Multiwall Carbon Nanotubes Directly Promote Fibroblast–Myofibroblast and Epithelial–Mesenchymal Transitions through the Activation of the TGF‐β/Smad Signaling Pathway

A number of studies have demonstrated that MWCNTs induce granuloma formation and fibrotic responses in vivo, and it has been recently reported that MWCNT‐induced macrophage activation and subsequent TGF‐β secretion contribute to pulmonary fibrotic responses. However, their direct effects against alveolar type‐II epithelial cells and fibroblasts and the corresponding underlying mechanisms remain largely unaddressed. Here, MWCNTs are reported to be able to directly promote fibroblast‐to‐myofibroblast conversion and the epithelial–mesenchymal transition (EMT) through the activation of the TGF‐β/Smad signaling pathway. Both of the cell transitions may play important roles in MWCNT‐induced pulmonary fibrosis. Firstly, in‐vivo and in‐vitro data show that long MWCNTs can directly interact with fibroblasts and epithelial cells, and some of them may be uptaken into fibroblasts and epithelial cells by endocytosis. Secondly, long MWCNTs can directly activate fibroblasts and increase both the basal and TGF‐β1‐induced expression of the fibroblast‐specific protein‐1, α‐smooth muscle actin, and collagen III. Finally, MWCNTs can induce the EMT through the activation of TGF‐β/Smad2 signaling in alveolar type‐II epithelial cells, from which some fibroblasts involved in pulmonary fibrosis are thought to originate. These observations suggest that the activation of the TGF‐β/Smad2 signaling plays a critical role in the process of the fibroblast‐to‐myofibroblast transition and the EMT induced by MWCNTs.     

The Genetic Heterogeneity among Different Mouse Strains Impacts the Lung Injury Potential of Multiwalled Carbon Nanotubes

Genetic variation constitutes an important variable impacting the susceptibility to inhalable toxic substances and air pollutants, as reflected by epidemiological studies in humans and differences among animal strains. While multiwalled carbon nanotubes (MWCNTs) are capable of causing lung fibrosis in rodents, it is unclear to what extent the genetic variation in different mouse strains influence the outcome. Four inbred mouse strains, including C57Bl/6, Balb/c, NOD/ShiLtJ, and A/J, to test the pro‐fibrogenic effects of a library of MWCNTs in vitro and in vivo are chosen. Ex vivo analysis of IL‐1β production in bone marrow‐derived macrophages (BMDMs) as molecular initiating event (MIE) is performed. The order of cytokine production (Balb/c > A/J > C57Bl/6 > NOD/ShiLtJ) in BMDMs is also duplicated during assessment of IL‐1β production in the bronchoalveolar lavage fluid of the same mouse strains 40 h after oropharyngeal instillation of a representative MWCNT. Animal test after 21 d also confirms a similar hierarchy in TGF‐β1 production and collagen deposition in the lung. Statistical analysis confirms a correlation between IL‐1β production in BMDM and the lung fibrosis. All considered, these data demonstrate that genetic background indeed plays a major role in determining the pro‐fibrogenic response to MWCNTs in the lung.     

TGF‐β1 Conjugated to Gold Nanoparticles Results in Protein Conformational Changes and Attenuates the Biological Function

Gold nanoparticles (AuNPs) are widely used as carriers or therapeutic agents due to their great biocompatibility and unique physical properties. Transforming growth factor‐beta 1 (TGF‐β1), a member of the cysteine‐knot structural superfamily, plays a pivotal role in many diseases and is known as an immunosuppressive agent that attenuates immune response resulting in tumor growth. The results reported herein reflect strong interactions between TGF‐β1 and the surface of AuNPs when incubated with serum‐containing medium, and demonstrate a time‐ and dose‐dependent pattern. Compared with other serum proteins that can also bind to the AuNP surface, AuNP–TGFβ1 conjugate is a thermodynamically favored compound. Epithelial cells undergo epithelial–mesenchymal transition (EMT) upon treatment with TGF‐β1; however, treatment with AuNPs reverses this effect, as detected by cell morphology and expression levels of EMT markers. TGF‐β1 is found to bind to AuNPs through S–Au bonds by X‐ray photoelectron spectroscopy. Fourier transform infrared spectroscopy is employed to analyze the conformational changes of TGF‐β1 on the surface of AuNPs. The results indicate that TGF‐β1 undergoes significant conformational changes at both secondary and tertiary structural levels after conjugation to the AuNP surface, which results in the deactivation of TGF‐β1 protein. An in vivo experiment also shows that addition of AuNPs attenuates the growth of TGF‐β1‐secreting murine bladder tumor 2 cells in syngeneic C3H/HeN mice, but not in immunocompromised NOD‐SCID mice, and this is associated with an increase in the number of tumor‐infiltrating CD4⁺ and CD8⁺ T lymphocytes and a decrease in the number of intrasplenic Foxp3(+) lymphocytes. The findings demonstrate that AuNPs may be a promising agent for modulating tumor immunity through inhibiting immunosuppressive TGF‐β1 signaling.     

