Early signs of multi-walled carbon nanotbues degradation in macrophages, via an intracellular pH-dependent biological mechanism; importance of length and functionalization.

BACKGROUND: Carbon nanotubes (CNT) can interact with the biological environment, which could participate in their associated toxicity. We recently demonstrated that pH is an important player of CNT fate inside macrophages. We wanted to further characterize such process, and therefore designed a study dedicated to decipher CNT biodegradation by macrophages, as a function of two major physico-chemical properties in regard with nanotoxicology; length and degree of functionalization. To achieve our aim, we synthesized, following a single initial production process, four MWCNT differing in length and/or surface chemistry: S-CNT (short), SF-CNT (short functionalized), L-CNT (long) and LF-CNT (long functionalized). RESULTS: Raman spectroscopy analysis performed on CNT recovered after exposure of RAW 264.7 macrophages for 6, 24, or 48 h demonstrate that CNT show early signs of biodegradation over time inside macrophages. The modulation of CNT length and functionalization, resulting in the modification of iron accessibility, both represent critical determinants of the biodegradation process; short pristine CNT were more prone to biodegradation than long CNT (pristine or functionalized), while short functionalized CNT were protected. Incubation of cells with Concanamycin completely prevents CNT from being modified, demonstrating that this biodegradation process is dependent on an intracellular pH-dependent mechanism. Interestingly, and despite evidence of degradation via Raman spectroscopy, the CNT length and diameter were not altered during the course of the study. CONCLUSIONS: In conclusion, our results identify a new mechanism of CNT biodegradation inside macrophages. This could give new insights for the understanding of CNT-associated toxicity, and represent important tools to develop safe(r)-by-design nanomaterials.

High-intensity interval and endurance training are associated with divergent skeletal muscle adaptations in a rodent model of hypertension.

Skeletal muscle is extremely adaptable to a variety of metabolic challenges, as both traditional moderate-intensity endurance (ET) and high-intensity interval training (HIIT) increases oxidative potential in a coordinated manner. Although these responses have been clearly demonstrated in healthy individuals, it remains to be determined whether both produce similar responses in the context of hypertension, one of the most prevalent and costly diseases worldwide. Therefore, in the current study, we used the Dahl sodium-sensitive rat, a model of hypertension, to determine the molecular responses to 4 wk of either ET or HIIT in the red (RG) and white gastrocnemius (WG) muscles. In the RG, both ET and HIIT increased the content of electron transport chain proteins and increased succinate dehydrogenase (SDH) content in type I fibers. Although both intensities of exercise shifted fiber type in RG (increased IIA, decreased IIX), only HIIT was associated with a reduction in endothelial nitric oxide synthase and an increase in HIF-1α proteins. In the WG, both ET and HIIT increased markers of the electron transport chain; however, HIIT decreased SDH content in a fiber-specific manner. ET increased type IIA, decreased IIB fibers, and increased capillarization, while, in contrast, HIIT increased the percentage of IIB fibers, decreased capillary-to-fiber ratios, decreased endothelial nitric oxide synthase, and increased hypoxia inducible factor-1α (HIF-1α) protein. Altogether, these data show that unlike in healthy animals, ET and HIIT have divergent effects in the skeletal muscle of hypertensive rats. This suggests ET may be optimal at improving the oxidative capacity of skeletal muscle in animals with hypertension.

Determination of carbon nanotubes concentration by tabletop dynamic susceptometer.

Carbon nanotubes (CNTs) can be incorporated in various materials to enhance their mechanical or electrical properties. Information on their precise concentration and local distribution is difficult to access non-invasively. For example, electron microscopy studies require cutting of samples. Another way to measure the concentration of CNTs is by the magnetic susceptibility of the ferrocene present in the CNTs by the synthesis process, which can be performed on sample coupons on a vibrating sample magnetometer (VSM); VSM is a bulky laboratory instrument, and the size of the samples studied is constrained. In order to provide a technique that is fast, easy, cheap, and adaptable to the size of the samples, we have developed a benchtop device that measures the CNT concentration through an original inductive dynamic measurement of the ferrocene magnetic susceptibility. We present the method for extracting CNT concentrations and show the results obtained on cement matrices with CNT concentrations of the order of a few percent.

