k is the response at x=0 and x₅₀ is the gold concentration at which the cell viability is reduced by 50% compared to control response. Finally, in the second equation, g represents the hormesis parameter, modelling a potential stimulatory effect of low gold concentrations.