Beyond Unpredictability: The Importance of Reproducibility in Understanding the Protein Corona of Nanoparticles.

While it is well established that the surface of a nanoparticle plays a pivotal role for the protein corona, the vast number of proteins present in biological media render general conclusions about affinities between nanoparticle surfaces and proteins nontrivial. Recently published articles increasingly reveal differences between systems and an ever increasing number of influencing factors for the protein corona. In contrast, the present study posits that the reported differences may, at least in part, be due to poor experimental design, which leads to biased results. The present study investigates protein adsorption onto silica nanoparticles with different chemical groups on the surface by the statistical analysis of triplicate measurements as well as control measurements. We demonstrate that 60% of the identified protein types did not have any significant affinities for the nanoparticles. Of the remaining 40%, 60% were driven by surface charges and only 40% preferentially adsorbed onto specific surface groups. Furthermore, we found that of the 20 most abundant proteins in the serum, only five bound to the nanoparticles studied here. We illustrate the importance of control replicate experiments to avoid exaggerated differences between systems and to properly quantify the differences and similarities between comparable systems.

Biological effects of four iron-containing nanoremediation materials on the green alga Chlamydomonas sp.

As nanoremediation strategies for in-situ groundwater treatment extend beyond nanoiron-based applications to adsorption and oxidation, ecotoxicological evaluations of newly developed materials are required. The biological effects of four new materials with different iron (Fe) speciations ([i] FerMEG12 - pristine flake-like milled Fe(0) nanoparticles (nZVI), [ii] Carbo-Iron® - Fe(0)-nanoclusters containing activated carbon (AC) composite, [iii] Trap-Ox® Fe-BEA35 (Fe-zeolite) - Fe-doped zeolite, and [iv] Nano-Goethite - 'pure' FeOOH) were studied using the unicellular green alga Chlamydomonas sp. as a model test system. Algal growth rate, chlorophyll fluorescence, efficiency of photosystem II, membrane integrity and reactive oxygen species (ROS) generation were assessed following exposure to 10, 50 and 500 mg L-1 of the particles for 2 h and 24 h. The particles had a concentration-, material- and time-dependent effect on Chlamydomonas sp., with increased algal growth rate after 24 h. Conversely, significant intracellular ROS levels were detected after 2 h, with much lower levels after 24 h. All Fe-nanomaterials displayed similar Z-average sizes and zeta-potentials at 2 h and 24 h. Effects on Chlamydomonas sp. decreased in the order FerMEG12 > Carbo-Iron® > Fe-zeolite > Nano-Goethite. Ecotoxicological studies were challenged due to some particle properties, i.e. dark colour, effect of constituents and a tendency to agglomerate, especially at high concentrations. All particles exhibited potential to induce significant toxicity at high concentrations (500 mg L-1), though such concentrations would rapidly decrease to mg or µg L-1 in aquatic environments, levels harmless to Chlamydomonas sp. The presented findings contribute to the practical usage of particle-based nanoremediation in environmental restoration.

Non-invasive continuous monitoring of pro-oxidant effects of engineered nanoparticles on aquatic microorganisms.

Engineered nanomaterials (ENMs) are key drivers for the development of highly sophisticated new technologies. As all new attainments, the rapidly increasing used of ENMs raise concerns about their safety for the environment and humans. There is growing evidence showing that if engineered nanomaterials are released into the environment, there is a possibility that they could cause harm to aquatic microorganisms. Among the divers effects triggering their toxicity the ability of ENMs to generate reactive oxygen species (ROS) capable of oxidizing biomolecules is currently considered a central mechanism of toxicity. Therefore, development of sensitive tools for quantification of the ROS generation and oxidative stress are highly sought. After briefly introducing ENMs-induced ROS generation and oxidative stress in the aquatic microorganisms (AMOs), this overview paper focuses on a new optical biosensor allowing sensitive and dynamic measurements of H2O2 in real-time using multiscattering enhanced absorption spectroscopy. Its principle is based on sensitive absorption measurements of the heme protein cytochrome c whose absorption spectrum alters with the oxidation state of constituent ferrous FeII and ferric FeIII. For biological applications cytochrome c was embedded in porous random media resulting in an extended optical path length through multiple scattering of light, which lowers the limit of detection to a few nM of H2O2. The sensor was also integrated in a microfluidic system containing micro-valves and sieves enabling more complex experimental conditions. To demonstrate its performance, abiotic absorption measurements of low concentrations of dye molecules and 10 nm gold particles were carried out achieving limits of detection in the low nM range. Other biologically relevant reactive oxygen species can be measured at sub-µM concentrations, which was shown for glucose and lactate through enzymatic reactions producing H2O2. In ecotoxicological investigations H2O2 excreted by aquatic microorganisms exposed to various stressors were measured. Pro-oxidant effects of nano-TiO2 and nano-CuO towards green alga Chlamydomonas reinhardtii were explored in various exposure media and under different light illuminations. Dynamics of Cd2+ induced effects on photosynthetic activity, sensitisation and recovery of cells of C. reinhardtii was also studied.

