Functionalised thin multi-walled carbon nanotubes are less toxic to lung cells in vitro than their native counterparts.

Some multi-walled carbon nanotubes (MWCNTs) induce mesothelioma in rodents, straight MWCNTs showing a more pronounced effect than tangled MWCNTs. As primary and secondary genotoxicity may play a role in MWCNT carcinogenesis, we used a battery of assays for DNA damage and micronuclei to compare the genotoxicity of straight (MWCNT-S) and tangled MWCNTs (MWCNT-T) in vitro (primary genotoxicity) and in vivo (primary or secondary genotoxicity). C57Bl/6 mice showed a dose-dependent increase in DNA strand breaks, as measured by the comet assay, in lung cells 24 h after a single pharyngeal aspiration of MWCNT-S (1–200 μg/mouse). An increase was also observed for DNA strand breaks in lung and bronchoalveolar lavage (BAL) cells and for micronucleated alveolar type II cells in mice exposed to aerosolized MWCNT-S (8.2–10.8 mg/m3) for 4 d, 4 h/d. No systemic genotoxic effects, assessed by the γ-H2AX assay in blood mononuclear leukocytes or by micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow or blood, were observed for MWCNT-S by either exposure technique. MWCNT-T showed a dose-related decrease in DNA damage in BAL and lung cells of mice after a single pharyngeal aspiration (1–200 μg/mouse) and in MNPCEs after inhalation exposure (17.5 mg/m3). In vitro in human bronchial epithelial BEAS-2B cells, MWCNT-S induced DNA strand breaks at low doses (5 and 10 μg/cm2), while MWCNT-T increased strand breakage only at 200 μg/cm2. Neither of the MWCNTs was able to induce micronuclei in vitro. Our findings suggest that both primary and secondary mechanisms may be involved in the genotoxicity of straight MWCNTs.

The influence of multiwalled carbon nanotubes on polycyclic aromatic hydrocarbon (PAH) bioavailability and toxicity to soil microbial communities in alfalfa rhizosphere

Preparation of multi-walled carbon nanotubes grafted with synthetic poly(l-lysine) through surface-initiated ring-opening polymerization

Fibrogenic carbon nanotubes (CNTs) induce the polarization of M1 and M2 macrophages in mouse lungs. Polarization of the macrophages differentiates the production of proinflammatory and pro‐resolving lipid mediators (LMs) to mediate acute inflammation and its resolution in a time‐dependent manner. In the present study, we examined the molecular mechanism by which multi‐walled CNTs (MWCNTs) induce M1 polarization in vitro, with focus on induction of arachidonate 5‐lipoxygenase (ALOX5), which is required to produce proinflammatory LMs, such as leukotriene B4 (LTB4). Treatment of J774A.1 macrophages with MWCNTs at a concentration of 2.5 µg/mL increased the expression of ALOX5 mRNA by 2.0‐fold at 1‐day and 2.5‐fold at 3‐day post‐exposure. At a concentration of 10 µg/mL, MWCNTs increased the ALOX5 mRNA expression by 3.5‐fold at 1‐day and 5.0‐fold at 3‐day post‐exposure. Immunoblotting shows that MWCNTs significantly increased the ALOX5 protein expression in macrophages. Luciferase reporter assays with the mouse ALOX5 promoter of 2 kilobase upstream of translation start codon demonstrate that the ALOX5 promoter activity increased more than 5‐fold over background. This activity was significantly elevated following 5’‐deletion analyses of ALOX5 promoter and further enhanced by MWCNT treatment, implicating a transcriptional mechanism in the induction of ALOX5. MWCNTs preferentially induced the expression of CD68, a cell surface marker of M1, by 2.5‐fold and induction was persistently high to 3‐day post‐exposure. Moreover, both the expression and activity of the inducible nitric oxide synthase, an intracellular marker of M1 macrophages, were increased by MWCNTs, indicating induction of M1 polarization. Consistent with this notion, MWCNTs increased the capacity of the macrophages to produce proinflammatory cytokines, such as tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β), and proinflammatory LMs, such as LTB4 and prostaglandin E2 (PGE2), as measured by enzyme‐linked immunosorbent assays. Taken together, these results support that persistent exposure to MWCNTs polarizes macrophages to M1 cells with induction of ALOX5 and elevated production of proinflammatory LMs to boost the acute inflammatory response to fibrogenic nanoparticles.

