Trine Berthing

Intact Mammalian Cell Function on Semiconductor Nanowire Arrays: New Perspectives for Cell‐Based Biosensing

Nanowires (NWs) are attracting more and more interest due to their potential cellular applications, such as delivery of compounds or sensing platforms. Arrays of vertical indium-arsenide (InAs) NWs are interfaced with human embryonic kidney cells and rat embryonic dorsal root ganglion neurons. A selection of critical cell functions and pathways are shown not to be impaired, including cell adhesion, membrane integrity, intracellular enzyme activity, DNA uptake, cytosolic and membrane protein expression, and the neuronal maturation pathway. The results demonstrate the low invasiveness of InAs NW arrays, which, combined with the unique physical properties of InAs, open up their potential for cellular investigations.

Exploring arrays of vertical one-dimensional nanostructures for cellular investigations

The endeavor of exploiting arrays of vertical one-dimensional (1D) nanostructures (NSs) for cellular applications has recently been experiencing a pronounced surge of activity. The interest is rooted in the intrinsic properties of high-aspect-ratio NSs. With a height comparable to a mammalian cell, and a diameter 100-1000 times smaller, NSs should intuitively reach far into a cell and, due to their small diameter, do so without compromising cell health. Single NSs would thus be expedient for measuring and modifying cell response. Further organization of these structures into arrays can provide up-scaled and detailed spatiotemporal information on cell activity, an achievement that would entail a massive leap forward in disease understanding and drug discovery. Numerous proofs-of-principle published recently have expanded the large toolbox that is currently being established in this rapidly advancing field of research. Encouragingly, despite the diversity of NS platforms and experimental conditions used thus far, general trends and conclusions from combining cells with NSs are beginning to crystallize. This review covers the broad spectrum of NS materials and dimensions used; the observed cellular responses with specific focus on adhesion, morphology, viability, proliferation, and migration; compares the different approaches used in the field to provide NSs with the often crucial cytosolic access; covers the progress toward biological applications; and finally, envisions the future of this technology. By maintaining the impressive rate and quality of recent progress, it is conceivable that the use of vertical 1D NSs may soon be established as a superior choice over other current techniques, with all the further benefits that may entail.

Templated Protein Assembly on Micro-Contact-Printed Surface Patterns. Use of the SNAP-tag Protein Functionality

Micro contact printing (microCP) has been established as a simple technique for high-resolution protein patterning for micro- and nanoarrays. However, as biochemical assays based on immobilized protein arrays progress from immunoassays to more delicate functional assays, the demand for methods of miniaturized, gentle, and oriented immobilization, which are applicable to many different target proteins, becomes larger. In this study, we present a novel microCP templated assembly approach, based on a recombinant SNAP-FLAG-HIS 10 (SFH) immobilization vehicle, which exploits the recently developed SNAP-tag protein. The SNAP-tag is derived from the human DNA repair protein hAGT, which covalently transfers the alkyl group of benzyl guanine (BG) substrates onto itself. We have designed a model SFH cassette carrying three tags (SNAP-tag, FLAG-tag, and HIS-tag), each of which can be used for fluorescence labeling or surface immobilization. When patterns of streptavidin modified with BG-biotin (streptavidin-BG) are stamped onto a surface, the SFH can subsequently assemble on the ligand pattern from solution, functioning as a general immobilization vehicle for high-resolution patterning of any protein expressed in the SFH cassette, in a gentle and oriented manner. Alternatively, the SFH can be site-selectively biotinylated using BG-biotin and, subsequently, assemble on stamped streptavidin. We exploit several ways to biotinylate the SFH protein via the SNAP-tag, promoting its templated assembly on micropatterns of streptavidin in four complementary formats. Quantitative analysis of the obtained patterns, revealed by immunostaining, indicates that all four approaches resulted in proper SFH immobilization and antibody recognition, demonstrating the versatility of the SFH cassette and the potential for high resolution patterning applications. Also, our data confirm that streptavidin can be stamped directly on surfaces, without loss of activity. While three strategies resulted in similar patterning efficiencies, one particular approach--namely templated assembly of SFH directly on streptavidin-BG patterns--resulted in an order of magnitude increase in patterning efficiency.

