Probing the pH-dependency of DC-SIGN/R multivalent lectin-glycan interactions using polyvalent glycan-gold nanoparticles.

The dendritic cell tetrameric lectin, DC-SIGN, and its closely related endothelial cell lectin, DC-SIGNR (collectively abbreviated as DC-SIGN/R) play a key role in the binding and transmission of deadly viruses, including Ebola, HIV, HCV, and SARS-CoV-2. Their virus binding/release processes involve a gradually acidifying environment following the natural intracellular trafficking pathways. Therefore, understanding DC-SIGN/R's pH-dependent binding properties with glycan ligands is of great importance. We have recently developed densely glycosylated gold nanoparticles (glycan-GNPs) as a powerful new tool for probing DC-SIGN/R multivalent lectin-glycan interaction (MLGI) mechanisms. They can provide not only quantitative MLGI affinities but also important structural information, such as binding site orientation and binding modes. Herein, we further employ the glycan-GNP probes to investigate the pH dependency of DC-SIGN/R MLGI properties. We find that DC-SIGN/R MLGIs exhibit distinct pH dependence over the normal physiological (7.4) to lysosomal (∼4.6) pH range. DC-SIGN binds glycan-GNPs strongly and stably from pH 7.4 to ∼5.8, but the binding is weakened significantly as pH decreases to ≤5.4 and may be fully dissociated at pH 4.6. This behaviour is fully consistent with DC-SIGN's role as an endocytic recycling receptor. In contrast, DC-SIGNR's affinity with glycan-GNPs is enhanced with the decreasing pH from 7.4 to 5.4, peaking at pH 5.4, and then reduced as pH is further lowered. Interestingly, both DC-SIGN/R binding with glycan-GNPs are found to be partially reversible in a pH-dependent manner.

Lithiated porous silicon nanowires stimulate periodontal regeneration.

Periodontal disease is a significant burden for oral health, causing progressive and irreversible damage to the support structure of the tooth. This complex structure, the periodontium, is composed of interconnected soft and mineralised tissues, posing a challenge for regenerative approaches. Materials combining silicon and lithium are widely studied in periodontal regeneration, as they stimulate bone repair via silicic acid release while providing regenerative stimuli through lithium activation of the Wnt/ß-catenin pathway. Yet, existing materials for combined lithium and silicon release have limited control over ion release amounts and kinetics. Porous silicon can provide controlled silicic acid release, inducing osteogenesis to support bone regeneration. Prelithiation, a strategy developed for battery technology, can introduce large, controllable amounts of lithium within porous silicon, but yields a highly reactive material, unsuitable for biomedicine. This work debuts a strategy to lithiate porous silicon nanowires (LipSiNs) which generates a biocompatible and bioresorbable material. LipSiNs incorporate lithium to between 1% and 40% of silicon content, releasing lithium and silicic acid in a tailorable fashion from days to weeks. LipSiNs combine osteogenic, cementogenic and Wnt/ß-catenin stimuli to regenerate bone, cementum and periodontal ligament fibres in a murine periodontal defect.

Impact of Surface Ligand on the Biocompatibility of InP/ZnS Quantum Dots with Platelets.

InP/ZnS quantum dots (QDs) have received a large focus in recent years as a safer alternative to heavy metal-based QDs. Given their intrinsic fluorescent imaging capabilities, these QDs can be potentially relevant for in vivo platelet imaging. The InP/ZnS QDs are synthesized and their biocompatibility investigated through the use of different phase transfer agents. Analysis of platelet function indicates that platelet-QD interaction can occur at all concentrations and for all QD permutations tested. However, as the QD concentration increases, platelet aggregation is induced by QDs alone independent of natural platelet agonists. This study helps to define a range of concentrations and coatings (thioglycolic acid and penicillamine) that are biocompatible with platelet function. With this information, the platelet-QD interaction can be identified using multiple methods. Fluorescent lifetime imaging microscopy (FLIM) and confocal studies have shown QDs localize on the surface of the platelet toward the center while showing evidence of energy transfer within the QD population. It is believed that these findings are an important stepping point for the development of fluorescent probes for platelet imaging.

Impact of Porous Silica Nanosphere Architectures on the Catalytic Performance of Supported Sulphonic Acid Sites for Fructose Dehydration to 5-Hydroxymethylfurfural.

