Avoiding drying-artifacts in transmission electron microscopy: Characterizing the size and colloidal state of nanoparticles

Standard transmission electron microscopy nanoparticle sample preparation generally requires the complete removal of the suspending liquid. Drying often introduces artifacts, which can obscure the state of the dispersion prior to drying and preclude automated image analysis typically used to obtain number-weighted particle size distribution. Here we present a straightforward protocol for prevention of the onset of drying artifacts, thereby allowing the preservation of in-situ colloidal features of nanoparticles during TEM sample preparation. This is achieved by adding a suitable macromolecular agent to the suspension. Both research- and economically-relevant particles with high polydispersity and/or shape anisotropy are easily characterized following our approach (http://bsa.bionanomaterials.ch), which allows for rapid and quantitative classification in terms of dimensionality and size: features that are major targets of European Union recommendations and legislation

Assessing the impact of the physical properties of industrially produced carbon nanotubes on their interaction with human primary macrophages in vitro

Currently it is not fully understood how carbon nanotubes (CNTs) may affect human health. Despite this, CNTs are produced at a tonne mass scale yearly. Due to their large production and intended use within a variety of applications it is imperative that a clear understanding of the hazard potential of CNTs is gained. The aim of this study therefore was to assess the impact of five different industrially produced CNTs which varied in their physical properties on the viability of human monocyte derived macrophages (MDM), and subsequently, at sub-lethal concentrations (0.005-0.02 mg/mL), their ability to cause oxidative stress and a pro-inflammatory response in these important immune cells over a 24-h period. None of the CNTs caused significant cytotoxicity up to 0.02 mg/mL after 24 h. Only the long multi-walled CNTs (MWNCTs) caused a significant, dose-dependent (0.005-0.02 mg/mL) reactive oxygen species production, whilst bundled MWCNTs showed a significant tumor necrosis factor alpha release after 24 h exposure at 0.02 mg/mL. No effects were observed for either tangled MWCNTs or short MWCNTs. It can be concluded from the findings of the present study that the industrially produced CNTs studied can cause hazardous effects in vitro that may be associated with their physical propertie

A Comparative Study of Different In Vitro Lung Cell Culture Systems to Assess the Most Beneficial Tool for Screening the Potential Adverse Effects of Carbon Nanotubes

To determine the potential inhalatory risk posed by carbon nanotubes (CNTs), a tier-based approach beginning with an in vitro assessment must be adopted. The purpose of this study therefore was to compare 4 commonly used in vitro systems of the human lung (human blood monocyte-derived macrophages [MDM] and monocyte-derived dendritic cells [MDDC], 16HBE14o- epithelial cells, and a sophisticated triple cell co-culture model [TCC-C]) via assessment of the biological impact of different CNTs (single-walled CNTs [SWCNTs] and multiwalled CNTs [MWCNTs]) over 24h. No significant cytotoxicity was observed with any of the cell types tested, although a significant (p < .05), dose-dependent increase in tumor necrosis factor (TNF)-α following SWCNT and MWCNT exposure at concentrations up to 0.02mg/ml to MDM, MDDC, and the TCC-C was found. The concentration of TNF-α released by the MDM and MDDC was significantly higher (p < .05) than the TCC-C. Significant increases (p < .05) in interleukin (IL)-8 were also found for both 16HBE14o- epithelial cells and the TCC-C after SWCNTs and MWCNTs exposure up to 0.02mg/ml. The TCC-C, however, elicited a significantly (p < .05) higher IL-8 release than the epithelial cells. The oxidative potential of both SWCNTs and MWCNTs (0.005-0.02mg/ml) measured by reduced glutathione (GSH) content showed a significant difference (p < .05) between each monoculture and the TCC-C. It was concluded that because only the co-culture system could assess each endpoint adequately, that, in comparison with monoculture systems, multicellular systems that take into consideration important cell type-to-cell type interactions could be used as predictive in vitro screening tools for determining the potential deleterious effects associated with CNT

Risk assessment of released cellulose nanocrystals – mimicking inhalatory exposure

Cellulose nanocrystals (CNCs) exhibit advantageous chemical and mechanical properties that render them attractive for a wide range of applications. During the life-cycle of CNC containing materials the nanocrystals could be released and become airborne, posing a potential inhalatory exposure risk towards humans. Absent reliable and dose-controlled models that mimic this exposure in situ is a central issue in gaining an insight into the CNC-lung interaction. Here, an Air Liquid Interface Cell Exposure system (ALICE), previously designed for studies of spherical nanoparticles, was used for the first time to establish a realistic physiological exposure test method for inhaled fiber shaped nano-objects; in this case, CNCs isolated from cotton. Applying a microscopy based approach the spatially homogenous deposition of CNCs was demonstrated as a prerequisite of the functioning of the ALICE. Furthermore, reliability and controllability of the system to nebulise high aspect ratio nanomaterials (HARN, e.g. CNCs) was shown. This opens the potential to thoroughly investigate the inhalatory risk of CNCs in vitro using a realistic exposure system

"It‘s Hard to Keep Track": Mapping a Shifting Nation in Dylan Horrocks‘s Hicksville

