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

Nonhlanhla Tlotleng,Mary Gulumian,Melissa A Vetten,Robert Tshikhudo,Frankline K Keter,Charlene Andraos,Kailen Boodhia,Delia Tanner Rascher,Amanda Skepu,Leigh-Anne Koekemoer

doi:10.1186/1743-8977-10-50

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.

Highlights

Reliable in vitro toxicity testing is needed prior to the commencement of in vivo testing necessary for hazard identification and risk assessment of nanoparticles
We demonstrate the use of two label-free technologies to investigate the cytotoxicity and uptake of 14 nm and 20 nm citrate stabilized Gold nanoparticle (AuNP) in three cell lines
A correlation was found in the toxicity of drugs when assessed by either neutral red uptake assay or the Real-TimeCell Analyzer (RTCA) [29], or in the cytotoxicity of sodium arsenite, cadmium chloride and cis-platinum assessed by MTT and RTCA [33], or in the immunocytotoxicity of baboon sera on pig cells with the MTT and RTCA [40] or cell toxicity assay using the Sulforhodamine B for staining proteins and measuring absorbance at a wavelength of 565 nm in correlation to RTCA [41]. Such correlation was even observed between impedance-based technology and the MTS assay for the assessment of CTAB-capped gold nanoparticles of difference shapes [36,37]. The difference between those reported in the literature and those observed in the present experiments is that we have found that the materials tested have shown absorbance or luminescence (Figure 3) and have interfered in the assessment of the final products produced from these test systems and subsequently no such correlation could be observed between our data generated by the conventional assay systems and that of the RTCA

Summary

Introduction

Reliable in vitro toxicity testing is needed prior to the commencement of in vivo testing necessary for hazard identification and risk assessment of nanoparticles. Further cytotoxicity studies with the MTT assay have shown no effect on the viability of the cells exposed to citrate stabilized AuNPs of sizes 3 nm, 5 nm, 12 nm, 17 nm, 37 nm, 50 nm, and 100 nm in HeLa cells [4], no effect of 20 nm AuNPs on the viability of human dermal fibroblasts-fetal cells [5] and no effect of 9.5 nm, 11.19 nm, and 25 nm citrate stabilised AuNPs on A549 and NCIH441 cells with the exception of the 11.19 nm and 25 nm at the highest concentration in A549 cells which displayed mild toxicity [6] In this latter study, a dose- and time-dependant increase of LDH release was observed [6]. The 17 nm citrate capped AuNPs had shown toxicity to A549 cells assessed by both MTT and LDH assays as well as by the ATP depletion measurements [9]

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Discussion

Conclusion