5-hydroxymethylfurfural represents a key chemical in the drive towards a sustainable circular economy within the chemical industry. The final step in 5-hydroxymethylfurfural production is the acid catalysed dehydration of fructose, for which supported organoacids are excellent potential catalyst candidates. Here we report a range of solid acid catalysis based on sulphonic acid grafted onto different porous silica nanosphere architectures, as confirmed by TEM, N2 porosimetry, XPS and ATR-IR. All four catalysts display enhanced active site normalised activity and productivity, relative to alternative silica supported equivalent systems in the literature, with in-pore diffusion of both substrate and product key to both performance and humin formation pathway. An increase in-pore diffusion coefficient of 5-hydroxymethylfurfural within wormlike and stellate structures results in optimal productivity. In contrast, poor diffusion within a raspberry-like morphology decreases rates of 5-hydroxymethylfurfural production and increases its consumption within humin formation.

Gradient Boosted Machine Learning Model to Predict H2, CH4, and CO2 Uptake in Metal-Organic Frameworks Using Experimental Data.

Predictive screening of metal-organic framework (MOF) materials for their gas uptake properties has been previously limited by using data from a range of simulated sources, meaning the final predictions are dependent on the performance of these original models. In this work, experimental gas uptake data has been used to create a Gradient Boosted Tree model for the prediction of H2, CH4, and CO2 uptake over a range of temperatures and pressures in MOF materials. The descriptors used in this database were obtained from the literature, with no computational modeling needed. This model was repeated 10 times, showing an average R2 of 0.86 and a mean absolute error (MAE) of ±2.88 wt % across the runs. This model will provide gas uptake predictions for a range of gases, temperatures, and pressures as a one-stop solution, with the data provided being based on previous experimental observations in the literature, rather than simulations, which may differ from their real-world results. The objective of this work is to create a machine learning model for the inference of gas uptake in MOFs. The basis of model development is experimental as opposed to simulated data to realize its applications by practitioners. The real-world nature of this research materializes in a focus on the application of algorithms as opposed to the detailed assessment of the algorithms.

Structure of polymeric nanoparticles encapsulating a drug - pamoic acid ion pair by scanning transmission electron microscopy.

Drug-delivery systems based on polymeric nanoparticles are useful for improving drug bioavailability and/or delivery of the active ingredient for example directly to the cancerous tumour. The physical and chemical characterization of a functionalized nanoparticle system is required to measure drug loading and dispersion but also to understand and model the rate and extent of drug release to help predict performance. Many techniques can be used, however, difficulties related to structure determination and identifying the precise location of the drug fraction make mathematical prediction complex and in many published examples the final conclusions are based on assumptions regarding an expected structure. Cryogenic scanning transmission electron microscopy imaging in combination with electron energy loss spectroscopy techniques are used here to address this issue and provide a multi-modal approach to the characterisation of a self-assembled polymeric nanoparticle system based upon a polylactic acid - polyethylene glycol (PLA-PEG) block copolymer containing a hydrophobic ion-pair between pamoic acid and an active pharmaceutical ingredient (API). Results indicate a regular dispersion of spherical nanoparticles of 88 ± 9 nm diameter. The particles are shown to have a multi-layer structure consisting of a 25 nm radius hydrophobic core of PLA and pamoic acid-API material with additional enrichment of the pamoic acid-API material within the inner core (that can be off-centre), surrounded by a 9 nm dense PLA-PEG layer all with a low-density PEG surface coating of around 10 nm thickness. This structure suggests that release of the API can only occur by diffusion through or degradation of the dense, 9 nm thick PLA-PEG layer either of which is a process consistent with the previously reported steady release kinetics of the API and counter ion from these nanoparticle formulations. Establishing accurate measures of product structure enables a link to performance by providing appropriate physical parameters for future mathematical modelling of barriers controlling API release in these nanoparticle formulations.

Polyvalent Glycan Functionalized Quantum Nanorods as Mechanistic Probes for Shape-Selective Multivalent Lectin-Glycan Recognition.