Tuning InAs Nanowire Density for HEK293 Cell Viability, Adhesion, and Morphology: Perspectives for Nanowire-Based Biosensors

Arrays of nanowires (NWs) are currently being established as vehicles for molecule delivery and electrical- and fluorescence-based platforms in the development of biosensors. It is conceivable that NW-based biosensors can be optimized through increased understanding of how the nanotopography influences the interfaced biological material. Using state-of-the-art homogenous NW arrays allow for a systematic investigation of how the broad range of NW densities used by the community influences cells. Here it is demonstrated that indium arsenide NW arrays provide a cell-promoting surface, which affects both cell division and focal adhesion up-regulation. Furthermore, a systematic variation in NW spacing affects both the detailed cell morphology and adhesion properties, where the latter can be predicted based on changes in free-energy states using the proposed theoretical model. As the NW density influences cellular parameters, such as cell size and adhesion tightness, it will be important to take NW density into consideration in the continued development of NW-based platforms for cellular applications, such as molecule delivery and electrical measurements.

Multi-walled carbon nanotube physicochemical properties predict pulmonary inflammation and genotoxicity

Abstract Lung deposition of multi-walled carbon nanotubes (MWCNT) induces pulmonary toxicity. Commercial MWCNT vary greatly in physicochemical properties and consequently in biological effects. To identify determinants of MWCNT-induced toxicity, we analyzed the effects of pulmonary exposure to 10 commercial MWCNT (supplied in three groups of different dimensions, with one pristine and two/three surface modified in each group). We characterized morphology, chemical composition, surface area and functionalization levels. MWCNT were deposited in lungs of female C57BL/6J mice by intratracheal instillation of 0, 6, 18 or 54 μg/mouse. Pulmonary inflammation (neutrophil influx in bronchoalveolar lavage (BAL)) and genotoxicity were determined on day 1, 28 or 92. Histopathology of the lungs was performed on day 28 and 92. All MWCNT induced similar histological changes. Lymphocytic aggregates were detected for all MWCNT on day 28 and 92. Using adjusted, multiple regression analyses, inflammation and genotoxicity were related to dose, time and physicochemical properties. The specific surface area (BET) was identified as a positive predictor of pulmonary inflammation on all post-exposure days. In addition, length significantly predicted pulmonary inflammation, whereas surface oxidation (–OH and –COOH) was predictor of lowered inflammation on day 28. BET surface area, and therefore diameter, significantly predicted genotoxicity in BAL fluid cells and lung tissue such that lower BET surface area or correspondingly larger diameter was associated with increased genotoxicity. This study provides information on possible toxicity-driving physicochemical properties of MWCNT. The results may contribute to safe-by-design manufacturing of MWCNT, thereby minimizing adverse effects.

Differences in inflammation and acute phase response but similar genotoxicity in mice following pulmonary exposure to graphene oxide and reduced graphene oxide

We investigated toxicity of 2–3 layered >1 μm sized graphene oxide (GO) and reduced graphene oxide (rGO) in mice following single intratracheal exposure with respect to pulmonary inflammation, acute phase response (biomarker for risk of cardiovascular disease) and genotoxicity. In addition, we assessed exposure levels of particulate matter emitted during production of graphene in a clean room and in a normal industrial environment using chemical vapour deposition. Toxicity was evaluated at day 1, 3, 28 and 90 days (18, 54 and 162 μg/mouse), except for GO exposed mice at day 28 and 90 where only the lowest dose was evaluated. GO induced a strong acute inflammatory response together with a pulmonary (Serum-Amyloid A, Saa3) and hepatic (Saa1) acute phase response. rGO induced less acute, but a constant and prolonged inflammation up to day 90. Lung histopathology showed particle agglomerates at day 90 without signs of fibrosis. In addition, DNA damage in BAL cells was observed across time points and doses for both GO and rGO. In conclusion, pulmonary exposure to GO and rGO induced inflammation, acute phase response and genotoxicity but no fibrosis.

Identification of Gene Transcription Start Sites and Enhancers Responding to Pulmonary Carbon Nanotube Exposure in Vivo

Increased use of nanomaterials in industry, medicine, and consumer products has raised concerns over their toxicity. To ensure safe use of nanomaterials, understanding their biological effects at the molecular level is crucial. In particular, the regulatory mechanisms responsible for the cascade of genes activated by nanomaterial exposure are not well-characterized. To this end, we profiled the genome-wide usage of gene transcription start sites and linked active enhancer regions in lungs of C57BL/6 mice 24 h after intratracheal instillation of a single dose of the multiwalled carbon nanotube (MWCNT) Mitsui-7. Our results revealed a massive gene regulatory response, where expression of key inflammatory genes (e.g., Csf3, Il24, and Fgf23) was increased >100-fold 24 h after Mitsui-7 exposure. Many of the Mitsui-7-responsive transcription start sites were alternative transcription start sites for known genes, and the number of alternative transcription start sites used in a given gene was correlated with overall Mitsui-7 response. Strikingly, genes that were up-regulated after Mitsui-7 exposure only through their main annotated transcription start site were linked to inflammatory and defense responses, while genes up-regulated only through alternative transcription start sites were functionally heterogeneous and not inflammation-associated. Furthermore, we identified almost 12 000 active enhancers, many of which were Mitsui-7-responsive, and we identified similarly responding putative target genes. Overall, our study provides the location and activity of Mitsui-7-induced enhancers and transcription start sites, providing a useful resource for targeted experiments elucidating the biological effects of nanomaterials and the identification of biomarkers for early detection of MWCNT-induced inflammation.

