Tunable electrospun scaffolds of polyacrylonitrile loaded with carbon nanotubes: from synthesis to biological applications.

Growing cells in a biomimetic environment is critical for tissue engineering as well as for studying the cell biology underlying disease mechanisms. To this aim a range of 3D matrices have been developed, from hydrogels to decellularized matrices. They need to mimic the extracellular matrix to ensure the optimal growth and function of cells. Electrospinning has gained in popularity due to its capacity to individually tune chemistry and mechanical properties and as such influence cell attachment, differentiation or maturation. Polyacrylonitrile (PAN) derived electrospun fibres scaffolds have shown exciting potential due to reports of mechanical tunability and biocompatibility. Building on previous work we fabricate here a range of PAN fibre scaffolds with different concentrations of carbon nanotubes. We characterize them in-depth in respect to their structure, surface chemistry and mechanical properties, using scanning electron microscopy, image processing, ultramicrotomic transmission electron microscopy, x-ray nanotomography, infrared spectroscopy, atomic force microscopy and nanoindentation. Together the data demonstrate this approach to enable finetuning the mechanical properties, while keeping the structure and chemistry unaltered and hence offering ideal properties for comparative studies of the cellular mechanobiology. Finally, we confirm the biocompatibility of the scaffolds using primary rat cardiomyocytes, vascular smooth muscle (A7r5) and myoblast (C2C12) cell lines.

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.

Development of a Mass Spectrometry Imaging Method for Detecting and Mapping Graphene Oxide Nanoparticles in Rodent Tissues.

Graphene-based nanoparticles are continuously being developed for biomedical applications, and their use raises concerns about their environmental and biological impact. In the literature, some imaging techniques based on fluorescence and radioimaging have been used to explore their fate in vivo. Here, we report on the use of label-free mass spectrometry and mass spectrometry imaging (MSI) for graphene oxide (GO) and reduced graphene oxide (rGO) analyses in rodent tissues. Thereby, we extend previous work by focusing on practical questions to obtain reliable and meaningful images. Specific radical anionic carbon clusters ranging from C2-• to C9-• were observed for both GO and rGO species, with a base peak at m/z 72 under negative laser desorption ionization mass spectrometry (LDI-MS) conditions. Extension to an LDI-MSI method was then performed, thus enabling the efficient detection of GO nanoparticles in lung tissue sections of previously exposed mice. The possibility of quantifying those nanoparticles on tissue sections has also been investigated. Two different ways of building calibration curves (i.e., GO suspensions spotted on tissue sections, or added to lung tissue homogenates) were evaluated and returned similar results, with linear dynamic concentration ranges over at least 2 orders of magnitude. Moreover, intra- and inter-day precision studies have been assessed, with relative standard deviation below 25% for each concentration point of a calibration curve. In conclusion, our study confirms that LDI-MSI is a relevant approach for biodistribution studies of carbon-based nanoparticles, as quantification can be achieved, provided that nanoparticle suspension and manufacturing are carefully controlled.

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.

Bifunctional coatings: coupling an organic adhesion promoter with an anticorrosion inorganic layer.

In this work, a multifunctional non-toxic chromium free treatment is proposed. Hexavalent chromium, largely used for anticorrosion surface treatments of aluminum alloys in aeronautics, will soon be completely banned due to its high toxicity (European REACH regulation) and new solutions are required. Here, in a first step, a polymeric film was grafted at the aluminum surface by the surface induced reduction of a diazonium salt. In a second step, the grafted surface was submitted to an anodization treatment, forming a thick aluminum oxide layer protecting the underlying metal against corrosion. No change in the organic coating was detected after the second step of the process. This leads to a multilayer coating, which provides competitive results regarding both the adhesion of paint and corrosion protection.

Carbon nanotubes, but not spherical nanoparticles, block autophagy by a shape-related targeting of lysosomes in murine macrophages.

Nanoparticles (NPs) can be toxic, depending on their physico-chemical characteristics. Macroautophagy/autophagy could represent a potential underlying mechanism of this toxicity. We therefore set up a study aimed to characterize in depth the effects, on autophagy, of macrophage exposure to NPs, with a particular attention paid to the role of NP physico-chemical characteristics (specifically chemical composition, shape, size, length, crystal phase, and/or surface properties). We demonstrate that exposure to carbon nanotubes (CNT) but not to spherical NPs leads to the blockage of the autophagic flux. We further identified lysosomal dysfunction, in association with the downregulation of SNAPIN expression, as the underlying mechanism responsible for the CNT-induced autophagy blockade. These results identify for the first time the shape as a major determinant of the interaction of NPs with the autophagy pathway. Moreover, identifying the lysosomes and SNAPIN as primary targets of MWCNT toxicity opens new directions in the interpretation and understanding of nanomaterial toxicity.

