M. Johnson, S. Hanna, A. Montoro Bustos, N. Sharp, J. Bennett, A. Kolmakov, C. Sims, R. Holbrook, K. Scott, K. Murphy, E. Petersen, L. Yu, B. Nelson
National Institute of Standards & Technology,
Keywords: C. elegans, gold nanoparticles, size-dependent uptake, imaging techniques
Summary:The prevalent use and incorporation of engineered nanomaterials and nanoparticles (ENPs) in consumer products increases the likelihood of their interaction with environmental and biological systems, and thus has fostered research efforts to understand potential risks and implications. Caenorhabditis elegans (C. elegans) has become an ideal model organism for nanotoxicity studies mainly due to their short life span, ease of culturing in a laboratory, transparency for visualization of changes in morphology. Several research efforts have been focused toward the uptake and effects of gold nanoparticles (AuNPs) on C. elegans. Here we present the results of the size dependent uptake of gold nanoparticles by C. elegans. Nematodes were exposed to 80 nm, 100 nm, and 150 nm citrate-stabilized AuNPs for 24 h at both equal Au mass fraction and equal particle number conditions. Common washing procedures have been previously determined as insufficient in removing excess suspended AuNPs after exposure, therefore, a sucrose density gradient centrifugation protocol1 was employed to separate the nematodes from AuNPs freely suspended in the exposure media. Imaging analysis of nematode samples was conducted using a variety of techniques including scanning electron microscopy combined with energy dispersive X-ray microscopy (SEM/EDS), secondary ion mass spectrometry (SIMS), and hyperspectral imaging to assess the efficacy of the C. elegans/AuNP separation, as well as to visualize any observable uptake. Quantification of total Au uptake in dry non-exposed and Au exposed C. elegans was determined following acid-assisted microwave digestions by conventional inductively coupled plasma mass spectrometry (ICP-MS) analyses. Alternatively, alkaline digestions of biological tissue were utilized for particle sizing in single particle (sp) ICP-MS analyses. SEM/EDS analysis revealed the presence of AuNPs within the intestinal track of AuNP-exposed nematodes. SIMS analysis provided visual confirmation of uptake. Nanoparticulate body burdens were measured for each AuNP uptake condition, including the number of particles ingested per nematode. Finally, size distributions and particle number concentrations were determined for all AuNPs dispersed in water and AuNPs taken up by C. elegans. spICP-MS analysis showed a significant broadening of the particle size distribution of NPs extracted from all AuNP-exposed nematodes which is indicative of transformation after ingestion. Our elemental mass spectrometry results suggest a size dependent AuNP uptake. The analysis of the liberated AuNPs in C. elegans revealed an alteration in the size distribution of NPs, which may be a result of the NPs being exposed to physiological processes (i.e. gut compaction or destruction of the particles during the digestion process) within the nematode.