Precise and Arbitrary Deposition of Biomolecules onto Biomimetic Fibrous Matrices for Spatially Controlled Cell Distribution and Functions

Advances in nano‐/microfabrication allow the fabrication of biomimetic substrates for various biomedical applications. In particular, it would be beneficial to control the distribution of cells and relevant biomolecules on an extracellular matrix (ECM)‐like substrate with arbitrary micropatterns. In this regard, the possibilities of patterning biomolecules and cells on nanofibrous matrices are explored here by combining inkjet printing and electrospinning. Upon investigation of key parameters for patterning accuracy and reproducibility, three independent studies are performed to demonstrate the potential of this platform for: i) transforming growth factor (TGF)‐β1‐induced spatial differentiation of fibroblasts, ii) spatiotemporal interactions between breast cancer cells and stromal cells, and iii) cancer‐regulated angiogenesis. The results show that TGF‐β1 induces local fibroblast‐to‐myofibroblast differentiation in a dose‐dependent fashion, and breast cancer clusters recruit activated stromal cells and guide the sprouting of endothelial cells in a spatially resolved manner. The established platform not only provides strategies to fabricate ECM‐like interfaces for medical devices, but also offers the capability of spatially controlling cell organization for fundamental studies, and for high‐throughput screening of various biomolecules for stem cell differentiation and cancer therapeutics.     

Morphological and compositional engineering of Ni/carbon nanotube composite film via a novel cyclic voltammetric route

Ni/multi-walled carbon nanotubes (MWCNTs) composite films were deposited on the glassy carbon electrode (GCE) by a Ni plating bath containing homogeneously dispersed MWCNTs using polyvinylpyrrolidone (PVP) as dispersion additive. Incorporation of MWCNTs into Ni matrix was greatly enhanced by the application of cyclic voltammetric (CV) deposition technique. The structure and nature of the Ni/MWCNT were characterized by field emission scanning electron microscope (FE-SEM) and X-ray diffraction (XRD). The results show that the content of MWCNT and the morphology of the deposited Ni/MWCNT composite film can be controlled by selecting the appropriate electroplating conditions. Further study indicates that the obtained Ni/MWCNT showed excellent electro-catalytic activity for the oxidation of ethanol in alkaline solution.     

Biologically Inspired,Cell‐Selective Release of Aptamer‐Trapped Growth Factors by Traction Forces

Biomaterial scaffolds that are designed to incorporate dynamic, spatiotemporal information have the potential to interface with cells and tissues to direct behavior. Here, a bioinspired, programmable nanotechnology‐based platform is described that harnesses cellular traction forces to activate growth factors, eliminating the need for exogenous triggers (e.g., light), spatially diffuse triggers (e.g., enzymes, pH changes), or passive activation (e.g., hydrolysis). Flexible aptamer technology is used to create modular, synthetic mimics of the Large Latent Complex that restrains transforming growth factor‐β1 (TGF‐β1). This flexible nanotechnology‐based approach is shown here to work with both platelet‐derived growth factor‐BB (PDGF‐BB) and vascular endothelial growth factor (VEGF‐165), integrate with glass coverslips, polyacrylamide gels, and collagen scaffolds, enable activation by various cells (e.g., primary human dermal fibroblasts, HMEC‐1 endothelial cells), and unlock fundamentally new capabilities such as selective activation of growth factors by differing cell types (e.g., activation by smooth muscle cells but not fibroblasts) within clinically relevant collagen sponges.     

