Boron Nitride Nanosheets Can Induce Water Channels Across Lipid Bilayers Leading to Lysosomal Permeabilization.

While the interaction between 2D materials and cells is of key importance to the development of nanomedicines and safe applications of nanotechnology, still little is known about the biological interactions of many emerging 2D materials. Here, an investigation of how hexagonal boron nitride (hBN) interacts with the cell membrane is carried out by combining molecular dynamics (MD), liquid-phase exfoliation, and in vitro imaging methods. MD simulations reveal that a sharp hBN wedge can penetrate a lipid bilayer and form a cross-membrane water channel along its exposed polar edges, while a round hBN sheet does not exhibit this behavior. It is hypothesized that such water channels can facilitate cross-membrane transport, with important consequences including lysosomal membrane permeabilization, an emerging mechanism of cellular toxicity that involves the release of cathepsin B and generation of radical oxygen species leading to cell apoptosis. To test this hypothesis, two types of hBN nanosheets, one with a rhomboidal, cornered morphology and one with a round morphology, are prepared, and human lung epithelial cells are exposed to both materials. The cornered hBN with lateral polar edges results in a dose-dependent cytotoxic effect, whereas round hBN does not cause significant toxicity, thus confirming our premise.

Nanomechanical mechanism for lipid bilayer damage induced by carbon nanotubes confined in intracellular vesicles.

Understanding the behavior of low-dimensional nanomaterials confined in intracellular vesicles has been limited by the resolution of bioimaging techniques and the complex nature of the problem. Recent studies report that long, stiff carbon nanotubes are more cytotoxic than flexible varieties, but the mechanistic link between stiffness and cytotoxicity is not understood. Here we combine analytical modeling, molecular dynamics simulations, and in vitro intracellular imaging methods to reveal 1D carbon nanotube behavior within intracellular vesicles. We show that stiff nanotubes beyond a critical length are compressed by lysosomal membranes causing persistent tip contact with the inner membrane leaflet, leading to lipid extraction, lysosomal permeabilization, release of cathepsin B (a lysosomal protease) into the cytoplasm, and cell death. The precise material parameters needed to activate this unique mechanical pathway of nanomaterials interaction with intracellular vesicles were identified through coupled modeling, simulation, and experimental studies on carbon nanomaterials with wide variation in size, shape, and stiffness, leading to a generalized classification diagram for 1D nanocarbons that distinguishes pathogenic from biocompatible varieties based on a nanomechanical buckling criterion. For a wide variety of other 1D material classes (metal, oxide, polymer), this generalized classification diagram shows a critical threshold in length/width space that represents a transition from biologically soft to stiff, and thus identifies the important subset of all 1D materials with the potential to induce lysosomal permeability by the nanomechanical mechanism under investigation.

Chemical Dissolution Pathways of MoS2 Nanosheets in Biological and Environmental Media.

Material stability and dissolution in aqueous media are key issues to address in the development of a new nanomaterial intended for technological application. Dissolution phenomena affect biological and environmental persistence; fate, transport, and biokinetics; device and product stability; and toxicity pathways and mechanisms. This article shows that MoS2 nanosheets are thermodynamically and kinetically unstable to O2-oxidation under ambient conditions in a variety of aqueous media. The oxidation is accompanied by nanosheet degradation and release of soluble molybdenum and sulfur species, and generates protons that can colloidally destabilize the remaining sheets. The oxidation kinetics are pH-dependent, and a kinetic law is developed for use in biokinetic and environmental fate modeling. MoS2 nanosheets fabricated by chemical exfoliation with n-butyl-lithium are a mixture of 1T (primary) and 2H (secondary) phases and oxidize rapidly with a typical half-life of 1-30 days. Ultrasonically exfoliated sheets are in pure 2H phase, and oxidize much more slowly. Cytotoxicity experiments on MoS2 nanosheets and molybdate ion controls reveal the relative roles of the nanosheet and soluble fractions in the biological response. These results indicate that MoS2 nanosheets will not show long-term persistence in living systems and oxic natural waters, with important implications for biomedical applications and environmental risk.

Biological and environmental interactions of emerging two-dimensional nanomaterials.