One-year post-exposure assessment of 14C-few-layer graphene biodistribution in mice: single versus repeated intratracheal administration.

Research on graphene-based nanomaterials has experienced exponential growth in the last few decades, driven by their unique properties and their future potential impact on our everyday life. With the increasing production and commercialization of these materials, there is significant interest in understanding their fate in vivo. Herein, we investigated the distribution of 14C-few-layer graphene (14C-FLG) flakes (lat. dim. ∼ 500 nm) in mice over a period of one year. Furthermore, we compared the effects of repeated low-dose and acute high-dose exposure by tracheal administration. The results showed that most of the radioactivity was found in the lungs in both cases, with longer elimination times in the case of acute high-dose administration. In order to gain deeper insights into the distribution pattern, we conducted ex vivo investigations using µ-autoradiography on tissue sections, revealing the heterogeneous distribution of the material following administration. For the first time, µ-autoradiography was used to conduct a comprehensive investigation into the distribution and potential presence of FLG within lung cells isolated from the exposed lungs. The presence of radioactivity in lung cells strongly suggests internalization of the 14C-FLG particles. Overall these results show the long-term accumulation of the material in the lungs over one year, regardless of the administration protocol, and the higher biopersistence of FLG in the case of an acute exposure. These findings highlight the importance of the exposure scenario in the context of intratracheal administration, which is of interest in the evaluation of the potential health risks of graphene-based nanomaterials.

Correlative radioimaging and mass spectrometry imaging: a powerful combination to study 14C-graphene oxide in vivo biodistribution.

Research on graphene based nanomaterials has flourished in the last decade due their unique properties and emerging socio-economic impact. In the context of their potential exploitation for biomedical applications, there is a growing need for the development of more efficient imaging techniques to track the fate of these materials. Herein we propose the first correlative imaging approach based on the combination of radioimaging and mass spectrometry imaging for the detection of Graphene Oxide (GO) labelled with carbon-14 in mice. In this study, 14C-graphene oxide nanoribbons were produced from the oxidative opening of 14C-carbon nanotubes, and were then intensively sonicated to provide nano-size 14C-GO flakes. After Intravenous administration in mice, 14C-GO distribution was quantified by radioimaging performed on tissue slices. On the same slices, MS-imaging provided a highly resolved distribution map of the nanomaterial based on the detection of specific radical anionic carbon clusters ranging from C2˙- to C9˙- with a base peak at m/z 72 (12C) and 74 (14C) under negative laser desorption ionization mass spectrometry (LDI-MS) conditions. This proof of concept approach synergizes the strength of each technique and could be advantageous in the pre-clinical development of future Graphene-based biomedical applications.

Growth of long and aligned multi-walled carbon nanotubes on carbon and metal substrates.

Well aligned, long and dense multi-walled carbon nanotubes (CNT) can be grown on both carbon fibres and any metal substrates compatible with the CNT synthesis temperature. The injection-CVD process developed involves two stages, including fibre pretreatment by depositing a SiO(2)-based sub-layer from an organometallic precursor followed by CNT growth from toluene/ferrocene precursor mixture. Carbon substrates, as well as metals, can easily be treated with this process, which takes place in the same reactor and does not need any handling in between the two stages. The aligned CNT carpets obtained are similar to the ones grown on reference quartz substrates. The CNT growth rate is fairly high (ca. 30 µm min(-1)) and it is possible to control CNT length by varying the CNT synthesis duration. The thickness of the SiO(2)-based sub-layer can be varied and is shown to have an influence on the CNT growth. This layer is assumed to play a diffusion barrier layer role between the substrate and the iron based catalyst nanoparticles producing CNT. The CNT anchorage to the carbon fibres has been checked and good overall adhesion proved, which is in favour of a good transfer of electrical charge and heat between the nanotubes and fibre.