Carbon Nanotubes Disrupt Iron Homeostasis and Induce Anemia of Inflammation through Inflammatory Pathway as a Secondary Effect Distant to Their Portal‐of‐Entry

Although numerous toxicological studies have been performed on carbon nanotubes (CNTs), a few studies have investigated their secondary and indirect effects beyond the primary target tissues/organs. Here, a cascade of events are investigated: the initiating event and the subsequent key events necessary for the development of phenotypes, namely CNT‐induced pro‐inflammatory effects on iron homeostasis and red blood cell formation, which are linked to anemia of inflammation (AI). A panel of CNTs are prepared including pristine multiwall CNTs (P‐MWCNTs), aminated MWCNTs (MWCNTs‐NH₂), polyethylene glycol MWCNTs (MWCNTs‐PEG), polyethyleneimine MWCNTs (MWCNTs‐PEI), and carboxylated MWCNTs (MWCNTs‐COOH). It has been demonstrated that all CNT materials provoke inflammatory cytokine interleukin‐6 (IL‐6) production and stimulate hepcidin induction, associated with disordered iron homeostasis, irrespective of exposure routes including intratracheal, intravenous, and intraperitoneal administration. Meanwhile, PEG and COOH modifications can ameliorate the activation of IL‐6‐hepcidin signaling. Long‐term exposure of MWCNTs results in AI and extramedullary erythropoiesis. Thus, an adverse outcome pathway is identified: MWCNT exposure leads to inflammation, hepatic hepcidin induction, and disordered iron metabolism. Together, the combined data depict the hazardous secondary toxicity of CNTs in incurring anemia through inflammatory pathway. This study will also open a new avenue for future investigations on CNT‐induced indirect and secondary adverse effects.     

Human‐Finger Electronics Based on Opposing Humidity‐Resistance Responses in Carbon Nanofilms

Carbon nanomaterials have excellent humidity sensing properties. Here, it is demonstrated that multiwalled carbon‐nanotube (MWCNT)‐ and reduced‐graphene‐oxide (rGO)‐based conductive films have opposite humidity/electrical resistance responses: MWCNTs increase their electrical resistance (positive response) and rGOs decrease their electrical resistance (negative response). The authors propose a new phenomenology that describes a “net”‐like model for MWCNT films and a “scale”‐like model for rGO films to explain these behaviors based on contributions from junction resistances (at interparticle junctions) and intrinsic resistances (of the particles). This phenomenology is accordingly validated via a series of experiments, which complement more classical models based on proton conductivity. To explore the practical applications of the converse humidity/resistance responses, a humidity‐insensitive MWCNT/rGO hybrid conductive films is developed, which has the potential to greatly improve the stability of carbon‐based electrical device to humidity. The authors further investigate the application of such films to human‐finger electronics by fabricating transparent flexible devices consisting of a polyethylene terephthalate substrate equipped with an MWCNT/rGO pattern for gesture recognition, and MWCNT/rGO/MWCNT or rGO/MWCNT/rGO patterns for 3D noncontact sensing, which will be complementary to existing 3D touch technology.     

NADPH Oxidase‐Dependent NLRP3 Inflammasome Activation and its Important Role in Lung Fibrosis by Multiwalled Carbon Nanotubes

The purpose of this paper is to elucidate the key role of NADPH oxidase in NLRP3 inflammasome activation and generation of pulmonary fibrosis by multi‐walled carbon nanotubes (MWCNTs). Although it is known that oxidative stress plays a role in pulmonary fibrosis by single‐walled CNTs, the role of specific sources of reactive oxygen species, including NADPH oxidase, in inflammasome activation remains to be clarified. In this study, three long aspect ratio (LAR) materials (MWCNTs, single‐walled carbon nanotubes, and silver nanowires) are used to compare with spherical carbon black and silver nanoparticles for their ability to trigger oxygen burst activity and NLRP3 assembly. All LAR materials but not spherical nanoparticles induce robust NADPH oxidase activation and respiratory burst activity in THP‐1 cells, which are blunted in p22^phox‐deficient cells. The NADPH oxidase is directly involved in lysosomal damage by LAR materials, as demonstrated by decreased cathepsin B release and IL‐1β production in p22^phox‐deficient cells. Reduced respiratory burst activity and inflammasome activation are also observed in bone marrow‐derived macrophages from p47^phox‐deficient mice. Moreover, p47^phox‐deficient mice have reduced IL‐1β production and lung collagen deposition in response to MWCNTs. Lung fibrosis is also suppressed by N‐acetyl‐cysteine in wild‐type animals exposed to MWCNTs.     