In vivo-induced size transformation of cerium oxide nanoparticles in both lung and liver does not affect long-term hepatic accumulation following pulmonary exposure

Recent findings show that cerium oxide (CeO2) nanoparticles may undergo in vivo-induced size transformation with the formation of smaller particles that could result in a higher translocation following pulmonary exposure compared to virtually insoluble particles, like titanium dioxide (TiO2). Therefore, we compared liver deposition of CeO2 and TiO2 nanoparticles of similar primary sizes 1, 28 or 180 days after intratracheal instillation of 162 μg of NPs in female C57BL/6 mice. Mice exposed to 162 μg CeO2 or TiO2 nanoparticles by intravenous injection or oral gavage were included as reference groups to assess the amount of NPs that reach the liver bypassing the lungs and the translocation of NPs from the gastrointestinal tract to the liver, respectively. Pulmonary deposited CeO2 nanoparticles were detected in the liver 28 and 180 days post-exposure and TiO2 nanoparticles 180 days post-exposure as determined by darkfield imaging and by the quantification of Ce and Ti mass concentration by inductively coupled plasma-mass spectrometry (ICP-MS). Ce and Ti concentrations increased over time and 180 days post-exposure the translocation to the liver was 2.87 ± 3.37% and 1.24 ± 1.98% of the initial pulmonary dose, respectively. Single particle ICP-MS showed that the size of CeO2 nanoparticles in both lung and liver tissue decreased over time. No nanoparticles were detected in the liver following oral gavage. Our results suggest that pulmonary deposited CeO2 and TiO2 nanoparticles translocate to the liver with similar calculated translocation rates despite their different chemical composition and shape. The observed particle size distributions of CeO2 nanoparticles indicate in vivo processing over time both in lung and liver. The fact that no particles were detected in the liver following oral exposure showed that direct translocation of nanoparticles from lung to the systemic circulation was the most important route of translocation for pulmonary deposited particles.

Physicochemical predictors of Multi-Walled Carbon Nanotube-induced pulmonary histopathology and toxicity one year after pulmonary deposition of 11 different Multi-Walled Carbon Nanotubes in mice

Multi‐walled carbon nanotubes (MWCNT) are widely used nanomaterials that cause pulmonary toxicity upon inhalation. The physicochemical properties of MWCNT vary greatly, which makes general safety evaluation challenging to conduct. Identification of the toxicity‐inducing physicochemical properties of MWCNT is therefore of great importance. We have evaluated histological changes in lung tissue 1 year after a single intratracheal instillation of 11 well‐characterized MWCNT in female C57BL/6N BomTac mice. Genotoxicity in liver and spleen was evaluated by the comet assay. The dose of 54 μg MWCNT corresponds to three times the estimated dose accumulated during a work life at a NIOSH recommended exposure limit (0.001 mg/m3). Short and thin MWCNT were observed as agglomerates in lung tissue 1 year after exposure, whereas thicker and longer MWCNT were detected as single fibres, suggesting biopersistence of both types of MWCNT. The thin and entangled MWCNT induced varying degree of pulmonary inflammation, in terms of lymphocytic aggregates, granulomas and macrophage infiltration, whereas two thick and straight MWCNT did not. By multiple regression analysis, larger diameter and higher content of iron predicted less histopathological changes, whereas higher cobalt content significantly predicted more histopathological changes. No MWCNT‐related fibrosis or tumours in the lungs or pleura was found. One thin and entangled MWCNT induced increased levels of DNA strand breaks in liver; however, no physicochemical properties could be related to genotoxicity. This study reveals physicochemical‐dependent difference in MWCNT‐induced long‐term, pulmonary histopathological changes. Identification of diameter size and cobalt content as important for MWCNT toxicity provides clues for designing MWCNT, which cause reduced human health effects following pulmonary exposure.