Size-dependent toxicity and cell interaction mechanisms of gold nanoparticles on mouse fibroblasts
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.
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These authors contributed equally.