Identification of the anthelix motif in the TGF‐β superfamily by molecular 3D‐Rapid Prototyping

3D‐Rapid Prototyping (3D‐RP) is a novel technique for the construction of highly accurate three‐dimensional polyamide models of biomolecules. This method has been shown to be a valuable tool in the modeling of protein‐protein‐interactions as well as in the analysis of surface topography. Using this technique we were recently able to identify a so far unknown structure on the concave side of bone morphogenetic protein 2 (BMP‐2). Since this structure is the imprint of a left‐handed helix we have called this negative an unthelix. Obviously this novel structural feature of BMP‐2 may act as a binding side for endogenous ligands. BMP‐2 belongs to the highly conserved Transforming Growth Factor‐β (TGF‐β) superfamily, a large group of multifunctional peptides controlling differentiation, proliferation and repair in multicellular organism. The protomer structures of all members share a cystine‐knot motif as a characteristic feature. The question therefore arose whether a) the novel anthelical motif found in BMP‐2 is a common structural feature of this family and b) if there are any differences in terms of pitch and radius of the anthelix. As anthelical structures are difficult to visualize and nearly impossible to quantify using 3D molecular visualization software we constructed models of BMP‐2, BMP‐7 and TGF‐β2 from X‐ray crystallographic data by 3D‐Rapid Prototyping (3D‐RP). The anthelix motif was found in BMP‐2, BMP‐7 and TGF‐β2 with similar values for pitch (ca. 8‐10 nm) and radius (ca. 0.5‐0.7 nm). In contrast the anthelical motif was not found in a 3D‐RP model of human chorionic gonadotropin (HCG) which is also a member of the cystine‐knot family but doesn’t belong to the TGF‐β superfamily. These results were corroborated by measurements of the intersubunit angle of these dimeric proteins (141‐149°) and the distances between the center of mass (1.68‐1.96 nm) of the subunits both of which appear to be determinants of the anthelical pitch. We conclude that the anthelical groove on the concave side is a common structural motif of BMP‐2, BMP‐7 and TGF‐β2 and maybe of the whole group of the TGF‐β superfamily.     

A Novel Microwave‐Assisted Carbothermic Route for the Production of Copper‐Carbon Nanotube Metal Matrix Composites Directly from Copper Oxide

Cu₂O was reduced to copper via a microwave‐assisted carbothermic route using multi‐walled carbon nanotubes (MWCNTs) as the carbon source. The reaction atmosphere as well as the degree of mixing of Cu₂O and MWCNTs was varied, and the resulting products were characterized as a function of microwave exposure time. Irradiation of thoroughly mixed Cu₂O and MWCNTs under argon for 45 s produced Cu‐MWCNT composites with high MWCNT loading and high hardness. This new approach for fabricating carbon nanotube‐reinforced metal matrix composites eliminates many of the challenges associated with traditional methods while requiring a fraction of the time and energy.     

Effective load transfer by a chromium carbide nanostructure in a multi-walled carbon nanotube/copper matrix composite

Multi-walled carbon nanotube (MWCNT) reinforced copper (Cu) matrix composites, which exhibit chromium (Cr) carbide nanostructures at the MWCNT/Cu interface, were prepared through a carbide formation using CuCr alloy powder. The fully densified and oriented MWCNTs dispersed throughout the composites were prepared using spark plasma sintering (SPS) followed by hot extrusion. The tensile strengths of the MWCNT/CuCr composites increased with increasing MWCNTs content, while the tensile strength of MWCNT/Cu composite decreased from that of monolithic Cu. The enhanced tensile strength of the MWCNT/CuCr composites is a result of possible load-transfer mechanisms of the interfacial Cr carbide nanostructures. The multi-wall failure of MWCNTs observed in the fracture surface of the MWCNT/CuCr composites indicates an improvement in the load-bearing capacity of the MWCNTs. This result shows that the Cr carbide nanostructures effectively transferred the tensile load to the MWCNTs during fracture through carbide nanostructure formation in the MWCNT/Cu composite.     