Two-dimensional materials have become a major focus in materials chemistry research worldwide with substantial efforts centered on synthesis, property characterization, and technological application. These high-aspect ratio sheet-like solids come in a wide array of chemical compositions, crystal phases, and physical forms, and are anticipated to enable a host of future technologies in areas that include electronics, sensors, coatings, barriers, energy storage and conversion, and biomedicine. A parallel effort has begun to understand the biological and environmental interactions of synthetic nanosheets, both to enable the biomedical developments and to ensure human health and safety for all application fields. This review covers the most recent literature on the biological responses to 2D materials and also draws from older literature on natural lamellar minerals to provide additional insight into the essential chemical behaviors. The article proposes a framework for more systematic investigation of biological behavior in the future, rooted in fundamental materials chemistry and physics. That framework considers three fundamental interaction modes: (i) chemical interactions and phase transformations, (ii) electronic and surface redox interactions, and (iii) physical and mechanical interactions that are unique to near-atomically-thin, high-aspect-ratio solids. Two-dimensional materials are shown to exhibit a wide range of behaviors, which reflect the diversity in their chemical compositions, and many are expected to undergo reactive dissolution processes that will be key to understanding their behaviors and interpreting biological response data. The review concludes with a series of recommendations for high-priority research subtopics at the "bio-nanosheet" interface that we hope will enable safe and successful development of technologies related to two-dimensional nanomaterials.

Biological and environmental transformations of copper-based nanomaterials.

Copper-based nanoparticles are an important class of materials with applications as catalysts, conductive inks, and antimicrobial agents. Environmental and safety issues are particularly important for copper-based nanomaterials because of their potential large-scale use and their high redox activity and toxicity reported from in vitro studies. Elemental nanocopper oxidizes readily upon atmospheric exposure during storage and use, so copper oxides are highly relevant phases to consider in studies of environmental and health impacts. Here we show that copper oxide nanoparticles undergo profound chemical transformations under conditions relevant to living systems and the natural environment. Copper oxide nanoparticle (CuO-NP) dissolution occurs at lysosomal pH (4-5), but not at neutral pH in pure water. Despite the near-neutral pH of cell culture medium, CuO-NPs undergo significant dissolution in media over time scales relevant to toxicity testing because of ligand-assisted ion release, in which amino acid complexation is an important contributor. Electron paramagnetic resonance (EPR) spectroscopy shows that dissolved copper in association with CuO-NPs are the primary redox-active species. CuO-NPs also undergo sulfidation by a dissolution-reprecipitation mechanism, and the new sulfide surfaces act as catalysts for sulfide oxidation. Copper sulfide NPs are found to be much less cytotoxic than CuO-NPs, which is consistent with the very low solubility of CuS. Despite this low solubility of CuS, EPR studies show that sulfidated CuO continues to generate some ROS activity due to the release of free copper by H2O2 oxidation during the Fenton-chemistry-based EPR assay. While sulfidation can serve as a natural detoxification process for nanosilver and other chalcophile metals, our results suggest that sulfidation may not fully and permanently detoxify copper in biological or environmental compartments that contain reactive oxygen species.

Graphene microsheets enter cells through spontaneous membrane penetration at edge asperities and corner sites.

Understanding and controlling the interaction of graphene-based materials with cell membranes is key to the development of graphene-enabled biomedical technologies and to the management of graphene health and safety issues. Very little is known about the fundamental behavior of cell membranes exposed to ultrathin 2D synthetic materials. Here we investigate the interactions of graphene and few-layer graphene (FLG) microsheets with three cell types and with model lipid bilayers by combining coarse-grained molecular dynamics (MD), all-atom MD, analytical modeling, confocal fluorescence imaging, and electron microscopic imaging. The imaging experiments show edge-first uptake and complete internalization for a range of FLG samples of 0.5- to 10-µm lateral dimension. In contrast, the simulations show large energy barriers relative to kBT for membrane penetration by model graphene or FLG microsheets of similar size. More detailed simulations resolve this paradox by showing that entry is initiated at corners or asperities that are abundant along the irregular edges of fabricated graphene materials. Local piercing by these sharp protrusions initiates membrane propagation along the extended graphene edge and thus avoids the high energy barrier calculated in simple idealized MD simulations. We propose that this mechanism allows cellular uptake of even large multilayer sheets of micrometer-scale lateral dimension, which is consistent with our multimodal bioimaging results for primary human keratinocytes, human lung epithelial cells, and murine macrophages.

Cell entry of one-dimensional nanomaterials occurs by tip recognition and rotation.

