Elsevier

Toxicology Letters

Volume 217, Issue 3, 13 March 2013, Pages 205-216
Toxicology Letters

Size-dependent toxicity and cell interaction mechanisms of gold nanoparticles on mouse fibroblasts

https://doi.org/10.1016/j.toxlet.2012.11.022Get rights and content

Abstract

Gold nanoparticles (AuNPs) are currently used in several fields including biomedical applications, although no conclusive information on their cytotoxicity is available. For this reason this work has investigated the effects of AuNPs in vitro on Balb/3T3 mouse fibroblasts. Results obtained exposing cells for 72 h to AuNPs 5 and 15 nm citrate stabilized, revealed cytotoxic effects only for AuNPs 5 nm at concentration  50 μM if measured by colony forming efficiency (CFE). To understand the differences in cytotoxicity observed for the two AuNPs sizes, we investigated the uptake and the intracellular distribution of the nanoparticles. By TEM it was observed that 5 and 15 nm AuNPs are internalized by Balb/3T3 cells and located within intracellular endosomal compartments. Quantification of the uptake by ICP-MS showed that AuNPs internalization enhanced even up to 72 h. Disruption of the actin cytoskeleton was evident, with cell footprints narrow and contracted; effects more remarkable in cells exposed to 5 nm AuNP. The mechanism of NPs cell internalization was investigated using immunocytochemistry and western blot. No significant effect was observed in the expression level of caveolin, while reduction of the expression and degradation of the clathrin heavy chain was observed in cells exposed for 72 h to AuNPs.

Highlights

► AuNPs remain stable in culture medium. ► We showed size-dependent cytotoxic effect of AuNPs. ► We observed autophagosomes after cell exposure to AuNPs 5 nm. ► Clathrin expression is reduced and protein cleavage was observed. ► A time-dependent increasing Au uptake is observed.

Introduction

The last decade has seen an important growth in the production of nanoscale materials as a result of their attractiveness for a large range of applications, for instance in biomedicine (Barreto et al., 2011), biosensing (Rivas et al., 2007, Zhao et al., 2011), microelectronics (Seker and Demir, 2011), material engineering (Peralta-Videa et al., 2011), energy production (Saunders, 2012, Valdés et al., 2012) or environment remediation (Bootharaju and Pradeep, 2012, Ojea-Jiménez et al., 2012). Among the many types of nanomaterials, gold nanoparticles (AuNPs) have attracted particular scientific and technological interest due to their unique optical properties, chemical stability, easy synthesis and functionalization, all of which make AuNPs attractive candidates for use in biomedicine including chemical sensing, biological imaging, drug delivery and cancer treatment. However, knowledge about AuNPs and health impact is essential before these nanomaterials can be used in real clinical settings (Alkilany and Murphy, 2010). An increasing number of scientific reports have been published addressing this issue, with the goal of understanding the interactions between nanoparticles (NPs) and cells as function of their size, shape, and surface chemistry (Lewinski et al., 2008, Zhao et al., 2011). Although AuNPs are considered inert particles and regarded as biocompatible, there are contradictory results concerning their toxicity (Sperling et al., 2008). Recently, AuNPs have been inserted in the list of nanomaterials investigated in the “Sponsorship Program” (SG3) of the Organization for Economic Co-operation and Development (OECD) working Party for Manufactured Nanomaterials (WPMN).

The goal of this work is to provide additional data on the toxic potential exerted by AuNPs of different sizes. We investigated the effects of AuNPs 5 and 15 nm on Balb/3T3 mouse fibroblast cell line, an in vitro model previously used to assess concurrent cytotoxicity and morphological transformation of nanomaterials (Ponti et al., 2012, Ponti et al., 2010, Uboldi et al., 2012). In addition in order to unveil differences among the two different AuNPs size, the uptake mechanisms of AuNPs in Balb/3T3 and the modification of the cellular cytoskeleton has been investigated. Moreover, changes in the expression of clathrin and caveolin due to their possible role in AuNPs uptake were evaluated.

