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.

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.

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.

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).

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.

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.

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.

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.

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization.

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.

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.

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.

Quantitative evaluation of multi-walled carbon nanotube uptake in wheat and rapeseed.

Environmental contamination with carbon nanotubes would lead to plant exposure and particularly exposure of agricultural crops. The only quantitative exposure data available to date which can be used for risk assessment comes from computer modeling. The aim of this study was to provide quantitative data relative to multi-walled carbon nanotube (MWCNT) uptake and distribution in agricultural crops, and to correlate accumulation data with impact on plant development and physiology. Roots of wheat and rapeseed were exposed in hydroponics to uniformly (14)C-radiolabeled MWCNTs. Radioimaging, transmission electron microscopy and raman spectroscopy were used to identify CNT distribution. Radioactivity counting made it possible absolute quantification of CNT accumulation in plant leaves. Impact of CNTs on seed germination, root elongation, plant biomass, evapotranspiration, chlorophyll, thiobarbituric acid reactive species and H(2)O(2) contents was evaluated. We demonstrate that less than 0.005‰ of the applied MWCNT dose is taken up by plant roots and translocated to the leaves. This accumulation does not impact plant development and physiology. In addition, it does not induce any modifications in photosynthetic activity nor cause oxidative stress in plant leaves. Our results suggest that if environmental contamination occurs and MWCNTs are in the same physico-chemical state than the ones used in the present article, MWCNT transfer to the food chain via food crops would be very low.

Size-, composition- and shape-dependent toxicological impact of metal oxide nanoparticles and carbon nanotubes toward bacteria.

Ecotoxicological effects of nanoparticles (NP) are still poorly documented while their commercialization for industrial and household applications increases. The aim of this study was to evaluate the influence of physicochemical characteristics on metal oxide NP and carbon nanotubes toxicological effects toward bacteria. Two strains of bacteria, Cupriavidus metallidurans CH34 and Escherichia coli MG1655 were exposed to TiO(2) or Al(2)O(3) NP or to multiwalled-carbon nanotubes (MWCNT). Particular attention was paid on optimizing NP dispersion to obtain nonagglomerated suspensions. Our results show that NP toxicity depends on their chemical composition, size, surface charge, and shape but not on their crystalline phase. MWCNT toxicity does not depend on their purity. Toxicity also depends on the bacterial strain: E. coli MG1655 is sensitive to NP, whereas C. metallidurans CH34 is not. Interestingly, NP are accumulated in both bacterial strains, and association between NP and bacteria is necessary for bacterial death to occur. NP may then represent a danger for the environment, causing the disappearance of some sensitive bacterial strains such as E. coli MG1655, but also being mobilized by nonsensitive strains such as C. metallidurans CH34 and transported through the whole ecosystem.

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.

Carbon nanotubes in macrophages: imaging and chemical analysis by X-ray fluorescence microscopy.

X-ray fluorescence microscopy (microXRF) is applied for the first time to study macrophages exposed to unpurified and purified single-walled (SW) and multiwalled (MW) carbon nanotubes (CNT). Investigating chemical elemental distributions allows one to (i) image nanotube localization within a cell and (ii) detect chemical modification of the cell after CNT internalization. An excess of calcium is detected for cells exposed to unpurified SWCNT and MWCNT and related toxicological assays are discussed.

Evidence of sequential lift in growth of aligned multiwalled carbon nanotube multilayers.

We synthesized aligned multiwalled carbon nanotube multilayers by aerosol-assisted catalytic chemical vapor deposition through sequential injections of aerosols containing both carbon and catalyst precursors. Each sequence was traced by a specific duration or precursor mixture, with the carbon source being possibly enriched in (13)C isotope labels. We discovered that any sequence involved the growth of a new layer on the substrate surface, under any pre-existing one by lifting it up, giving definitive evidence of a base-growth mechanism.