Annette von dem Bussche

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.

Adsorption of essential micronutrients by carbon nanotubes and the implications for nanotoxicity testing.

Nanotoxicology and nanomedicine make extensive use of in vitro cellular assays that were developed prior to the nanotechnology era. The introduction of nanomaterials to these standard assays causes problems that are currently limiting progress in the field.[1] Nanoparticles are often difficult to disperse;[2] they can interfere with optical measurements through light absorption, and they can interact with dyes used as molecular probes of cellular integrity.[3] In some cases the resulting artifacts can lead to gross misinterpretation of effects on cell viability and cytotoxicity.[4] Because sp2-hybridized carbon materials are near-universal sorbents for organic compounds in aqueous phases, and in light of a recent report of favorable noncovalent interactions between small-aromatic-molecule therapeutic agents and single-walled carbon nanotubes (SWNTs),[5] we hypothesize that SWNTs will adsorb a wide variety of small organic solutes from biological media, not limited to indicator dyes or their water-insoluble reduction products.

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.

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.

Nitric Oxide Synthase-3 Overexpression Causes Apoptosis and Impairs Neuronal Mitochondrial Function: Relevance to Alzheimer's-Type Neurodegeneration

Dementia in Alzheimers 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 H2O2 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.

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.

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

Significance Recent experimental studies report correlations between carbon nanotube toxicity and tube length and stiffness. Very little is known, however, about the actual behavior of these fibrous nanomaterials inside living cells following uptake, and the fundamental mechanistic link between stiffness and toxicity is unclear. Here we reveal a nanomechanical mechanism by which sufficiently long and stiff carbon nanotubes damage lysosomes, a class of membrane-enclosed organelles found inside cells that are responsible for breaking down diverse biomolecules and debris. The precise material parameters needed to activate this unique mechanical toxicity pathway are identified through coupled theoretical modeling, molecular dynamics simulations, and experimental studies, leading to a predictive pathogenicity classification diagram that distinguishes toxic from biocompatible nanomaterials based on their geometry and stiffness. 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.