Using an art form that justifiably lays claim to both visual and literary genealogies—the graphic novel—Dylan Horrocks's Hicksville advances, rather than strictly challenges, many of the discussions which have informed the local manufacture of art and literature. My purpose in this thesis is to explore Horrocks's deployment of the critical perspectives of both art historical and literary discourse as they have developed from the pre-colonial to the twenty-first century in New Zealand, especially those associated with cultural nationalism. Hicksville claims a particular relation to the existing traditions within both art-historical and literary lines wherein they are conjoined in practice; integrated into the formal properties of Horrocks's work, the traditional concerns of local art and literature are not only subject matter but guide Horrocks‘s approach to narrative. The tension between art and place—the responsibility of the artist to the nation and its referents—appears in Hicksville as a structuring device rather than polemic via its concern with the economisation of art—or global capitalism—as it bears upon particular places and art practices. Yet Horrocks‘s handling of this theme upholds neither aestheticism nor populism. Rather, he invites the reader to make sense of extensive references to a range of artistic figures, from Heaphy to Hergé to Hotere, in a way that accounts for their equal force. Hicksville thus deliberately destabilises the joint histories of art and literary history to pointed effect, valuing its range of artistic and cultural inheritances—whether the visual or literary, the highbrow or lowbrow—for how they can remind us that contemporary artistic accounts of New Zealand must also consider the various ways the country has been constructed throughout its wide

Assessment of a panel of interleukin-8 reporter lung epithelial cell lines to monitor the pro-inflammatory response following zinc oxide nanoparticle exposure under different cell culture conditions

Stably transfected lung epithelial reporter cell lines pose an advantageous alternative to replace complex experimental techniques to monitor the pro-inflammatory response following nanoparticle (NP) exposure. Previously, reporter cell lines have been used under submerged culture conditions, however, their potential usefulness in combination with air-liquid interface (ALI) exposures is currently unknown. Therefore, the aim of the present study was to compare a panel of interleukin-8 promoter (pIL8)-reporter cell lines (i.e. green or red fluorescent protein (GFP, RFP), and luciferase (Luc)), originating from A549 lung epithelial type II-like cells cells, following NPs exposure under both submerged and ALI conditions. All cell lines were exposed to zinc oxide (ZnO) NPs at 0.6 and 6.2 μg/cm 2 for 3 and 16 hours under both submerged and ALI conditions. Following physicochemical characterization, the cytotoxic profile of the ZnO-NPs was determined for each exposure scenario. Expression of IL-8 from all cell types was analyzed at the promoter level and compared to the mRNA (qRT-PCR) and protein level (ELISA). In summary, each reporter cell line detected acute pro-inflammatory effects following ZnO exposure under each condition tested. The pIL8-Luc cell line was the most sensitive in terms of reporter signal strength and onset velocity following TNF-α treatment. Both pIL8-GFP and pIL8-RFP also showed a marked signal induction in response to TNF-α, although only after 16 hrs. In terms of ZnO-NP-induced cytotoxicity pIL8-RFP cells were the most affected, whilst the pIL8-Luc were found the least responsive. In conclusion, the use of fluorescence-based reporter cell lines can provide a useful tool in screening the pro-inflammatory response following NP exposure in both submerged and ALI cell cultures. The online version of this article (doi:10.1186/s12989-015-0104-6) contains supplementary material, which is available to authorized users

A comparative study of different in vitro lung cell culture systems to assess the most beneficial tool for screening the potential adverse effects of carbon nanotubes

To determine the potential inhalatory risk posed by carbon nanotubes (CNTs), a tier-based approach beginning with an in vitro assessment must be adopted. The purpose of this study therefore was to compare 4 commonly used in vitro systems of the human lung (human blood monocyte-derived macrophages [MDM] and monocyte-derived dendritic cells [MDDC], 16HBE14o- epithelial cells, and a sophisticated triple cell co-culture model [TCC-C]) via assessment of the biological impact of different CNTs (single-walled CNTs [SWCNTs] and multiwalled CNTs [MWCNTs]) over 24h. No significant cytotoxicity was observed with any of the cell types tested, although a significant (p p p p p in vitro screening tools for determining the potential deleterious effects associated with CNTs

Can the Ames test provide an insight into nano-object mutagenicity? Investigating the interaction between nano-objects and bacteria

The aim of this study was to assess the interaction of a series of well characterised nano-objects with the Gram negative bacterium Salmonella typhimurium, and how such an interaction may relate to the potential mutagenicity of nano-objects. Transmission electron microscopy showed that nano-objects (Au-PMA-ATTO NPs, CeO₂ NPs, SWCNTs and MWCNTs), as well as CAFs entered S. typhimurium. Only DEPs did not penetrate/enter the bacteria, however, were the only particle stimulus to induce any significant mutagenicity through the Ames test. Comparison with a sophisticated 3D in vitro cell model showed CAFs, DEPs, SWCNTs and MWCNTs to cause a significant increase in mammalian cell proliferation, whilst both the Au-PMA-ATTO NPs and CeO₂ NPs had not significant adverse effects. In conclusion, these results indicate that various of different nano-objects are able to penetrate the double-lipid bilayer of Gram negative bacteria, although the Ames test may not be a good indicator for nano-object mutagenicity

Elucidating the Potential Biological Impact of Cellulose Nanocrystals

Cellulose nanocrystals exhibit an interesting combination of mechanical properties and physical characteristics, which make them potentially useful for a wide range of consumer applications. However, as the usage of these bio-based nanofibers increases, a greater understanding of human exposure addressing their potential health issues should be gained. The aim of this perspective is to highlight how knowledge obtained from studying the biological impact of other nanomaterials can provide a basis for future research strategies to deduce the possible human health risks posed by cellulose nanocrystals