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.

Single-step preparation of TiO2/MWCNT Nanohybrid materials by laser pyrolysis and application to efficient photovoltaic energy conversion.

This paper presents the continuous-flowand single-step synthesis of a TiO2/MWCNT (multiwall carbon nanotubes) nanohybrid material. The synthesis method allows achieving high coverage and intimate interface between the TiO2particles and MWCNTs, together with a highly homogeneous distribution of nanotubes within the oxide. Such materials used as active layer in theporous photoelectrode of solid-state dye-sensitized solar cells leads to a substantial performance improvement (20%) as compared to reference devices.

Carbon nanotube translocation to distant organs after pulmonary exposure: insights from in situ (14)C-radiolabeling and tissue radioimaging.

Few approaches are available to investigate the potential of carbon nanotubes (CNTs) to translocate to distant organs following lung exposure, although this needs to be taken into account to evaluate potential CNT toxicity. Here, we report a method for quantitative analysis of the tissue biodistribution of multiwalled CNTs (MWCNTs) as a function of time. The method relies on the use of in situ (14)C-radiolabeled MWCNTs and combines radioimaging of organ tissue sections to ex vivo analysis of MWCNTs by electron microscopy. To illustrate the usefulness of this approach, mice were exposed to a single dose of 20 µg of (14)C-labeled MWCNTs by pharyngeal aspiration and were subjected to a follow-up study over one year. After administration, MWCNT were cleared from the lungs, but there was a concomitant relocation of these nanoparticles to distant organs starting throughout the follow-up period, with nanoparticle accumulation increasing with time. After one year, accumulation of MWCNTs was documented in several organs, including notably the white pulp of the spleen and the bone marrow. This study shows that the proposed method may be useful to complement other approaches to address unresolved toxicological issues associated with CNTs. These issues include their persistence over long periods in extrapulmonary organs, the relationship between the dose and the extent of translocation, and the effects of "safety by design" on those processes. The same approach could be used to study the translocation propensity of other nanoparticles containing carbon atoms.

Intracellular fate of carbon nanotubes inside murine macrophages: pH-dependent detachment of iron catalyst nanoparticles.

BACKGROUND: Carbon nanotubes (CNT) are a family of materials featuring a large range of length, diameter, numbers of walls and, quite often metallic impurities coming from the catalyst used for their synthesis. They exhibit unique physical properties, which have already led to an extensive development of CNT for numerous applications. Because of this development and the resulting potential increase of human exposure, an important body of literature has been published with the aim to evaluate the health impact of CNT. However, despite evidences of uptake and long-term persistence of CNT within macrophages and the central role of those cells in the CNT-induced pulmonary inflammatory response, a limited amount of data is available so far on the CNT fate inside macrophages. Therefore, the overall aim of our study was to investigate the fate of pristine single walled CNT (SWCNT) after their internalization by macrophages. METHODS: To achieve our aim, we used a broad range of techniques that aimed at getting a comprehensive characterization of the SWCNT and their catalyst residues before and after exposure of murine macrophages: X-ray diffraction (XRD), High Resolution (HR) Transmission Electron Microscopy (TEM), High Angle Annular Dark Field-Scanning TEM (HAADF-STEM) coupled to Electron Energy Loss Spectroscopy (EELS), as well as micro-X-ray fluorescence mapping (µXRF), using synchrotron radiation. RESULTS: We showed 1) the rapid detachment of part of the iron nanoparticles initially attached to SWCNT which appeared as free iron nanoparticles in the cytoplasm and nucleus of CNT-exposed murine macrophages, and 2) that blockade of intracellular lysosomal acidification prevented iron nanoparticles detachment from CNT bundles and protected cells from CNT downstream toxicity. CONCLUSIONS: The present results, while obtained with pristine SWCNT, could likely be extended to other catalyst-containing nanomaterials and surely open new ways in the interpretation and understanding of CNT toxicity.

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.

Metal-free nitrogen-containing carbon nanotubes prepared from triazole and tetrazole derivatives show high electrocatalytic activity towards the oxygen reduction reaction in alkaline media.

High-performance oxygen reduction reaction (ORR) catalysts based on metal-free nitrogen-containing precursors and carbon nanotubes are reported. The investigated systems allow the evaluation of the effect of nitrogen-containing groups towards ORR and the results show that the catalysts are compatible with the conditions encountered in alkaline fuel cells, exhibiting good catalytic activity and stability compared with conventional Pt/C electrocatalyst.

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.