Materials with high aspect ratio, such as carbon nanotubes and asbestos fibres, have been shown to cause length-dependent toxicity in certain cells because these long materials prevent complete ingestion and this frustrates the cell. Biophysical models have been proposed to explain how spheres and elliptical nanostructures enter cells, but one-dimensional nanomaterials have not been examined. Here, we show experimentally and theoretically that cylindrical one-dimensional nanomaterials such as carbon nanotubes enter cells through the tip first. For nanotubes with end caps or carbon shells at their tips, uptake involves tip recognition through receptor binding, rotation that is driven by asymmetric elastic strain at the tube-bilayer interface, and near-vertical entry. The precise angle of entry is governed by the relative timescales for tube rotation and receptor diffusion. Nanotubes without caps or shells on their tips show a different mode of membrane interaction, posing an interesting question as to whether modifying the tips of tubes may help avoid frustrated uptake by cells.

Hepatitis C virus NS2 protein triggers endoplasmic reticulum stress and suppresses its own viral replication.

BACKGROUND & AIMS: We previously reported that the NS2 protein of hepatitis C virus (HCV) inhibits the expression of reporter genes driven by a variety of cellular and viral promoters. The aim of the study was to determine whether the broad transcriptional repression is caused by endoplasmic reticulum (ER) stress. METHODS: Phosphorylation of the translation initiation factor eIF2α and HCV replication was detected by Western and Northern blot, respectively. De novo protein synthesis was measured by metabolic labeling. Activation of ER stress responsive genes was determined by promoter reporter assay, as well as mRNA and protein measurement by real time PCR and Western blot. RESULTS: Transient or inducible NS2 protein expression increased eIF2α phosphorylation and reduced de novo protein synthesis. It up-regulated promoter activities and transcript levels of ER stress inducible genes including GRP78, ATF6, and GADD153, as well as GRP78 protein level. The same effect was observed when NS2 was synthesized as part of the core-E1-E2-p7-NS2 polypeptide. NS2 protein also inhibited reporter gene expression from the HCV internal ribosome entry site and consequently reduced HCV replication. The full-length HCV replicon activated GRP78, ATF6, and GADD153 promoters more efficiently than the subgenomic replicon lacking the coding sequence for both the structural proteins and NS2. Abrogation of HCV infection/replication, by an inhibitor of the NS3 protease, relieved ER stress. CONCLUSIONS: HCV infection can induce ER stress, with NS2 protein being a major mediator. The stress can be relieved by a feedback mechanism.

Tocopheryl Polyethylene Glycol Succinate as a Safe, Antioxidant Surfactant for Processing Carbon Nanotubes and Fullerenes.

This work investigates the physical interactions between carbon nanomaterials and tocopheryl polyethylene glycol succinate (TPGS). TPGS is a synthetic amphiphile that undergoes enzymatic cleavage to deliver the lipophilic antioxidant, alpha-tocopherol (vitamin E) to cell membranes, and is FDA approved as a water-soluble vitamin E nutritional supplement and drug delivery vehicle. Here we show that TPGS 1000 is capable of dispersing multi-wall and single-wall carbon nanotubes in aqueous media, and for multiwall tubes is more effective than the commonly used non-ionic surfactant Triton X-100. TPGS is also capable of solubilizing C(60) in aqueous phases by dissolving fullerene in the core of its spherical micelles. Drying of these solutions leads to fullerene/TPGS phase separation and the self-assembly of highly ordered asymmetric nanoparticles, with fullerene nanocrystals attached to the hydrophobic end of crystalline TPGS nanobrushes. The article discusses surface charge, colloidal stability, and the potential applications of TPGS as a safe surfactant for "green" processing of carbon nanomaterials.

Functional consequences of frizzled-7 receptor overexpression in human hepatocellular carcinoma.

BACKGROUND & AIMS: The molecular pathogenesis of human hepatocellular carcinoma (HCC) is understood poorly. In some tumors, activation of the Wnt/beta-catenin pathway as a result of beta-catenin gene mutations has been found. However, in many other HCCs, activation of the Wnt/beta-catenin pathway has been shown in the absence of such mutations. METHODS: We previously have identified the upstream human Frizzled-7 receptor (FZD7) gene of this pathway. In the present study, a quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) assay for FZD7 was developed and overexpression of FZD7 was detected in 90% of tumors, most of which were related to chronic hepatitis B virus infection. FZD7 also was overexpressed in the 6 HCC cell lines tested and functional analysis showed that FZD7 messenger RNA (mRNA) levels correlated with enhanced cellular motility. RESULTS: Transfection of HCC cells with dominant-negative mutant constructs encoding a C-terminally truncated FZD7 protein decreased wild-type beta-catenin protein accumulation and reduced cell motility. More importantly, we observed beta-catenin accumulation in human HCC tumors containing the wild-type beta-catenin gene in the context of high-level FZD7 expression. CONCLUSIONS: These observations suggest that the Wnt/beta-catenin signal transduction pathway is involved much more commonly in the molecular pathogenesis of HCC than previously recognized because FZD7 overexpression occurred early in the disease process, stabilized wild-type beta-catenin levels, and contributed to enhanced tumor cell migration.