Toward achieving harmonization in a nano-cytotoxicity assay measurement through an interlaboratory comparison study.

Development of reliable cell-based nanotoxicology assays is important for evaluation of potentially hazardous engineered nanomaterials. Challenges to producing a reliable assay protocol include working with nanoparticle dispersions and living cell lines, and the potential for nano-related interference effects. Here we demonstrate the use of a 96-well plate design with several measurement controls and an interlaboratory comparison study involving five laboratories to characterize the robustness of a nanocytotoxicity MTS cell viability assay based on the A549 cell line. The consensus EC50 values were 22.1 mg/L (95% confidence intervals 16.9 mg/L to 27.2 mg/L) and 52.6 mg/L (44.1 mg/L to 62.6 mg/L) for positively charged polystyrene nanoparticles for the serum-free and serum conditions, respectively, and 49.7 µmol/L (47.5 µmol/L to 51.5 µmol/L) and 77.0 µmol/L (54.3 µmol/L to 99.4 µmol/L) for positive chemical control cadmium sulfate for the serum-free and serum conditions, respectively. Results from the measurement controls can be used to evaluate the sources of variability and their relative magnitudes within and between laboratories. This information revealed steps of the protocol that may need to be modified to improve the overall robustness and precision. The results suggest that protocol details such as cell line ID, media exchange, cell handling, and nanoparticle dispersion are critical to ensure protocol robustness and comparability of nanocytotoxicity assay results. The combination of system control measurements and interlaboratory comparison data yielded insights that would not have been available by either approach by itself.

New insights into ROS dynamics: a multi-layered microfluidic chip for ecotoxicological studies on aquatic microorganisms.

Reactive oxygen species (ROS) play an important role in the life of every cell, including cellular defense and signaling mechanisms. Continuous and quantitative ROS sensing can provide valuable information about the cell state, but it remains a challenge to measure. Here, we introduce a multi-layered microfluidic chip with an integrated optical sensor for the continuous sensitive detection of extracellular hydrogen peroxide (H2O2), one of the most stable ROS. This platform includes hydraulically controlled microvalves and microsieves, which enable the precise control of toxicants and complex exposure sequences. In particular, we use this platform to study the dynamics of toxicity-induced ROS generation in the green microalga Chlamydomonas reinhardtii during short-term exposures, recovery periods, and subsequent re-exposures. Two cadmium-based toxicants with distinct internalization mechanisms are used as stress inducers: CdSe/ZnS quantum dots (Qdots) and ionic cadmium (Cd(2+)). Our results show the quantitative dynamics of ROS generation by the model microalga, the recovery of cell homeostasis after stress events and the cumulative nature of two consecutive exposures. The dissolution of quantum dots and its possible influence on toxicity and H2O2 depletion is discussed. The obtained insights are relevant from ecotoxicological and physiological perspectives.

Dynamics of sub-lethal effects of nano-CuO on the microalga Chlamydomonas reinhardtii during short-term exposure.

Though nano-CuO has been classified as toxic toward aquatic microorganisms and its use in various applications is expected to increase in near future, its ecotoxicity is currently poorly understood. The aim of this study was to investigate the mechanisms of nano-CuO toxicity based on the paradigm of oxidative stress, the dynamics of response over 24h, and the modulating effect of exposure conditions on toxicity and responses. To this end, the model microalga Chlamydomonas reinhardtii was exposed to 10mgL(-1) nano-CuO in five different exposure media, including two different growth media, two Good's buffers, and natural lake water. The measured endpoints included cell growth, morphological aspect, chlorophyll autofluorescence, oxidative stress, and membrane permeabilization. The results suggest that agglomerated nano-CuO is toxic and that exposure media are decisive in whether or not particles or free Cu ions are the main mediators of toxicity. A significant particle effect was only observed in the Good's buffer 3-(N-morpholino) propanesulfonic acid. However, nano-CuO particles especially influenced the dynamics of response early in exposure, between 0h and 5h, suggesting that an adaptation to particle stress may occur or that particles modify the bioavailability of the free Cu ions in early exposure.

Effects of copper-oxide nanoparticles, dissolved copper and ultraviolet radiation on copper bioaccumulation, photosynthesis and oxidative stress in the aquatic macrophyte Elodea nuttallii.

