Angela E. Goode

pH-Dependent Toxicity of High Aspect Ratio ZnO Nanowires in Macrophages Due to Intracellular Dissolution

High-aspect ratio ZnO nanowires have become one of the most promising products in the nanosciences within the past few years with a multitude of applications at the interface of optics and electronics. The interaction of zinc with cells and organisms is complex, with both deficiency and excess causing severe effects. The emerging significance of zinc for many cellular processes makes it imperative to investigate the biological safety of ZnO nanowires in order to guarantee their safe economic exploitation. In this study, ZnO nanowires were found to be toxic to human monocyte macrophages (HMMs) at similar concentrations as ZnCl(2). Confocal microscopy on live cells confirmed a rise in intracellular Zn(2+) concentrations prior to cell death. In vitro, ZnO nanowires dissolved very rapidly in a simulated body fluid of lysosomal pH, whereas they were comparatively stable at extracellular pH. Bright-field transmission electron microscopy (TEM) showed a rapid macrophage uptake of ZnO nanowire aggregates by phagocytosis. Nanowire dissolution occurred within membrane-bound compartments, triggered by the acidic pH of the lysosomes. ZnO nanowire dissolution was confirmed by scanning electron microscopy/energy-dispersive X-ray spectrometry. Deposition of electron-dense material throughout the ZnO nanowire structures observed by TEM could indicate adsorption of cellular components onto the wires or localized zinc-induced protein precipitation. Our study demonstrates that ZnO nanowire toxicity in HMMs is due to pH-triggered, intracellular release of ionic Zn(2+) rather than the high-aspect nature of the wires. Cell death had features of necrosis as well as apoptosis, with mitochondria displaying severe structural changes. The implications of these findings for the application of ZnO nanowires are discussed.

Uptake of Noncytotoxic Acid-Treated Single-Walled Carbon Nanotubes into the Cytoplasm of Human Macrophage Cells

Water-soluble single-walled nanotubes (SWNTs) are being tested as contrast agents for medical imaging and for the delivery of therapeutically active molecules to target cells. However, before they become used commercially, it will be essential to establish their subcellular distribution and whether they are cytotoxic. Here we characterize uptake of unlabeled, acid-treated, water-soluble SWNTs by human monocyte derived macrophage cells using a combination of Raman spectroscopy and analytical electron microscopy and compare our findings to previous work on unpurified SWNTs. Raman spectroscopy demonstrated that acid-treated SWNTs had a greater number of functional groups on the carbon walls than nontreated SWNT. The acid-treated SWNTs were less aggregated within cells than unpurified SWNTs. Bundles, and also individual acid-treated SWNTs, were found frequently inside lysosomes and also the cytoplasm, where they caused no significant changes in cell viability or structure even after 4 days of exposure.

Chemical speciation of nanoparticles surrounding metal-on-metal hips

Spectromicroscopy of tissue surrounding failed CoCr metal-on-metal hip replacements detected corroded nanoscale debris in periprosthetic tissue in two chemical states, with concomitant mitochondrial damage. The majority of debris contained Cr(3+), with trace amounts of oxidised cobalt. A minority phase containing a core of metallic chromium and cobalt was also observed.

Silver nanoparticles reduce brain inflammation and related neurotoxicity through induction of H2S-synthesizing enzymes

Silver nanoparticles (AgNP) are known to penetrate into the brain and cause neuronal death. However, there is a paucity in studies examining the effect of AgNP on the resident immune cells of the brain, microglia. Given microglia are implicated in neurodegenerative disorders such as Parkinson’s disease (PD), it is important to examine how AgNPs affect microglial inflammation to fully assess AgNP neurotoxicity. In addition, understanding AgNP processing by microglia will allow better prediction of their long term bioreactivity. In the present study, the in vitro uptake and intracellular transformation of citrate-capped AgNPs by microglia, as well as their effects on microglial inflammation and related neurotoxicity were examined. Analytical microscopy demonstrated internalization and dissolution of AgNPs within microglia and formation of non-reactive silver sulphide (Ag2S) on the surface of AgNPs. Furthermore, AgNP-treatment up-regulated microglial expression of the hydrogen sulphide (H2S)-synthesizing enzyme cystathionine-γ-lyase (CSE). In addition, AgNPs showed significant anti-inflammatory effects, reducing lipopolysaccharide (LPS)-stimulated ROS, nitric oxide and TNFα production, which translated into reduced microglial toxicity towards dopaminergic neurons. Hence, the present results indicate that intracellular Ag2S formation, resulting from CSE-mediated H2S production in microglia, sequesters Ag+ ions released from AgNPs, significantly limiting their toxicity, concomitantly reducing microglial inflammation and related neurotoxicity.

Contamination of holey/lacey carbon films in STEM.

In many cases, the key to obtaining good TEM results is in the sample preparation itself. Even once a thin specimen is achieved, other factors determine how well the sample will behave in the microscope. One of the main hindrances to TEM and STEM-EELS analysis is the build up of carbon contamination on the sample under the electron beam. This process may occur due to the nature of the sample itself or the support grids or films on which the sample sits. Here, we investigate contamination on holey and lacey carbon films from three different suppliers. We find that all grids have a large amount of mobile hydrocarbon contamination on them, as well as other larger contaminant species on the surface. Even after a variety of cleaning routines, none of the films are clean enough for STEM-EELS experiments requiring long acquisition times.

