Size-dependent cytotoxicity of silver nanoparticles in human lung cells: the role of cellular uptake, agglomeration and Ag release

Abstract

BackgroundSilver nanoparticles (AgNPs) are currently one of the most manufactured nanomaterials. A wide range of toxicity studies have been performed on various AgNPs, but these studies report a high variation in toxicity and often lack proper particle characterization. The aim of this study was to investigate size- and coating-dependent toxicity of thoroughly characterized AgNPs following exposure of human lung cells and to explore the mechanisms of toxicity.MethodsBEAS-2B cells were exposed to citrate coated AgNPs of different primary particle sizes (10, 40 and 75 nm) as well as to 10 nm PVP coated and 50 nm uncoated AgNPs. The particle agglomeration in cell medium was investigated by photon cross correlation spectroscopy (PCCS); cell viability by LDH and Alamar Blue assay; ROS induction by DCFH-DA assay; genotoxicity by alkaline comet assay and γH2AX foci formation; uptake and intracellular localization by transmission electron microscopy (TEM); and cellular dose as well as Ag release by atomic absorption spectroscopy (AAS).ResultsThe results showed cytotoxicity only of the 10 nm particles independent of surface coating. In contrast, all AgNPs tested caused an increase in overall DNA damage after 24 h assessed by the comet assay, suggesting independent mechanisms for cytotoxicity and DNA damage. However, there was no γH2AX foci formation and no increased production of intracellular reactive oxygen species (ROS). The reasons for the higher toxicity of the 10 nm particles were explored by investigating particle agglomeration in cell medium, cellular uptake, intracellular localization and Ag release. Despite different agglomeration patterns, there was no evident difference in the uptake or intracellular localization of the citrate and PVP coated AgNPs. However, the 10 nm particles released significantly more Ag compared with all other AgNPs (approx. 24 wt% vs. 4–7 wt%) following 24 h in cell medium. The released fraction in cell medium did not induce any cytotoxicity, thus implying that intracellular Ag release was responsible for the toxicity.ConclusionsThis study shows that small AgNPs (10 nm) are cytotoxic for human lung cells and that the toxicity observed is associated with the rate of intracellular Ag release, a ‘Trojan horse’ effect.

Different responses of Caco-2 and MCF-7 cells to silver nanoparticles are based on highly similar mechanisms of action

“…Also intracellular degradation of AgNPs was demonstrated, supporting the previously proposed ''Trojan horse'' mechanism of dissolving AgNPs inside the cell (Bouwmeester et al, 2011;Gliga et al, 2014). It proved difficult to detect the Ag-20 NPs with CM or TEM, due to their small size and degradation.…”

“…However, the kinetics behind the biological activity have not fully been elucidated: i.e. Ag + uptake, or a ''Trojan horse'' mechanism in which AgNPs shed Ag + in lysosomes following cellular uptake (Bouwmeester et al, 2011;Gliga et al, 2014).…”

Capillary electrophoresis coupled with inductively coupled mass spectrometry as an alternative to cloud point extraction based methods for rapid quantification of silver ions and surface coated silver nanoparticles

“…Human exposure to Ag is growing as the availability of Ag NPs containing products continuously expands. Recent studies have suggested that both Ag NPs and Ag ions can induce cytotoxicity through different mechanism [2][3][4]. Additionally, numerous studies have suggested that Ag NPs are prone to oxidation and can release Ag ions to the surrounding environment, and that reverse formation to NPs from Ag ions is also a possibility [5][6][7][8].…”

Silver nanoparticle toxicity is related to coating materials and disruption of sodium concentration regulation

“…Cit-AgNPs had high polydispersity index despite having a relatively average stable aggregation size (Table 1), possibly indicative of a small number of large aggregates in the test suspension. Size dependent toxicity of AgNPs has been reported in vitro (e.g., Gilga et al 2014;Park et al 2011) and some in vivo tests (e.g., Asghari et al 2014;Gorth et al 2011). …”

“…These studies compared AgNPs with large difference in diameters (e.g., 20-30nm, 100nm and >500nm in Gorth et al 2014; 10nm, 40nm and 75nm in Gilga et al 2014). However, when comparing particles with the smaller difference in diameters (20nm, 40nm, 60nm, 80nm and 100nm), size dependent toxicity was not observed (Ivask et al 2014).…”