Critical role of surface chemical modifications induced by length shortening on multi-walled carbon nanotubes-induced toxicity.

Given the increasing use of carbon nanotubes (CNT) in composite materials and their possible expansion to new areas such as nanomedicine which will both lead to higher human exposure, a better understanding of their potential to cause adverse effects on human health is needed. Like other nanomaterials, the biological reactivity and toxicity of CNT were shown to depend on various physicochemical characteristics, and length has been suggested to play a critical role. We therefore designed a comprehensive study that aimed at comparing the effects on murine macrophages of two samples of multi-walled CNT (MWCNT) specifically synthesized following a similar production process (aerosol-assisted CVD), and used a soft ultrasonic treatment in water to modify the length of one of them. We showed that modification of the length of MWCNT leads, unavoidably, to accompanying structural (i.e. defects) and chemical (i.e. oxidation) modifications that affect both surface and residual catalyst iron nanoparticle content of CNT. The biological response of murine macrophages to the two different MWCNT samples was evaluated in terms of cell viability, pro-inflammatory cytokines secretion and oxidative stress. We showed that structural defects and oxidation both induced by the length reduction process are at least as responsible as the length reduction itself for the enhanced pro-inflammatory and pro-oxidative response observed with short (oxidized) compared to long (pristine) MWCNT. In conclusion, our results stress that surface properties should be considered, alongside the length, as essential parameters in CNT-induced inflammation, especially when dealing with a safe design of CNT, for application in nanomedicine for example.

Conducting Interface for Efficient Growth of Vertically Aligned Carbon Nanotubes: Towards Nano-Engineered Carbon Composite.

Vertically aligned carbon nanotubes (VACNT) are manufactured nanomaterials with excellent properties and great potential for numerous applications. Recently, research has intensified toward achieving VACNT synthesis on different planar and non-planar substrates of various natures, mainly dependent on the user-defined application. Indeed, VACNT growth has to be adjusted and optimized according to the substrate nature and shape to reach the requirements for the application envisaged. To date, different substrates have been decorated with VACNT, involving the use of diffusion barrier layers (DBLs) that are often insulating, such as SiO2 or Al2O3. These commonly used DBLs limit the conducting and other vital physico-chemical properties of the final nanomaterial composite. One interesting route to improve the contact resistance of VACNT on a substrate surface and the deficient composite properties is the development of semi-/conducting interlayers. The present review summarizes different methods and techniques for the deposition of suitable conducting interfaces and controlled growth of VACNT on diverse flat and 3-D fibrous substrates. Apart from exhibiting a catalytic efficiency, the DBL can generate a conducting and adhesive interface involving performance enhancements in VACNT composites. The abilities of different conducting interlayers are compared for VACNT growth and subsequent composite properties. A conducting interface is also emphasized for the synthesis of VACNT on carbonaceous substrates in order to produce cost-effective and high-performance nano-engineered carbon composites.

Critical Role of the Acetylene Content and Fe/C Ratio on the Thickness and Density of Vertically Aligned Carbon Nanotubes Grown at Low Temperature by a One-Step Catalytic Chemical Vapor Deposition Process.

The present work explores the role of the carbon source content and the Fe/C ratio on the synthesis of vertically aligned carbon nanotubes (VACNTs) by one-step aerosol-assisted CCVD operated at a medium temperature (615 °C) on aluminum substrates. The main objective was to overcome the limitations of VACNT growth, constituting a drawback for applications requiring thick VACNTs. By using acetylene as carbon feedstock and ferrocene as a catalyst precursor, we demonstrate that when acetylene content is reduced to 1.5 vol%, it is possible to grow VACNT carpets up to 700 µm thick while maintaining constant VACNT growth for a long duration (up to 160 min). The carbon conversion yield is significantly improved when the acetylene content reaches 1.5 vol%. The Al surface roughness also influences VACNT growth. An optimum Fe/C ratio of 0.8 wt.% coupled with a low acetylene content gives the highest growth rate (5.4 µm/min) ever reported for a thermal aerosol-assisted CCVD process operated at such a low temperature. The CNT number density can be controlled by varying the Fe/C ratio, enabling high density growth (e.g., 1.3 × 1011 CNT/cm2).