Presently, the effects of crop roots on crop root zone thermal characteristics are poorly understood, and new fertilizers are rarely considered from the perspective of changing crop root zone thermal characteristics. This study explored the effect of applying two new fertilizers, multiwalled carbon nanotubes (MWCNTs) and Bacillus atrophaeus (B. atrophaeus), on the crop root zone thermal characteristics of saline farmland soils through in situ measurements. The results showed that MWCNTs and B. atrophaeus could indirectly affect crop root zone thermal characteristics by changing the crop root growth. Combined application of MWCNTs and B. atrophaeus could promote both to induce positive effects, promote crop root growth, and significantly alleviate the adverse effects of soil salinization. The thermal conductivity and heat capacity of the shallow root zone were reduced due to the presence of crop roots, while the opposite was true in the deep root zone. For example, the thermal conductivity of the 0–5 cm rich root zone in the MWCNT treatment was 0.8174 W m−1 ·K−1, and the thermal conductivity of the poor root zone was 13.42% higher than that of the rich root zone. MWCNTs and B. atrophaeus can also change the spatial distribution of soil moisture, soil salt, and soil particle size characteristics by influencing the root-soil interactions and indirectly affecting crop root zone thermal characteristics. In addition, MWCNTs and B. atrophaeus could directly affect the root zone thermal characteristics by changing the soil properties. The higher the soil salt content was, the more obvious the effect of the MWCNTs and B. atrophaeus on the crop root zone thermal characteristics. The thermal conductivity and heat capacity of the crop root zone were positively correlated with the soil moisture content, soil salt content and soil particle specific surface area and negatively correlated with the soil particle size and the fresh and dry root weights. In summary, MWCNTs and B. atrophaeus significantly affected crop root zone thermal characteristics directly and indirectly and could adjust the temperature of the crop root zone.

Toxicity of carbon nanotubes to the activated sludge process

The usage of multi-walled carbon nanotubes (MWCNT) has increased exponentially in the past years, but, potential toxicity mechanisms are not clear. We studied the transcriptomic alterations induced by one multi-walled carbon nanotube (MWCNT) and its -OH and -COOH functionalized derivatives in human HepG2 cells. We showed that all three MWCNT treatments induced alterations in stress-related signaling pathways, inflammation-related signaling pathways, cholesterol synthesis pathways, proliferation-related pathways, senescence-related pathways and cancer-related pathways. In stress-related pathways, the acute phase response was induced in all three MWCNTs and all doses treated and ranked high. Other stress-related pathways were also related to the oxidative-induced signaling pathways, such as NRF-2 mediated oxidative stress response, hepatic fibrosis/Stella cell activation, iNOS signaling, and Hif1α signaling. Many inflammation-related pathways were altered, such as IL-8, IL-6, TNFR1, TNFR2, and NF-κB signaling pathways. These results were consistent with our previous results with exposures to the same three multi-walled carbon nanotubes in human lung BEAS-2B and also with results in mice and rats. From the microRNA target filter analysis, TXNIP & miR-128-3p interaction was present in all three MWCNT treatments, and maybe important for the induction of oxidative stress. CXCL-8 & miR-146-5p and Wee1 & miR-128-3p were only present in the cells treated with the parent and the OH-functionalized MWCNTs. These mRNA-miRNA interactions were involved in oxidative stress, inflammation, cell cycle, cholesterol biosynthesis and cancer related pathways. Target filter analysis also showed altered liver hyperplasia/hyperproliferation and hepatic cancer pathways. In short, target filter analysis complemented the transcriptomic analysis and pointed to specific gene/microRNA interactions that can help inform mechanism of action. Moreover, our study showed that the signaling pathways altered in HepG2 cells correlated well with the toxicity and carcinogenicity observed in vivo, indicating that HepG2 may be a good in vitro predictive model for MWCNT toxicity studies.