Section snippets

Gold nanoparticles synthesis and characterization

AuNPs of 5 nm size were produced by NaBH4 (Sigma–Aldrich, Italy) reduction of a starting solution of 2.5 mM sodium citrate (Sigma–Aldrich, Italy) and 0.5 mM HAuCl4 (Sigma–Aldrich, Italy) in water. Typically, 100 mL of the starting solution was cooled in ice bath to 4 °C before the reduction was initiated by the addition of 1 mL of freshly prepared ice-cold NaBH4 solution (0.1 M) under rapid stirring. AuNPs formation was observed to occur immediately as shown by the rapid color change from pale yellow

Characterization of AuNPs

In the present work, 5 and 15 nm diameter AuNPs were used. AuNPs were in situ synthesized and characterized as previously described (Section 2.1). UV–vis spectrophotometry of AuNPs showed different absorption peaks at wavelengths 508 and 520 nm (Fig. 1), consistent with their different diameters and with previously reported results (Oh et al., 2011). The size of the NPs was verified by three complementary techniques: electron microscopy, DLS, and CPS. Fig. 2 shows scanning transmission electron

Discussion

Citrate stabilized AuNPs 5 and 15 nm were synthesized by wet chemical synthesis based on the reduction of tetrachloroauric acid. A specific characterization is needed to correlate the biological effects induced by NPs (Bouwmeester et al., 2011); therefore one aim of the study was the evaluation of AuNPs morphology, purity, behavior in culture medium, and surface charge. By three complementary size characterization techniques, we confirmed the presence of a single peak in the size distribution,

Conclusions

We propose CFE assay on Balb/3T3 cells as a valuable test to screen NPs toxicity due to the reliability and reproducibility of the assay and of the sensitivity of the cell model used. Our results revealed cytotoxicity only in cells treated for 72 h with AuNPs 5 nm at concentration higher than 50 μM, while no cytotoxic effects was found in cells exposed to AuNPs 15 nm. We highlighted that cytotoxicity results can be better understood, thus more correctly interpreted when combined with cell

Conflict of interest statement

All authors declare they have no conflict of interests.

The authors alone are responsible for content and writing of the paper.

Acknowledgments

The authors would like to thank Dr. Annarita Stringaro and Dr. Agnese Molinari for the access and use of the TEM instrument at the Istituto Superiore di Sanita’ (ISS) in Rome, we would like to thank Fabio Villa from Zeiss for his assistance. We acknowledge Dr. Edyta Bajak, Dr. Chiara Uboldi and Dr. Hubert Rauscher for helpful discussions. The CPS analyses were carried out by Robin Capomaccio and Claudia Cascio.

References (41)

  • A.M. Alkilany et al.

    Toxicity and cellular uptake of gold nanoparticles: what we have learned so far?

    Journal of Nanoparticle Research

    (2010)
  • Arnida et al.

    Cellular uptake and toxicity of gold nanoparticles in prostate cancer cells: a comparative study of rods and spheres

    Journal of Applied Toxicology

    (2010)
  • J.A. Barreto et al.

    Nanomaterials: applications in cancer imaging and therapy

    Advanced Materials

    (2011)
  • M.S. Bootharaju et al.

    Understanding the degradation pathway of the pesticide, chlorpyrifos by noble metal nanoparticles

    Langmuir

    (2012)
  • H. Bouwmeester et al.

    Characterization of translocation of silver nanoparticles and effects on whole-genoma gene expression using an in vitro intestinal epithelium coculture model

    ACS Nano

    (2011)
  • C. Brandenberger et al.

    Quantitative evaluation of cellular uptake and trafficking of plain and polyethylene glycol-coated gold nanoparticles

    Small

    (2010)
  • B.D. Chithrani et al.

    Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells

    Nano Letters

    (2006)
  • E.C. Cho et al.

    The effect of sedimentation and diffusion on cellular uptake of gold nanoparticles

    Nature Nanotechnology

    (2011)
  • E.E. Connor et al.

    Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity

    Small

    (2005)
  • C. Freese et al.

    Uptake and cytotoxicity of citrate-coated gold nanospheres: comparative studies on human endothelial and epithelial cells

    Particle and Fibre Toxicology

    (2012)
  • Cited by (0)

    1

    These authors contributed equally.

    View full text