Analysis of the Continuous Feeding of Catalyst Particles during the Growth of Vertically Aligned Carbon Nanotubes by Aerosol-Assisted CCVD.

Aerosol-assisted catalytic chemical vapor deposition (AACCVD) is a powerful one-step process to produce vertically aligned carbon nanotubes (VACNTs), characterized by the continuous supply of the catalyst precursor (metallocene). The behavior of catalyst species all along the synthesis is essential for the continuous growth of VACNTs. It is there investigated through detailed observations and elemental analyses at scales of VACNT carpets and of individual CNTs. Our approach is based on two complementary experiments: quenching of the sample cooling, and sequential injection of two distinct metallocenes. Metal-based nanoparticles nucleated in the gas-phase during the whole synthesis duration are shown to diffuse in between the growing VACNTs from the top of the CNT carpet towards the substrate. They are much smaller than the catalyst particles formed on the substrate in the initial steps of the process and evidences are given that they continuously feed these catalyst particles at the VACNT roots. Particularly, the electron energy-loss spectroscopy (EELS) analyses of metal-based segments found into a single CNT show that the second injected metal is very gradually incorporated in the particle initially formed from the metal firstly injected. The feeding of the catalyst particles by the nanoparticles continuously nucleated in the gas-phase is therefore an essential feature of the base-growth of CNTs by AACCVD.

Optimized network of multi-walled carbon nanotubes for chemical sensing.

This work reports the design of a resistive gas sensor based on 2D mats of multi-walled carbon nanotubes (MWCNTs) grown by aerosol-assisted chemical vapour deposition. The sensor sensitivity was optimized using chlorine as analyte by tuning both CNT network morphology and CNT electronic properties. Optimized devices, operating at room temperature, have been calibrated over a large range of concentration and are shown to be sensitive down to 27 ppb of chlorine. The as-grown MWCNT response is compared with responses of 2000 °C annealed CNTs, as well as of nitrogen-doped CNTs and CNTs functionalized with polyethyleneimine (PEI). Under chlorine exposure, the resistance decrease of as-grown and annealed CNTs is attributed to charge transfer from chlorine to CNTs and demonstrates their p-type semiconductor behaviour. XPS analysis of CNTs exposed to chlorine shows the presence of chloride species that confirms electron charge transfer from chlorine to CNTs. By contrast, the resistance of nitrogen-doped and PEI functionalized CNTs exposed to chlorine increases, in agreement with their n-type semiconductor nature. The best response is obtained using annealed CNTs and is attributed to their higher degree of crystallinity.

Safe-by-design strategies for lowering the genotoxicity and pulmonary inflammation of multiwalled carbon nanotubes: Reduction of length and the introduction of COOH groups.

Potentially, the toxicity of multiwalled carbon nanotubes (MWCNTs) can be reduced in a safe-by-design strategy. We investigated if genotoxicity and pulmonary inflammation of MWCNTs from the same batch were lowered by a) reducing length and b) introducing COOH-groups into the structure. Mice were administered: 1) long and pristine MWCNT (CNT-long) (3.9 µm); 2) short and pristine CNT (CNT-short) (1 µm); 3) CNT modified with high ratio COOH-groups (CNT-COOH-high); 4) CNT modified with low ratio COOH-groups (CNT-COOH-low). MWCNTs were dosed by intratracheal instillation at 18 or 54 µg/mouse (∼0.9 and 2.7 mg/kg bw). Neutrophils numbers were highest after CNT-long exposure, and both shortening the MWCNT and addition of COOH-groups lowered pulmonary inflammation (day 1 and 28). Likewise, CNT-long induced genotoxicity, which was absent with CNT-short and after introduction of COOH groups. In conclusion, genotoxicity and pulmonary inflammation of MWCNTs were lowered, but not eliminated, by shortening the fibres or introducing COOH-groups.