Probing plasma-induced defect formation and oxidation in carbon nanotubes by Raman dispersion spectroscopy

The effect of high-temperature heat treatment on purity and structural changes of multiwalled carbon nanotubes (MWCNTs) were studied by subjecting the raw MWCNTs (pristine MWCNTs) to 2600∘C for 60 and 120 min. Thermogravimetric analysis (TGA), X-ray diffraction, Raman spectroscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to study the effect of heat-treatment duration on the purity and structural changes of MWCNTs. Results show that high-temperature heat treatment can be used to purify MWCNTs with proper optimization of treatment time. It was observed that 60 min heat treatment of raw MWCNTs imparts high purity and structural perfection to MWCNTs, while 120 min heat treatment imparts structural degradation to MWCNTs with collapse of the innermost shells. The present study indicates that metal impurities act as moderators in controlling the degradation of MWCNTs up to certain duration, and once the metal impurities escape completely, further heat treatment degrades the structure of MWCNTs.

The medical applications of carbon nanotubes (CNTs) have garnered much attention. However, evaluating the safety of CNTs remains difficult, and no consensus has been reached. Moreover, assessing the biosafety of multi-walled CNTs (MWCNTs), which can become tangled during manufacturing, is challenging because they do not readily disperse. We studied how the dispersion state of tangled MWCNTs affects their cytotoxicity, using three sonicators. Flotube 9110 (FT9110), tangled MWCNTs, were dispersed in two dispersants (fetal bovine serum and polysorbate 80) using a new type of sonicator (PR-1) and two conventional sonicators. The size and cytotoxicity of the dispersed FT9110 were measured using the BEAS-2B human bronchial epithelial cell line. The PR-1 dispersed the FT9110 to agglomerates <200 nm in diameter; FT9110 dispersed with the PR-1 did not show cytotoxicity regardless of dispersant. The other sonicators dispersed the FT9110 to particles >1000 nm in diameter, and cytotoxicity depended on the dispersant. We found that excluding cells adhered to agglomerated FT9110 before evaluating cytotoxicity can lead to false-positive results. The PR-1 sonicator dispersed tangled FT9110 to many single fibers, which showed lower cytotoxicity than conventionally-sonicated MWCNTs. We suggest that dispersion state should be accounted for when evaluating the cytotoxicity of MWCNTs.

BackgroundIn vitro models are widely used in nanotoxicology. In these assays, a careful documentation of the fraction of nanomaterials that reaches the cells, i.e. the in vitro delivered dose, is a critical element for the interpretation of the data. The in vitro delivered dose can be measured by quantifying the amount of material in contact with the cells, or can be estimated by applying particokinetic models. For carbon nanotubes (CNTs), the determination of the in vitro delivered dose is not evident because their quantification in biological matrices is difficult, and particokinetic models are not adapted to high aspect ratio materials. Here, we applied a rapid and direct approach, based on femtosecond pulsed laser microscopy (FPLM), to assess the in vitro delivered dose of multi-walled CNTs (MWCNTs).Methods and resultsWe incubated mouse lung fibroblasts (MLg) and differentiated human monocytic cells (THP-1) in 96-well plates for 24 h with a set of different MWCNTs. The cytotoxic response to the MWCNTs was evaluated using the WST-1 assay in both cell lines, and the pro-inflammatory response was determined by measuring the release of IL-1β by THP-1 cells. Contrasting cell responses were observed across the MWCNTs. The sedimentation rate of the different MWCNTs was assessed by monitoring turbidity decay with time in cell culture medium. These turbidity measurements revealed some differences among the MWCNT samples which, however, did not parallel the contrasting cell responses. FPLM measurements in cell culture wells revealed that the in vitro delivered MWCNT dose did not parallel sedimentation data, and suggested that cultured cells contributed to set up the delivered dose. The FPLM data allowed, for each MWCNT sample, an adjustment of the measured cytotoxicity and IL-1β responses to the delivered doses. This adjusted in vitro activity led to another toxicity ranking of the MWCNT samples as compared to the unadjusted activities. In macrophages, this adjusted ranking was consistent with existing knowledge on the impact of surface MWCNT functionalization on cytotoxicity, and might better reflect the intrinsic activity of the MWCNT samples.ConclusionThe present study further highlights the need to estimate the in vitro delivered dose in cell culture experiments with nanomaterials. The FPLM measurement of the in vitro delivered dose of MWCNTs can enrich experimental results, and may refine our understanding of their interactions with cells.