Single-step synthesis of MWCNT/ZnO nanocomposite using co-chemical vapor deposition method

Single-step synthesis of MWCNT and ZnO nanocomposite was conducted by co-chemical vapor deposition method. Electron microscopic analysis revealed that the fabricated nanostructures consisted of MWCNTs with a diameter of 60-70 nm which were coated with ZnO nanoparticles with an average size of 20-30 nm. The growth of ZnO nanoparticles took place after the formation of MWCNTs. EDS and XRD analyses could confirm the high crystallinity of ZnO deposited on the MWCNT surface. In comparison with pristine MWCNTs and ZnO nanoparticles, the UV absorption of MWCNT/ZnO nanocomposite was changed through modification with ZnO nanoparticles.     

In situ assessment of carbon nanotube flow and filtration monitoring through glass fabric using electrical resistance measurement

Filtration of nanofillers into porous fabric media is still an issue during the preparation of advanced fiber-reinforced composites. The assessment of resin/multiwall carbon nanotube (MWCNT) flow, MWCNT filtration, and the cure monitoring of glass fiber/carbon nanotube-polyester composites by means of the measurement of the electrical resistance was introduced. The vacuum-assisted resin transfer molding technique was used. The electrical resistances measured over the span of a composite were qualitatively correlated with MWCNT flow and the degree of MWCNT filtration. It was found that while the complexity of the fabrics could likely introduce preferential deposition of MWCNTs, their filtration is mainly affected by their dispersion state in the resin suspension. Relationships among critical parameters such as the lengths and diameters of MWCNTs, the inter- and intra-tow dimensions of glass fabrics, the dispersion level of MWCNTs, and the viscosity of nanocomposite samples are discussed and correlated to the filtration, cure, and flow phenomena. We showed that our method can also serve as an early warning to obviate defects in the resulting composite.     

Synthesis of polyaniline/multi-walled carbon nanotube nanocomposites in water/oil microemulsion

A water/oil microemulsion system having been successfully used for synthesizing polyaniline(PANi) nanoparticles, was employed for preparing PANi/multi-walled carbon nanotube (MWCNT) nanocomposites via in situ chemical oxidative polymerization. The structures and the electrical property of PANi/MWCNT nanocomposites were also studied. The studies showed that PANi could coat MWCNTs to form nanocables with core-shell structure, and the backbone structure of PANi was not damaged by the introduction of MWCNTs. The conductivities of PANi/MWCNT nanocomposites were higher than that of PANi. Moreover, a model was supposed to be used for describing a PANi/MWCNT nanocable formation by in situ microemulsion polymerization.     

Broad‐Spectrum Antibacterial Activity of Carbon Nanotubes to Human Gut Bacteria

Carbon nanotubes (CNTs) hold promise in manufacturing, environmental, and biomedical applications, as well as food and agricultural industries. Previous observations have shown that CNTs have antimicrobial activity; however, the impact of CNTs to human gut microbes has not been investigated. Here, the antibacterial activity of CNTs against the microbes commonly encountered in the human digestion system—L. acidophilus, B. adolescentis, E. coli, E. faecalis, and S. aureus—are evaluated. The bacteria studied include pathogenic and non‐pathogenic, gram‐positive and negative, and both sphere and rod strains. In this study, CNTs, including single‐walled CNTs (SWCNTs, 1–3 μm), short and long multi‐walled CNTs (s‐MWCNTs: 0.5–2 μm; l‐MWCNTs: >50 μm), and functionalized multi‐walled CNTs (hydroxyl‐ and carboxyl‐modification, 0.5–2 μm), all have broad‐spectrum antibacterial effects. Notably, CNTs may selectively lyse the walls and membranes of human gut microbes, depending on not only the length and surface functional groups of CNTs, but also the shapes of the bacteria. The mechanism of antibacterial activity is associated with their diameter‐dependent piercing and length‐dependent wrapping on the lysis of microbial walls and membranes, inducing release of intracellular components DNA and RNA and allowing a loss of bacterial membrane potential, demonstrating complete destruction of bacteria. Thin and rigid SWCNT show more effective wall/membrane piercing on spherical bacteria than MWCNTs. Long MWCNT may wrap around gut bacteria, increasing the area making contact with the bacterial wall. This work suggests that CNTs may be broad‐spectrum and efficient antibacterial agents in the gut, and selective application of CNTs could reduce the potential hazard to probiotic bacteria.     