In this study, the uptake and sub-toxic effects of CuO nanoparticles (CuO-NPs), dissolved Cu(II) alone or in combination with UV radiation on the aquatic macrophyte Elodea nuttallii were studied. Emphasis was on Cu accumulation, growth, photosynthesis and the oxidative stress related enzymes peroxidase (POD) and superoxide dismutase (SOD). The results showed stronger Cu accumulation in plants exposed to 10 mg L(-1) CuO-NPs, corresponding to 1.4-2 mg L(-1) dissolved Cu(II), than to 256 µg L(-1) Cu(II). However, the ratio between the accumulated Cu and dissolved Cu in CuO treatments was lower than in Cu(II) treatments. Additional UV exposure increased accumulation in both treatments, with the effect being stronger for Cu accumulation from CuO-NPs than for dissolved Cu(II). Photosynthetic capacity was strongly reduced by UV treatment, whereas remained unaffected by Cu(II) or CuO-NP treatments. Similarly, the increase of SOD activity was more pronounced in the UV treatments. On the other hand, POD activity enhancement was strongest in the plants exposed to CuO-NPs for 24 h. Expression of the copper transporter COPT1 as revealed by RT-qPCR was inhibited by Cu(II) and CuO-NP treatment, limiting the uptake of excess Cu into the cells. Overall, the combined exposure of E. nuttallii to UV radiation with CuO-NPs or Cu(II) has a higher impact than exposure to CuO-NPs or Cu(II) alone. The results imply that heavy pollution of natural water with CuO-NPs or dissolved Cu might have stronger effects in combination with natural UV irradiation on organisms in situ.

Portable oxidative stress sensor: dynamic and non-invasive measurements of extracellular H2O2 released by algae.

Reactive oxygen species (ROS) generated by aerobic organisms are essential for physiological processes such as cell signaling, apoptosis, immune defense and oxidative stress mechanisms. Unbalanced oxidant/antioxidant budgets are involved in many diseases and, therefore, the sensitive measurement of ROS is of great interest. Here, we present a new device for the real-time monitoring of oxidative stress by measuring one of the most stable ROS, namely hydrogen peroxide (H2O2). This portable oxidative stress sensor contains the heme protein cytochrome c (cyt c) as sensing element whose spectral response enables the detection of H2O2 down to a detection limit of 40 nM. This low detection limit is achieved by introducing cyt c in a random medium, enabling multiscattering that enhances the optical trajectory through the cyt c spot. A contact microspotting technique is used to produce reproducible and reusable cyt c spots which are stable for several days. Experiments in static and microfluidic regimes, as well as numerical simulations demonstrate the suitability of the cyt c/H2O2 reaction system for the real-time sensing of the kinetics of biological processes without H2O2 depletion in the measurement chamber. As an example, we detect the release of H2O2 from the green alga Chlamydomonas reinhardtii exposed to either 180 nM functionalized CdSe/ZnS core shell quantum dots, or to 10 mg/l TiO2 nanoparticles. The continuous measurement of extracellular H2O2 by this optical sensor with high sensitivity is a promising new means for real-time cytotoxicity tests, the investigation of oxidative stress and other physiological cell processes.

Oxidative stress induced by inorganic nanoparticles in bacteria and aquatic microalgae--state of the art and knowledge gaps.

Nanotechnology has revolutionised many areas of modern life, technology and research, which is reflected in the steadily increasing global demand for and consumption of engineered nanomaterials and the inevitable increase of their release into the environment by human activity. The overall long-term impact of engineered nanomaterials on ecosystems is still unknown. Various inorganic nanoparticles have been found to exhibit bactericidal properties and cause growth inhibition in model aquatic microalgae, but the mechanisms of toxicity are not yet fully understood. The causal link between particle properties and biological effects or reactive oxygen species generation is not well established and represents the most eminent quest of nanoecotoxicological investigation. In this review, the current mechanistic understanding of the toxicity of inorganic metal and metal oxide engineered nanomaterials towards bacterial and aquatic microalgal model organisms based on the paradigm of oxidative stress is presented along with a detailed compilation of available literature on the major toxicity factors and research methods.

Uptake and effects of microplastics on cells and tissue of the blue mussel Mytilus edulis L. after an experimental exposure.

In this study, we investigated if industrial high-density polyethylene (HDPE) particles, a model microplastic free of additives, ranging > 0-80 µm are ingested and taken up into the cells and tissue of the blue mussel Mytilus edulis L. The effects of exposure (up to 96 h) and plastic ingestion were observed at the cellular and subcellular level. Microplastic uptake into the gills and digestive gland was analyzed by a new method using polarized light microscopy. Mussel health status was investigated incorporating histological assessment and cytochemical biomarkers of toxic effects and early warning. In addition to being drawn into the gills, HDPE particles were taken up into the stomach and transported into the digestive gland where they accumulated in the lysosomal system after 3 h of exposure. Our results show notable histological changes upon uptake and a strong inflammatory response demonstrated by the formation of granulocytomas after 6 h and lysosomal membrane destabilization, which significantly increased with longer exposure times. We provide proof of principle that microplastics are taken up into cells and cause significant effects on the tissue and cellular level, which can be assessed with standard cytochemical biomarkers and polarized light microscopy for microplastic tracking in tissue.