Label-free in vitro toxicity and uptake assessment of citrate stabilised gold nanoparticles in three cell lines

Abstract

BackgroundReliable in vitro toxicity testing is needed prior to the commencement of in vivo testing necessary for hazard identification and risk assessment of nanoparticles. In this study, the cytotoxicity and uptake of 14 nm and 20 nm citrate stabilised gold nanoparticles (AuNPs) in the bronchial epithelial cell line BEAS-2B, the Chinese hamster ovary cell line CHO, and the human embryonic kidney cell line HEK 293 were investigated.MethodsCytotoxicity of the AuNPs was assessed via traditional XTT-, LDH-, and ATP-based assays, followed by cell impedance studies. Dark-field imaging and hyperspectral imaging were used to confirm the uptake of AuNPs into the cells.ResultsInterference of the AuNPs with the XTT- and ATP-based assays was overcome through the use of cell impedance technology. AuNPs were shown to be relatively non-toxic using this methodology; nevertheless CHO cells were the most sensitive cell type with 20 nm AuNPs having the highest toxicity. Uptake of both 14 nm and 20 nm AuNPs was observed in all cell lines in a time- and cell type-dependent manner.ConclusionsUsing the cell impedance and dark-field hyperspectral imaging technologies, it was possible to study the toxicity of AuNPs in different cell lines and show that these cells could internalize AuNPs with their subsequent intracellular aggregation. It was also possible to show that this toxicity would not correlate with the level of uptake but it would correlate with cell-type and the size of the AuNPs. Therefore, these two label-free methodologies used in this study are suitable for in vitro studies on the effects of AuNPs, and could present themselves as appropriate and valuable methodologies for future nanoparticle toxicity and uptake studies.

Gold nanoparticles prepared by laser ablation in aqueous biocompatible solutions: assessment of safety and biological identity for nanomedicine applications

“…At this high concentration, a decrease may reflect interference between Au-NPs and dye reagents rather than a cell death induction. 51 In fact, the conflicting results observed in literature on Au-NP toxicity may be due to the lack of normalized protocols, emphasizing the need to establish standards in nanotoxicology. In addition, cell morphology, cell cytoskeleton, and reactive oxygen species levels were unmodified by Au-NP exposure, indicating that cell proliferation and motility were not altered.…”

Aneuploidogenic effects and DNA oxidation induced in vitro by differently sized gold nanoparticles

“…PBL were treated at the same conditions of adherent Raw264.7 cells. PBL were collected in sterile tubes and resuspended in 0.5 mL complete MEM; erythrocyte lysis buffer (155 mM NH 4 Cl, 10 mM KHCO 3 , 1 mM Na 2 EDTA, pH 7.4; 3.5 mL/tube) was added, and after 90 seconds cells were collected by centrifuging (3,000 rpm, 5 minutes). PBL were washed in 3.5 mL of complete medium and then pelleted (3,000 rpm, 5 minutes).…”

“…3 However, the toxicity of Au NPs, as well as of others such as silver NPs, might not correlate with the level of uptake but cell-type and the size can play a role. 4,5 In fact, 20 nm citrated Au NPs resulted as more toxic than 14 nm ones in ovarian hamster's cells but not in bronchial and kidney derived cell cultures. 4 However, even if it is recognized that the cell type plays a role in the uptake of Au NPs, the size is not involved: differently sized Au NPs displayed a similar uptake in epithelial (A549 and NCIH441) and in endothelial (HDMEC and hCMEC) cells, but endothelial cells were shown to internalize a higher amount of Au NPs than epithelial ones.…”

“…4,5 In fact, 20 nm citrated Au NPs resulted as more toxic than 14 nm ones in ovarian hamster's cells but not in bronchial and kidney derived cell cultures. 4 However, even if it is recognized that the cell type plays a role in the uptake of Au NPs, the size is not involved: differently sized Au NPs displayed a similar uptake in epithelial (A549 and NCIH441) and in endothelial (HDMEC and hCMEC) cells, but endothelial cells were shown to internalize a higher amount of Au NPs than epithelial ones. 6,7 Despite these intravenously, the adsorption of blood proteins onto their surface is one of the most relevant and studied parameters.…”

Spectroscopic Insights into the Nano-Bio Interface

“…Hyperspectral imaging presents an alternative to traditional electron microscopy methods for the identiication of nanoparticles and protein corona inside the cell [27][28][29][30]. The exhaustive sample preparation needed for electron microscopy, such as encapsulation in a polymer followed by microtoming, often leads to artifacts.…”