Microfocus study of metal distribution and speciation in tissue extracted from revised metal on metal hip implants

Unexplained tissue inflammation in metal-on-metal hip replacements is suspected to be caused by implant-derived nanoparticles. The aim of this study was to investigate the nature of the metal particles in tissue surrounding metal-on-metal (MOM) hips that has been extracted during revision. Mapping of tissue surrounding the failed MOM hips was performed using microfocus X-ray Fluorescence (XRF). This revealed mainly Cr which was localized to the cellular regions. There was co-localisation of Co, were present, to areas of high Cr abundance. XANES of the tissue and appropriate standards revealed that the most common species were Cr(III) and Co(II). EXAFS analysis of the tissue and various metal standards revealed that the most abundant implant-related species was Cr(III) phosphate. Different tissue preparation methods, including frozen sectioning, were examined but were found not to affect the distribution or speciation of the metals in the tissue.

Direct in situ observation of ZnO nucleation and growth via transmission X-ray microscopy

The nucleation and growth of a nanostructure controls its size and morphology, and ultimately its functional properties. Hence it is crucial to investigate growth mechanisms under relevant growth conditions at the nanometer length scale. Here we image the nucleation and growth of electrodeposited ZnO nanostructures in situ, using a transmission X-ray microscope and specially designed electrochemical cell. We show that this imaging technique leads to new insights into the nucleation and growth mechanisms in electrodeposited ZnO including direct, in situ observations of instantaneous versus delayed nucleation.

Inhibition of Leptin-ObR Interaction Does not Prevent Leptin Translocation Across a Human Blood-Brain Barrier Model.

The adipocyte‐derived hormone leptin regulates appetite and energy homeostasis through the activation of leptin receptors (ObR) on hypothalamic neurones; hence, leptin must be transported through the blood–brain barrier (BBB) to reach its target sites in the central nervous system. During obesity, however, leptin BBB transport is decreased, in part precluding leptin as a viable clinical therapy against obesity. Although the short isoform of the ObR (ObRa) has been implicated in the transport of leptin across the BBB as a result of its elevated expression in cerebral microvessels, accumulating evidence indicates that leptin BBB transport is independent of ObRa. In the present study, we employed an ObR‐neutralising antibody (9F8) to directly examine the involvement of endothelial ObR in leptin transport across an in vitro human BBB model composed of the human endothelial cell line hCMEC/D3. Our results indicate that, although leptin transport across the endothelial monolayer was nonparacellular, and energy‐ and endocytosis‐dependent, it was not inhibited by pre‐treatment with 9F8, despite the ability of the latter to recognise hCMEC/D3‐expressed ObR, prevent leptin–ObR binding and inhibit leptin‐induced signal transducer and activator of transcription 3 (STAT‐3) phosphorylation in hCMEC/D3 cells. Furthermore, hCMEC/D3 cells expressed the transporter protein low‐density lipoprotein receptor‐related protein‐2 (LRP‐2), which is capable of binding and endocytosing leptin. In conclusion, our results demonstrate that leptin binding to and signalling through ObR is not required for efficient transport across human endothelial monolayers, indicating that ObR is not the primary leptin transporter at the human BBB, a role which may fall upon LRP‐2. A deeper understanding of leptin BBB transport will help clarify the exact causes for leptin resistance seen in obesity and aid in the development of more efficient BBB‐penetrating leptin analogues.

Avoiding artefacts during electron microscopy of silver nanomaterials exposed to biological environments

Electron microscopy has been applied widely to study the interaction of nanomaterials with proteins, cells and tissues at nanometre scale. Biological material is most commonly embedded in thermoset resins to make it compatible with the high vacuum in the electron microscope. Room temperature sample preparation protocols developed over decades provide contrast by staining cell organelles, and aim to preserve the native cell structure. However, the effect of these complex protocols on the nanomaterials in the system is seldom considered. Any artefacts generated during sample preparation may ultimately interfere with the accurate prediction of the stability and reactivity of the nanomaterials. As a case study, we review steps in the room temperature preparation of cells exposed to silver nanomaterials (AgNMs) for transmission electron microscopy imaging and analysis. In particular, embedding and staining protocols, which can alter the physicochemical properties of AgNMs and introduce artefacts thereby leading to a misinterpretation of silver bioreactivity, are scrutinized. Recommendations are given for the application of cryogenic sample preparation protocols, which simultaneously fix both particles and diffusible ions. By being aware of the advantages and limitations of different sample preparation methods, compromises or selection of different correlative techniques can be made to draw more accurate conclusions about the data.

Mapping functional groups on oxidised multi-walled carbon nanotubes at the nanometre scale

Despite voluminous research on the acid oxidation of carbon nanotubes (CNTs), there is a distinct lack of experimental results showing distributions of functional groups at the nanometre length scale. Here, functional peaks have been mapped across individual multi-walled CNTs with low-dose, monochromated electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM). Density functional theory simulations show that the EELS features are consistent with oxygenated functional groups, most likely carboxyl moieties.