Preparation of (14)C-labeled multiwalled carbon nanotubes for biodistribution investigations.

A new method allowing the (14)C-labeling of carboxylic acid functions of carbon nanotubes is described. The key step of the labeling process is a decarbonylation reaction that has been developed and optimized with the help of a screening method. The optimized process has been successfully applied to multiwalled carbon nanotubes (MWNTs), and the corresponding (14)C-labeled nanotubes were used to investigate their in vivo behavior. Preliminary results obtained after i.v. contamination of rats revealed liver as the main target organ. Radiolabeling of NTs with a long-life radioactive nucleus like (14)C, coupled to a highly sensitive autoradiographic method, that provides a unique detection threshold, will make it possible to determine for a long time period whether or not NTs remain in any organs after animal exposure.

Protein folding intermediates: native-state hydrogen exchange.

The hydrogen exchange behavior of native cytochrome c in low concentrations of denaturant reveals a sequence of metastable, partially unfolded forms that occupy free energy levels reaching up to the fully unfolded state. The step from one form to another is accomplished by the unfolding of one or more cooperative units of structure. The cooperative units are entire omega loops or mutually stabilizing pairs of whole helices and loops. The partially unfolded forms detected by hydrogen exchange appear to represent the major intermediates in the reversible, dynamic unfolding reactions that occur even at native conditions and thus may define the major pathway for cytochrome c folding.

Single-Step Synthesis of Vertically Aligned Carbon Nanotube Forest on Aluminium Foils.

Vertically aligned carbon nanotube (VACNT) forests are promising for supercapacitor electrodes, but their industrialisation requires a large-scale cost-effective synthesis process suitable to commercial aluminium (Al) foils, namely by operating at a low temperature (<660 °C). We show that Aerosol-Assisted Catalytic Chemical Vapour Deposition (CCVD), a single-step roll-to-roll compatible process, can be optimised to meet this industrial requirement. With ferrocene as a catalyst precursor, acetylene as a carbon source and Ar/H2 as a carrier gas, clean and dense forests of VACNTs of about 10 nm in diameter are obtained at 615 °C with a growth rate up to 5 µm/min. Such novel potentiality of this one-step CCVD process is at the state-of-the-art of the multi-step assisted CCVD processes. To produce thick samples, long synthesis durations are required, but growth saturation occurs that is not associated with a diffusion phenomenon of iron in aluminium substrate. Sequential syntheses show that the saturation trend fits a model of catalytic nanoparticle deactivation that can be limited by decreasing acetylene flow, thus obtaining sample thickness up to 200 µm. Cyclic voltammetry measurements on binder-free VACNT/Al electrodes show that the CNT surface is fully accessible to the ionic liquid electrolyte, even in these dense VACNT forests.

Alcohol induces DNA damage and the Fanconi anemia D2 protein implicating FANCD2 in the DNA damage response pathways in brain.

