Nanoparticle-assay marker interaction: effects on nanotoxicity assessment
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Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein–particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges.
KeywordsNanomaterial LDH assay TNF- α Toxicology Cytotoxicity Environmental and health effects
The authors would also like to acknowledge the financial support from the Agency for Science, Technology and Research (A*STAR) (Project No: 102 129 0098), the Singapore Centre on Environmental Life Sciences Engineering (SCELSE) (M4330001.C70.703012), the NTU-National Healthcare Group (NTU-NHG) grant (ARG/14012), and the School of Materials Science and Engineering (M020070110), Nanyang Technological University, Singapore.
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