Multivalent lectin-glycan interactions (MLGIs) are widespread in biology and hold the key to many therapeutic applications. However, the underlying structural and biophysical mechanisms for many MLGIs remain poorly understood, limiting our ability to design glycoconjugates to potently target specific MLGIs for therapeutic intervention. Glycosylated nanoparticles have emerged as a powerful biophysical probe for MLGIs, although how nanoparticle shape affects the MLGI molecular mechanisms remains largely unexplored. Herein, we have prepared fluorescent quantum nanorods (QRs), densely coated with α-1,2-manno-biose ligands (QR-DiMan), as multifunctional probes to investigate how scaffold geometry affects the MLGIs of a pair of closely related, tetrameric viral receptors, DC-SIGN and DC-SIGNR. We have previously shown that a DiMan-capped spherical quantum dot (QD-DiMan) gives weak cross-linking interactions with DC-SIGNR but strong simultaneous binding with DC-SIGN. Against the elongated QR-DiMan, DC-SIGN retains similarly strong simultaneous binding of all four binding sites with a single QR-DiMan (apparent K d ≈ 0.5 nM, ∼1.8 million-fold stronger than the corresponding monovalent binding), while DC-SIGNR gives both weak cross-linking and strong individual binding interactions, resulting in a larger binding affinity enhancement than that with QD-DiMan. S/TEM analysis of QR-DiMan-lectin assemblies reveals that DC-SIGNR's different binding modes arise from the different nanosurface curvatures of the QR scaffold. The glycan display at the spherical ends presents too high a steric barrier for DC-SIGNR to bind with all four binding sites; thus, it cross-links between two QR-DiMan to maximize binding multivalency, whereas the more planar character of the cylindrical center allows the glycans to bridge all binding sites in DC-SIGNR. This work thus establishes glycosylated QRs as a powerful biophysical probe for MLGIs not only to provide quantitative binding affinities and binding modes but also to demonstrate the specificity of multivalent lectins in discriminating different glycan displays in solution, dictated by the scaffold curvature.

Polyvalent Glycan Quantum Dots as a Multifunctional Tool for Revealing Thermodynamic, Kinetic, and Structural Details of Multivalent Lectin-Glycan Interactions.

Multivalent lectin-glycan interactions (MLGIs) are widespread and vital for biology. Their binding biophysical and structural details are thus highly valuable, not only for the understanding of binding affinity and specificity mechanisms but also for guiding the design of multivalent therapeutics against specific MLGIs. However, effective techniques that can reveal all such details remain unavailable. We have recently developed polyvalent glycan quantum dots (glycan-QDs) as a new probe for MLGIs. Using a pair of closely related tetrameric viral-binding lectins, DC-SIGN and DC-SIGNR, as model examples, we have revealed and quantified their large affinity differences in glycan-QD binding are due to distinct binding modes: with simultaneous binding for DC-SIGN and cross-linking for DC-SIGNR. Herein, we further extend the capacity of the glycan-QD probes by investigating the correlation between binding mode and binding thermodynamics and kinetics and further probing a structural basis of their binding nature. We reveal that while both lectins' binding with glycan-QDs is enthalpy driven with similar binding enthalpy changes, DC-SIGN pays a lower binding entropy penalty, resulting in a higher affinity than DC-SIGNR. We then show that DC-SIGN binding gives a single second-order kon rate, whereas DC-SIGNR gives a rapid initial binding followed by a much slower secondary interaction. We further identify a structural element in DC-SIGN, absent in DC-SIGNR, that plays an important role in maintaining DC-SIGN's MLGI character. Its removal switches the binding from being enthalpically to entropically driven and gives mixed binding modes containing both simultaneous and cross-linking binding behavior, without markedly affecting the overall binding affinity and kinetics.

Controlling adsorption density of polymer-stabilised metal nanoparticles at the oil-water interface.

Emulsion droplets offer an alternative to solid supports as templates for the deposition of metallic nanoparticles. An emulsion interface provides the opportunity to exploit both sides of the nanoparticles and to utilise the liquid core as a microreactor in addition to forming a scaffold for encapsulation. However, despite the extensive literature studying a very broad range of factors influencing the characteristics of particle-stabilised (Pickering) emulsions, most reports focus on particles of diameters >100 nm and a very small proportions consider particles of diameters <10 nm. For catalytic purposes of course, the latter species are of utmost interest. Here, we report the synthesis of poly(vinyl pyrrolidone) (PVP) stabilised platinum nanoparticles, where the platinum core ranges between 3 and 5 nm in diameter and their subsequent use as emulsifiers for the oil-water interface where they form a densely packed layer. The nanoparticle density at the interface is quantified by both measuring the remaining concentration of nanoparticles in the aqueous phase after adsorption and also directly at the oil-water interface via cryo-TEM. The effect of electrolyte concentration and of addition of excess PVP in the bulk aqueous nanoparticle dispersion prior to emulsification on the resulting nanoparticle density at the oil-water interface is also determined.