Hepatitis C virus NS2 protein inhibits gene expression from different cellular and viral promoters in hepatic and nonhepatic cell lines.

The mechanisms leading to viral persistence and hepatocarcinogenesis in hepatitis C virus (HCV) infection are not fully understood. Recently, evidence has been accumulated that HCV viral proteins might interfere with gene expression of host cells. Here, we demonstrate that the amino-terminal portion of HCV NS2 protein inhibits the expression of reporter genes driven by different promoters or enhancer elements and also inhibits hepatitis B virus (HBV) gene expression and HBV DNA replication. The inhibitory effect of HCV NS2 on liver and non-liver-specific promoters and enhancer elements might be relevant for the pathogenesis of chronic HCV infection.

Nitric oxide synthase-3 overexpression causes apoptosis and impairs neuronal mitochondrial function: relevance to Alzheimer's-type neurodegeneration.

Dementia in Alzheimer's disease (AD) is correlated with cell loss that is mediated by apoptosis, mitochondrial (Mt) dysfunction, and possibly necrosis. Previous studies demonstrated increased expression of the nitric oxide synthase 3 (NOS3) gene in degenerating neurons of AD brains. For investigating the role of NOS3 overexpression as a mediator of neuronal loss, human PNET2 central nervous system-derived neuronal cells were infected with recombinant adenovirus vectors that expressed either human NOS3 or green fluorescent protein cDNA under the control of a CMV promoter. NOS3 overexpression resulted in apoptosis accompanied by increased levels of p53, p21/Waf1, Bax, and CD95. In addition, NOS3 overexpression impaired neuronal Mt function as demonstrated by the reduced levels of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and nicotinamide adenine dinucleotide (reduced form)-tetrazolium reductase activities and MitoTracker Red fluorescence. These adverse effects of NOS3 were associated with increased cellular levels of reactive oxygen species and impaired membrane integrity and were not produced in cells that were transfected with a cDNA encoding catalytically inactive NOS3. Importantly, modest elevations in NOS3 expression, achieved by infection with low multiplicities of adenovirus-NOS3 infection, did not cause apoptosis but rendered the cells more sensitive to oxidative injury by H(2)O(2) or diethyldithiocarbamate. In contrast, treatment with NO donors did not enhance neuronal sensitivity to oxidative injury. These results suggest that NOS3-induced neuronal death is mediated by Mt dysfunction, oxidative injury, and impaired membrane integrity, rather than by NO production, and that neuroprotection from these adverse effects of NOS3 may be achieved by modulating intracellular levels of oxidative stress.

Cell activation by synthetic lipopeptides of the hepatitis C virus (HCV)--core protein is mediated by toll like receptors (TLRs) 2 and 4.

T cell epitopes coupled to a lipid moiety (lipopeptides) may be superior immunostimulants compared to peptide antigens and are currently studied as potential vaccines. The cause of enhanced immunogenicity of lipopeptides is largely unknown but members of the novel family of Toll like receptors (TLR) such as TLR2 and TLR4 have been shown to mediate activation of cells in response to bacterial lipopolysaccharide (LPS) and other lipidated bacterial or viral components. We studied TLR-mediated activation by 14 synthetic lipopeptides corresponding to T cell epitopes on hepatitis C virus (HCV) core in human embryonic kidney cells (HEK293) transiently over-expressing TLR2 and in Ba/F3 mouse bone marrow cells stably transfected with TLR4 and the adaptor molecule MD-2. Stimulation of transfected HEK293 or Ba/F3 cells was measured via luciferase activity as a reporter of nuclear factor kappaB activation. Free peptides, a non-HCV-related lipopeptide as well as LPS and the lipopeptide SK4 were used as controls. Ten of the 14 HCV core lipopeptides stimulated luciferase activity in TLR2-transfected HEK293 cells but not in mock-transfected control cells. Nine of the 14 lipopeptides also stimulated luciferase activity in the TLR4/MD-2 double-transfected Ba/F3 cells but not Ba/F3 control cells. Overall, there was a close statistical correlation between TLR2 and TLR4/MD-2-mediated cell activation by the lipopeptides. In contrast, the corresponding free peptides had no stimulatory effect on TLR2 nor on TLR4/MD-2 transfected cells. Thus, lipopeptides but not their corresponding free peptides can activate cells via TLRs 2 and 4. This activation is apparently affected by the amino acid sequence of the peptide moiety.