Elsevier

Biomaterials

Volume 31, Issue 23, August 2010, Pages 5996-6003
Biomaterials

Autophagy and oxidative stress associated with gold nanoparticles

https://doi.org/10.1016/j.biomaterials.2010.04.014Get rights and content

Abstract

Elemental metal nanoparticles like cadmium and silver are known to cause oxidative stress and are also highly toxic. Yet for gold nanoparticles (AuNPs), it is not well established whether these particles are biologically toxic. Here we show that AuNPs, which were taken up by MRC-5 human lung fibroblasts in vitro, induce autophagy concomitant with oxidative stress. We also observed formation of autophagosomes together with the uptake of AuNPs in the lung fibroblasts as well as upregulation of autophagy proteins, microtubule-associated protein 1 light chain 3 (MAP-LC3) and autophagy gene 7 (ATG 7) in treated samples. AuNP treated cells also generated significantly more lipid hydroperoxides (p-value < 0.05), a positive indication of lipid peroxidation. Verification with western blot analysis for malondialdehyde (MDA) protein adducts confirmed the presence of oxidative damage. In addition, AuNP treatment also induced upregulation of antioxidants, stress response genes and protein expression. Exposure to AuNPs is a potential source of oxidative stress in human lung fibroblasts and autophagy may be a cellular defence mechanism against oxidative stress toxicity.

Introduction

Nanotoxicity is an emerging field of research, a response to growing uses of nanosized materials in a slew of technological applications and consumer products [1]. Early research into toxic effects of ultrafine carbon particles and carbon nanotubes highlighted the potential health risks from exposure of these particles in our environment [2]. These particulates are potentially hazardous when it comes in contact with the human body, especially the respiratory system being the most vulnerable route of entry. In vivo studies in rats exposed to aerosols of gold nanoparticles (AuNPs) revealed that the nanoparticles were rapidly taken into the system with the highest accumulation in the lungs, aorta, oesaphagus and olfactory bulb [3]. Moreover, particles of nano-dimension are believed to be more biologically reactive than their bulk counterparts due to their small size and larger surface area to volume ratio [1].

Gold in its bulk form has long been considered an inert, noble metal with some therapeutic and even medicinal value, hence gold nanoparticles (AuNPs) are thought also to be relatively non-cytotoxic [4]. Yet there are differing reports of the extent of the toxic nature of these particles owing to the different modifications of the AuNP, surface functional attachments and the shape and diameter size of the nanospheres [5], [6]. Moreover, the metallic nature of the metal derived NPs and the presence of transition metals encourages the production of reactive oxygen species (ROS) leading to oxidative stress [7], [8], [9]. Elemental metal NPs like cadmium and silver are known to induce oxidative stress and apoptosis in various cell types [10], [11]. In spite of this, the link between AuNP and oxidative stress is not well established. Most often, the harmful effects of ROS may be manifested through damage of DNA, oxidations of polyunsaturated fatty acids in lipids and oxidations of amino acids in proteins [12]. Previously, we have shown that DNA damage occurs in AuNP treated lung fibroblast cells [13]. In this study, we evaluated the presence of oxidative damage in lipids and proteins of AuNP treated lung fibroblast cells, as well as gene profiling of oxidative stress markers and found evidence of autophagosome formation.

Section snippets

Cell culture

MRC-5 human fetal lung fibroblast cells (ATCC No.: CCL-171) were cultured in RPMI media supplemented with 10% fetal bovine serum (FBS) in 100 μg/ml penicillin/streptomycin in 37 °C 5% CO2 incubator.

AuNP synthesis and preparation

Gold nanoparticles (AuNPs), 20 nm in diameter, were prepared in citrate reduction of gold salts as previously described [13]. The citrate buffer was removed and the nanoparticles were coated with fetal bovine serum, washed and reconstituted in phosphate buffer saline (PBS) solution. The size and zeta

Internalization of AuNPs in cells

After 72 h of AuNP treatment, particles may be so highly aggregated in the cell cytoplasm that they appear as bright blue spots under light microscopy (Fig. 1A and B). Additionally, we noticed that AuNP clusters accumulated in endosomes and lysozomes in the cytoplasm (Fig. 1C and D), which is not surprising as these are the eventual endpoints of ingested materials marked for degradation [16]. The AuNP treated fibroblasts appeared to be highly active with many of them exhibiting large numbers of

Conclusion

We surmise that the effect of the treatment of AuNP induces oxidative damage in lung fibroblast cells, pulling together a myriad of antioxidants and stress response proteins in a defence pathway. While the presence of AuNP could create an oxidative environment, it also affects the regulation of cellular stress response mechanisms and at the same time induces the formation of autophagosomes, possibly to protect the cell from succumbing to oxidative stress.

Acknowledgement

This work was supported by research funding from the Singapore Ministry of Education Academic Research Fund Tier 1 via grant R279-000-205-112 and Tier 2 via grant MOE2008-T2-1-046, and the National University of Singapore (NUS) Environmental Research Institute (NERI) via grant R706-000-002-646.

References (37)

  • G. Oberdorster et al.

    Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles

    Environ Health Perspect

    (2005)
  • L.E. Yu et al.

    Translocation and effects of gold nanoparticles after inhalation exposure in rats

    Nanotoxicology

    (2007)
  • E.E. Connor et al.

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

    Small

    (2005)
  • H. Takahashi et al.

    Modification of gold nanorods using phosphatidylcholine to reduce cytotoxicity

    Langmuir

    (2006)
  • Y. Pan et al.

    Size-dependent cytotoxicity of gold nanoparticles

    Small

    (2007)
  • W. MacNee et al.

    Mechanism of lung injury caused by PM10 and ultrafine particles with special reference to COPD

    Eur Respir J

    (2003)
  • H.Y. Jia et al.

    Potential oxidative stress of gold nanoparticles by induced-NO releasing in serum

    J Am Chem Soc

    (2009)
  • S. Durocher et al.

    Disulfide-linked, gold nanoparticle based reagent for detecting small molecular weight thiols

    J Am Chem Soc

    (2009)
  • Cited by (450)

    View all citing articles on Scopus
    View full text