Industrial-relevant TiO2 types do not promote cytotoxicity in the A549 or TK6 cell lines regardless of cell specific interaction.

Due to the expansive application of TiO2 and its variance in physico-chemical characteristics, the toxicological profile of TiO2, in all its various forms, requires evaluation. This study aimed to assess the hazard of five TiO2 particle-types in relation to their cytotoxic profile correlated to their cellular interaction, specifically in human lymphoblast (TK6) and type-II alveolar epithelial (A549) cells. Treatment with the test materials was undertaken at a concentration range of 1-100 µg/cm2 over 24 and 72 h exposure. TiO2 interaction with both cell types was visualised by transmission electron microscopy, supported by energy-dispersive X-ray. None of the TiO2 materials tested promoted cytotoxicity in either cell type over the concentration and time range studied. All materials were observed to interact with the A549 cells and were further noted to be internalised following 24 h exposure. In contrast, only the pigmentary rutile was internalised by TK6 lymphoblasts after 24 h exposure. Where uptake was observed there was no evidence, as determined by 2D microscopy techniques, of particle localisation within the nucleus of either cell type. This study indicates that industrially relevant TiO2 particles demonstrate cell interactions that are cell-type dependent and do not induce cytotoxicity at the applied dose range.

Understanding stress-induced disorder and breakage in organic crystals: beyond crystal structure anisotropy.

Crystal engineering has advanced the strategies for design and synthesis of organic solids with the main focus being on customising the properties of the materials. Research in this area has a significant impact on large-scale manufacturing, as industrial processes may lead to the deterioration of such properties due to stress-induced transformations and breakage. In this work, we investigate the mechanical properties of structurally related labile multicomponent solids of carbamazepine (CBZ), namely the dihydrate (CBZ·2H2O), a cocrystal of CBZ with 1,4-benzoquinone (2CBZ·BZQ) and the solvates with formamide and 1,4-dioxane (CBZ·FORM and 2CBZ·DIOX, respectively). The effect of factors that are external (e.g. impact stressing) and/or internal (e.g. phase transformations and thermal motion) to the crystals are evaluated. In comparison to the other CBZ multicomponent crystal forms, CBZ·2H2O crystals tolerate less stress and are more susceptible to breakage. It is shown that this poor resistance to fracture may be a consequence of the packing of CBZ molecules and the orientation of the principal molecular axes in the structure relative to the cleavage plane. It is concluded, however, that the CBZ lattice alone is not accountable for the formation of cracks in the crystals of CBZ·2H2O. The strength and the temperature-dependence of electrostatic interactions, such as hydrogen bonds between CBZ and coformer, appear to influence the levels of stress to which the crystals are subjected that lead to fracture. Our findings show that the appropriate selection of coformer in multicomponent crystal forms, targetting superior mechanical properties, needs to account for the intrinsic stress generated by molecular vibrations and not solely by crystal anisotropy. Structural defects within the crystal lattice, although highly influenced by the crystallisation conditions and which are especially difficult to control in organic solids, may also affect breakage.

Exploring water in oil emulsions simultaneously stabilized by solid hydrophobic silica nanospheres and hydrophilic soft PNIPAM microgel.

A general drawback of microgels is that they do not stabilize water-in-oil (w/o) emulsions of non-polar oils. Simultaneous stabilization with solid hydrophobic nanoparticles and soft hydrophilic microgels overcomes this problem. For a fundamental understanding of this synergistic effect the use of well defined particle systems is crucial. Therefore, the present study investigates the stabilization of water droplets in a highly non-polar oil phase using temperature responsive, soft and hydrophilic PNIPAM microgel particles (MGs) and solid and hydrophobic silica nanospheres (SNs) simultaneously. The SNs are about 20 times smaller than the MGs. In a multiscale approach the resulting emulsions are studied from the nanoscale particle properties over microscale droplet sizes to macroscopic observations. The synergy of the particles allows the stabilization of water-in-oil (w/o) emulsions, which was not possible with MGs alone, and offers a larger internal interface than the stabilization with SNs alone. Furthermore, the incorporation of hydrophilic MGs into a hydrophobic particle layer accelerates the emulsions sedimentation speed. Nevertheless, the droplets are still sufficiently protected against coalescence even in the sediment and can be redispersed by gentle shaking. Based on droplet size measurements and cryo-SEM studies we elaborate a model, which explains the found phenomena.

