Nanotoxicology: 2D analysis in a 3D matrix

Engineered nanoparticles are the fastest growing nanotechnology product, and of increasing environmental concern. Examples include antimicrobial silver nanoparticles, iron oxide particles used in bulk scale for environmental remediation, and micro- and nano-plastics. The latter are added to a broad range of cosmetic products. Larger plastic pieces pollute the environment by breaking down naturally to form smaller particles. These particles may attract and concentrate hydrophobic persistent organic pollutants on their surface, which might later be released once inside a biological matrix, as has been shown indirectly in e.g. birds [1]. All these particles are found in nature and humans are exposed to them daily (e.g. in beer [2]). People who regularly consume cultured bivalves may ingest up to 11.000 micro plastic particles per year by this route alone, even ignoring particles smaller than 5 µm [3]. Classically, nanoparticle uptake studies have been performed by acid digestion and chemical analysis [4]. But how do these particles interact with organisms and their tissue? Are they taken up in the tissue [5], or are they passively passing through the digestion system? Such questions are interesting in particular with regard to cytotoxic particles, such as silver, or very small particles that might enter the nucleus and interfere with DNA replication. Particles with the capability to carry toxic compounds, such as plastic, also have the potential for causing disruption of normal cellular function. In order to further our understanding of how such particles interact with tissue at the cellular level, nanoscale analysis is required, either to verify the nature of the particle (STEM EDX point analysis of single particles, Figure 1 a-c) or to trace substances released from their surface (NanoSIMS). However, before such analysis, a first challenge is to locate the particles in the affected cells. This has proven a major obstacle because of the relatively large size of the organism investigated. Indeed, screening a large volume of the organism under investigation to locate nanoparticles before EDX and NanoSIMS analysis is thus often a large part of the work. We work with model organisms such as microalgae, daphnia and earthworms that are likely to come into contact with, and accumulate nanoparticles. During the past few years we have employed various methods to localize different particles inside the organisms, e.g. serial block-face imaging, FIB-SEM imaging, freeze-dried cryo-sections and serial sections (Figure 1 d). Correlation between screening- and analysis techniques is key to locate, and be able to investigate the effects of nanoparticles in a biological matrix. At the moment we are exploring methods to screen large amounts of serial sections made by a custom-made knife, and methods for transferring interesting sections to carriers compatible with either STEM EDX or NanoSIMS. References [1] K. Tanaka et al., Marine Poll Bull 69 (1-2) (2013) 219. [2] G. Liebezeit and E. Liebezeit, Food Addit. Contam. Part a Chem. Anal. Control Exposure Risk Assess (2014). [3] L. van Cauwenberghe, and C. R. Janssen. Environ Pollut 193 (2014) 65. [4] L. M. Skjolding et al., Ecotoxicology 23 (2014) 1172. [5] P. Rosenkrantz et al., Environ Toxicol Chem 28 (2009) 2142. [6] We would like to thank Irina Kolotueva for invaluable technical advice. This research was supported by The Society of Electron Microscope Technology, and the European Research Council (Grant no. 281579). FIG. 1. (a-c) HAADF STEM images of D. magna gut epithelia exposed to 10 nm gold nanoparticles (Au NP) (0.4 mg Au/L) for 24h. Corresponding EDX spectra are superimposed. For clarity the C-peaks are capped and only up to 2.5 keV is depicted. (a) Au NP at microvilli, scale bar = 100 nm, (b) Au NP at base of microvilli, scale bar = 100 nm, (c) Os-rich particles in gut cell lipid droplet, scale bar = 50 nm, (d) Serial sections of L. variegatus exposed to iron particles on Si-wafer.