Fabrication of multi-walled carbon nanotube–carbon fiber hybrid material via electrophoretic deposition followed by pyrolysis process

An integrated multi-walled carbon nanotube (MWCNT)–carbon fiber (CF) hybrid material has been fabricated by electrophoretic deposition of acid-functionalized MWCNTs on CF surface followed by soaking in a 10% solution of petroleum pitch in toluene, followed by pyrolysis in a nitrogen atmosphere. It has been revealed that MWCNTs entirely covered the CF surface. Mechanical properties of composites reinforced by MWCNT–CF hybrids were considerably enhanced (up to 120% in tensile strength and 100% in elastic modulus) compared to composites reinforce by as-received CFs. According to fractography observations, robust interlocking occurred between epoxy matrix and MWCNT–CF hybrids.     

Properties of polycarbonate (PC)/multi-wall carbon nanotube (MWCNT) nanocomposites prepared by melt blending

This investigation deals with an easy method to develop electrical conductivity in polycarbonate (PC)/multi-wall carbon nanotube (MWCNT) nanocomposites with low loading of MWCNT. This was achieved by melt-blending of in-situ bulk polymerized low molecular weight poly(methyl methacrylate) (PMMA)/MWCNT nanocomposites and PC in various compositions at 280 degrees C in internal mixer. Differential scanning calorimetry (DSC) study showed single Tg in (85/15 w/w) PC/PMMA blend, indicating miscibility of PC and PMMA in the blend. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) studies of the melt-blended PC/PMMA/MWCNT nanocomposites revealed homogeneous dispersion and distribution of MWCNTs in PC matrix. Finally, through optimizing the blending composition of PC and PMMA/MWCNT nanocomposites, electrical conductivity of 3.74 x 10(-7) S x cm(-1) was achieved in the (85/15 w/w) PC/PMMA/MWCNT nanocomposites with the MWCNTs loading as low as approximately 0.37 wt%. Storage modulus of PC was found to increase significantly in presence of small amount (0.37 wt%) of MWCNTs in the nanocomposites.     

Ultrahigh-current field emission from sandwich-grown well-aligned uniform multi-walled carbon nanotube arrays with high adherence strength

We describe a new method to grow multi-walled carbon nanotube (MWCNT) arrays, which enable very high and stable macroscopic emission current density of 3.55?A?cm(-2) along with a scalable total emission current of more than 710?mA. A sandwich-growth technology was employed to synthesize vertically well-aligned MWCNT arrays in large areas and patterned uniformly by using microwave plasma chemical vapour deposition. A thick nickel layer was inserted between the silicon substrate and catalyst layer to achieve good adhesion between the MWCNTs and the substrate. Scanning electron microscope and transmission electron microscope investigations showed that well-structured, vertically aligned and uniform MWCNTs with perfect crystal lattices had been grown on lithographically predetermined sites. The root ends of MWCNTs adhered firmly to the nickel layer, establishing high electrical and thermal conductance of the MWCNTs to the substrate. This feature largely explains the large and stable emission current density of the MWCNT arrays.     

Conducting polymer‐coated, palladium‐functionalized multi‐walled carbon nanotubes for the electrochemical sensing of hydroxylamine

Electrochemical sensors of hydroxylamine were fabricated on glassy carbon electrodes (GCEs) by the electropolymerization of 3,4‐ethylenedioxypyrrole (EDOP) and 3,4‐ethylenedioxythiophene (EDOT) on palladium (Pd) nanoparticles attached to thiolated multi‐walled carbon nanotubes (MWCNTs), denoted as PEDOP/MWCNT‐Pd/GCE and PEDOT/MWCNT‐Pd/GCE. The sensors were characterized by field emission scanning electron microscopy and electrochemical impedance spectroscopy. They showed strong catalytic activity toward the oxidation of hydroxylamine. Cyclic voltammetry and amperometry were used to characterize the sensors' performances. The detection limits of hydroxylamine by PEDOP/MWCNT‐Pd/GCE and PEDOT/MWCNT‐Pd/GCE were 0.22 and 0.24 μM (S/N = 3), respectively. The sensors' sensitivity, selectivity, and stability were also investigated.