Continuous microfluidic synthesis of zirconium-based UiO-67 using a coiled flow invertor reactor.

Metal-organic frameworks (MOFs), particularly Zirconium based, have a wide variety of potential applications, such as catalysis and separation. However, these are held back by traditionally only being synthesised in long batch reactions, which causes the process to be expensive and limit the amount of reaction control available, leading to potential batch to batch variation in the products, such as particle size distributions. Microfluidics allows for batch reactions to be performed with enhanced mass/heat transfer, with the coiled flow inverter reactor (CFIR) setup narrowing the residence time distribution, which is key in controlling the particle size and crystallinity. In this work, a Zirconium based MOF, UiO-67, has been synthesised continuously using a microfluidic CFIR, which has allowed for the product to be formed in 30 min, a fraction of the traditional batch heating time of 24 h. The microfluidicially synthesised UiO-67 is also smaller product with a narrower particle size distribution (≈200 nm to ≈400 nm) than its batch counterpart (~500 nm to over 3 µm).

Engineering of Microcage Carbon Nanotube Architectures with Decoupled Multimodal Porosity and Amplified Catalytic Performance.

New approaches for the engineering of the 3D microstructure, pore modality, and chemical functionality of hierarchically porous nanocarbon assemblies are key to develop the next generation of functional aerogel and membrane materials. Here, interfacially driven assembly of carbon nanotubes (CNT) is exploited to fabricate structurally directed aerogels with highly controlled internal architectures, composed of pseudo-monolayer, CNT microcages. CNT Pickering emulsions enable engineering at fundamentally different length scales, whereby the microporosity, mesoporosity, and macroporosity are decoupled and individually controlled through CNT type, CNT number density, and process energy, respectively. In addition, metal nanocatalysts (Cu, Pd, and Ru) are embedded within the architectures through an elegant sublimation and shock-decomposition approach; introducing the first approach that enables through-volume functionalization of intricate, pre-designed aerogels without microstructural degradation. Catalytic structure-function relationships are explored in a pharma-important amidation reaction; providing insights on how the engineered frameworks enhance catalyst activity. A sophisticated array of advanced tomographic, spectroscopic, and microscopic techniques reveal an intricate 3D assembly of CNT building-blocks and their influence on the functional properties of the enhanced nanocatalysts. These advances set a basis to modulate structure and chemistry of functional aerogel materials independently in a controlled fashion for a variety of applications, including energy conversion and storage, smart electronics, and (electro)catalysis.

Characterization of Amorphous Solid Dispersions and Identification of Low Levels of Crystallinity by Transmission Electron Microscopy.

Amorphous solid dispersions (ASDs) are used to increase the solubility of oral medicines by kinetically stabilizing the more soluble amorphous phase of an active pharmaceutical ingredient with a suitable amorphous polymer. Low levels of a crystalline material in an ASD can negatively impact the desired dissolution properties of the drug. Characterization techniques such as powder X-ray diffraction (pXRD), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR) are often used to detect and measure any crystallinity within ASDs. These techniques are unable to detect or quantify very low levels because they have limits of detection typically in the order of 1-5%. Herein, an ASD of felodipine (FEL) and polyvinylpyrrolidone/vinyl acetate copolymer (PVP/VA) prepared via a hot melt extrusion (HME) in a mass ratio of 30:70 was characterized using a range of techniques. No signs of residual crystallinity were found by pXRD, DSC, or FTIR. However, transmission electron microscopy (TEM) did identify two areas containing crystals at the edges of milled particles from a total of 55 examined. Both crystalline areas contained Cl Kα X-ray peaks when measured by energy-dispersive X-ray spectroscopy, confirming the presence of FEL (due to the presence of Cl atoms in FEL and not in PVP/VA). Further analysis was carried out by TEM using conical dark field (DF) imaging of a HME ASD of 50:50 FEL-PVP/VA to provide insights into the recrystallization process that occurs at the edges of particles during accelerated ageing conditions in an atmosphere of 75% relative humidity. Multiple metastable polymorphs of recrystallized FEL could be identified by selected area electron diffraction (SAED), predominately form II and the more stable form I. Conical DF imaging was also successful in spatially resolving and sizing crystals. This work highlights the potential for TEM-based techniques to improve the limit of detection of crystallinity in ASDs, while also providing insights into transformation pathways by identifying the location, size, and form of any crystallization that might occur on storage. This opens up the possibility of providing an enhanced understanding of a drug product's stability and performance.

