An effective method for distinguishing extracellular and intracellular nanoparticles through chemical extractions

Nanoparticles (NPs) can adsorb onto cell surfaces (i.e., extracellular NPs) and be internalized by cells (i.e., intracellular NPs), leading to their accumulation and potential toxicity. Therefore, distinguishing adsorbed and internalized NPs is crucial in studying their accumulation and toxicity. In this study, various washing agents (EDTA, sodium citrate, cysteine, and H⁺) were compared to differentiate internalized NPs from total accumulation in the protozoan Tetrahymena thermophila, by evaluating efficacies of extracting adsorbed NPs from cell surfaces. Key factors influencing the extraction procedures, including the type and concentration of washing agent, contact time, washing cycles, and effects of agents on the organism, were systematically optimized. Consequently, we identified an effective washing agent (i.e., a mixture of 3 mM EDTA, 10 mM sodium citrate, and 10 mM cysteine in Dryl's medium at pH 7) that efficiently extracted adsorbed metal NPs (Fe₂O₃-NPs, TiO₂-NPs, SiO₂-NPs, Ag-NPs, and Au-NPs modified by -COOH and -NH₂) without causing growth inhibition or cell lysis. Further, a washing procedure was proposed, involving the extraction of samples with the mixture twice for 5 min each. Our study represents the first systematic optimization of a washing protocol for extracting adsorbed NPs across diverse NP types. The developed methodology demonstrates broad applicability, minimal impact on cellular function, and enhanced extraction efficiency compared to existing methods. It will facilitate further investigation into the underlying mechanisms of NP bioaccumulation (including uptake and efflux) and associated toxicity in protozoa, providing critical insights for environmental safety assessments and advancing nanotoxicology research.