BACKGROUND: The largest cause of neurological damage to children is prenatal exposure to alcohol and chronic alcohol use in adults is associated with neurodegeneration, dementia and long-term behavioral changes. Microarray analysis identified the DNA damage response (DDR) gene, Fanconi anemia (Fanc) D2, to be robustly upregulated in mouse midbrain following 24-hour in vivo exposure to ethanol. In this study, we investigate the ability of ethanol to generate DNA strand breaks, predicted substrates for the Fanc pathway and the potential role of FANCD2 in the DDR to ethanol in brain. METHODS: The effect of ethanol on FANCD2 mRNA levels was measured by quantitative real time PCR using mouse brain and human neuronal cells. FANCD2 protein levels and ubiquitination were measured by Western blotting and immunocytochemistry. DNA damage induction by ethanol/acetaldehyde was measured using the Comet assay and gamma H2AX immunocytochemistry. Levels of DNA and RNA synthesis were measured in cell strains using (3)H-thymidine or (3)H-uridine up-take. RESULTS: Chronic exposure to ethanol induced FANCD2 in mouse midbrain in vivo and in the nucleus of human neuronal cells in culture. However, there was no concomitant increase in the amount of ubiquitinated FANCD2. Acetaldehyde also induced nonubiquitinated FANCD2 protein, and we were able to demonstrate the ability of acetaldehyde to generate DNA double strand breaks, lesions which normally induce ubiquitination of FANCD2. Ethanol also inhibited both RNA and DNA synthesis in proliferating cells consistent with effects on transcription and replication. CONCLUSION: In contrast to other DNA damaging agents, ethanol/acetaldehyde generated DNA strand breaks without inducing ubiquitination of FANCD2, despite increasing protein levels in the nucleus. These data are consistent with recent reports that suggest the Fanconi anemia pathway plays an important role in the adult brain in response to DNA damage. Further work is required to establish what this role is, in particular the potential function of nonubiquitinated FANCD2 and its role in the DNA damage response in postmitotic neurons and neural precursor cells.

In vitro investigation of oxide nanoparticle and carbon nanotube toxicity and intracellular accumulation in A549 human pneumocytes.

If released in the environment, nanomaterials might be inhaled by populations and cause damage to the deepest regions of the respiratory tract, i.e., the alveolar compartment. To model this situation, we studied the response of A549 human pneumocytes after exposure to aluminium oxide or titanium oxide nanoparticles, and to multi-walled carbon nanotubes. The influence of size, crystalline structure and chemical composition was investigated. After a detailed identification of nanomaterial physico-chemical characteristics, cells were exposed in vitro and viability and intracellular accumulation were assessed. In our conditions, carbon nanotubes were more toxic than metal oxide nanoparticles. Our results confirmed that both nanotubes and nanoparticles are able to rapidly enter into cells, and distribute in the cytoplasm and intracellular vesicles. Among nanoparticles, we demonstrate significant difference in biological response as a function of size, crystalline phase and chemical composition. Their toxicity was globally lower than nanotubes toxicity. Among nanotubes, the length did not influence cytotoxicity, neither the presence of metal catalyst impurities.

Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy: do the genes explain the diseases?

Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy are three distinct human syndromes associated with sensitivity to ultraviolet radiation. We review evidence that these syndromes overlap with each other and arise from mutations in genes involved in nucleotide-excision repair and RNA transcription. Attempts have been made to explain the syndromes in terms of defects in repair and transcription. These two biochemical pathways do not easily account for all the features of the syndromes. Therefore, we propose a third pathway, in which the syndromes are due, in part, to defects in a demethylation mechanism involving the excision of methylated cytosine. Perturbation of demethylation could affect the developmentally regulated expression of some genes.

Mechanisms and uses of hydrogen exchange.

Recent work has largely completed our understanding of the hydrogen-exchange chemistry of unstructured proteins and nucleic acids. Some of the high-energy structural fluctuations that determine the hydrogen-exchange behavior of native macromolecules have been explained; others remain elusive. A growing number of applications are exploiting hydrogen-exchange behavior to study difficult molecular systems and elicit otherwise inaccessible information on protein structure, dynamics and energetics.

Dispersion study of long and aligned multi-walled carbon nanotubes in water.

Dispersion of nanotubes is a crucial step for many applications. The properties of the final nanotube-based material are strongly dependent on the quality of nanotube suspensions. In this study, long and aligned multi-walled carbon nanotubes obtained by catalytic chemical vapour deposition were dispersed in water with different dispersing agents using high intensity ultrasounds. Among different additives, we selected sodium dodecyl sulfate (SDS) as dispersing agent to prepare suspensions of nanotubes. UV-Visible spectrometry method was used to measure the influence of dispersion parameters (power and duration of sonication) on dispersion state and suspension stability. Therefore, we demonstrated that, even if high intensity ultra-sounds are breaking nanotubes, it is possible to obtain stable water-based suspensions containing MWNTs which exhibit length up to 20 microm.