Nanomaterials such as single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) may repeatedly enter the soil environment with unknown adverse consequences. To provide the information on the effects of repeated exposure of CNTs, we determined the response of soil enzyme activity and soil basal respiration (SBR) through a two-week incubation of farmland soil repeatedly treated with different concentrations of CNTs (100, 200, 500 mg kg−1 for SWCNTs and 100, 500, 1000 mg Kg−1 for MWCNTs). The activities of catalase, alkaline phosphatase, and invertase and SBR were measured after one- and two-time treatments. The repeated contamination of SWCNTs and MWCNTs repressed the activity of alkaline phosphatase and invertase in the 14-day incubation. Alkaline phosphatase and invertase were more sensitive indicators of CNTs’ contamination than catalase and soil basal respiration. High concentration of the SWCNTs stimulated SBR while the lower concentration suppressed SBR. The recurred exposure of SWCNTs and MWCNTs repressed the activity of catalase and invertase. The obtained results indicated that the soil microorganisms were suppressed under repeated pollution, as suggested by the same suppressed response of SBR between SWCNTs and MWCNTs treatment, except for the concentration of 500 mg kg−1.

Cobalt (catalyst) is sputtered at different power levels (20, 50, and 80 W) on graphite foil on which multiwalled carbon nanotubes are subsequently grown directly by chemical vapor deposition. Next, the multiwalled carbon nanotube electrodes are immersed in a nitric acid solution and then annealed at different temperatures (200, 350, and ). At the 1000th cycle of potential cycling, a maximum specific capacitance of 109 F/g is obtained under the best conditions (Co sputtering and annealing ). These show high electrochemical stability and good specific capacitance. Furthermore, viewed under transmission electron microscope and measured by electrochemical analyzer, the higher the annealing temperature, the more integral the sidewall surface and the lower the equivalent series resistance of the multiwalled carbon nanotubes.

Synthesis and properties of poly(ether urethane) membranes filled with isophorone diisocyanate-grafted carbon nanotubes

The aim of this study was to evaluate adverse effects of multiwalled carbon nanotubes (MWCNT), produced for industrial purposes, on the human epithelial cell line A549. MWCNT were dispersed in dipalmitoyl lecithin (DPL), a component of pulmonary surfactant, and the effects of dispersion in DPL were compared to those in two other media: ethanol (EtOH) and phosphate-buffered saline (PBS). Effects of MWCNT were also compared to those of two asbestos fibers (chrysotile and crocidolite) and carbon black (CB) nanoparticles, not only in A549 cells but also in mesothelial cells (MeT5A human cell line), used as an asbestos-sensitive cell type. MWCNT formed agglomerates on top of both cell lines (surface area 15–35 μm2) that were significantly larger and more numerous in PBS than in EtOH and DPL. Whatever the dispersion media, incubation with 100 μg/ml MWCNT induced a similar decrease in metabolic activity without changing cell membrane permeability or apoptosis. Neither MWCNT cellular internalization nor oxidative stress was observed. In contrast, asbestos fibers penetrated into the cells, decreased metabolic activity but not cell membrane permeability, and increased apoptosis, without decreasing cell number. CB was internalized without any adverse effects. In conclusion, this study demonstrates that MWCNT produced for industrial purposes exert adverse effects without being internalized by human epithelial and mesothelial pulmonary cell lines.