Tuning stable noble metal nanoparticles dispersions to moderate their interaction with model membranes.

HYPOTHESIS: The properties of stable gold (Au) nanoparticle dispersions can be tuned to alter their activity towards biomembrane models. EXPERIMENTS: Au nanoparticle coating techniques together with rapid electrochemical screens of a phospholipid layer on fabricated mercury (Hg) on platinum (Pt) electrode have been used to moderate the phospholipid layer activity of Au nanoparticle dispersions. Screening results for Au nanoparticle dispersions were intercalibrated with phospholipid large unilamellar vesicle (LUV) interactions using a carboxyfluorescein (CF) leakage assay. All nanoparticle dispersions were characterised for size, by dynamic light scattering (DLS) and transmission electron microscopy (TEM). FINDINGS: Commercial and high quality home synthesised Au nanoparticle dispersions are phospholipid monolayer active whereas Ag nanoparticle dispersions are not. If Au nanoparticles are coated with a thin layer of Ag then the particle/lipid interaction is suppressed. The electrochemical assays of the lipid layer activity of Au nanoparticle dispersions align with LUV leakage assays of the same. Au nanoparticles of decreasing size and increasing dispersion concentration showed a stronger phospholipid monolayer/bilayer interaction. Treating Au nanoparticles with cell culture medium and incubation of Au nanoparticle dispersions in phosphate buffered saline (PBS) solutions removes their phospholipid layer interaction.

The quantitative impact of fluid vs. solid interfaces on the catalytic performance of pickering emulsions.

Pickering emulsions (PEs), i.e. particle stabilized emulsions, are used as reaction environments in biphasic catalysis for the hydroformylation of 1-dodecene into tridecanal using the catalyst rhodium (Rh)-sulfoxantphos (SX). The present study connects the knowledge about particle-catalyst interactions and PE structure with the reaction results. It quantifies the efficiency of the catalytic performance of the catalyst localized in the voids between the particles (liquid-liquid interface) and the catalyst adsorbed on the particle surface (liquid-solid interface) using a new numerical approach. First, it is ensured that the overall packing density and geometry at the droplet interface and the size of the water droplets of the resulting w/o PEs are predictable. Second, it is shown that approximately all particles assemble at the droplet surface after emulsion preparation and neither the packing parameter nor the droplet size change with the particle surface charge or size when the total particle cross section is kept constant. Third, studies on the influence of the catalyst on the emulsion structure reveal that irrespective of the particle charge the surface active and negatively charged catalyst Rh-SX reduces the PE droplet size significantly and decreases the particle packing parameter from s = 0.91 (hexagonal packing in 2D) to s = 0.69 (shattered structure). In this latter case, large voids of the free w/o interface form and become covered with the catalyst. With a deep knowledge about the PE structure the reaction efficiencies of the liquid-liquid vs. the solid-liquid interface are quantified. By excluding any other influence factors, it is shown that the activity of the catalyst is the same at the fluid and solid interface and the performance of the reaction is explained by the geometry of the system. After the reaction, the catalyst retention via membrane filtration is shown to be successfully achieved without damaging the emulsions. This enables the continuous recovery of the catalyst, i.e. the most expensive compound in PE-based catalytic reactions, being a crucial criterion for industrial applications.

Few-layer graphene induces both primary and secondary genotoxicity in epithelial barrier models in vitro.

BACKGROUND: Toxicological evaluation of engineered nanomaterials (ENMs) is essential for occupational health and safety, particularly where bulk manufactured ENMs such as few-layer graphene (FLG) are concerned. Additionally, there is a necessity to develop advanced in vitro models when testing ENMs to provide a physiologically relevant alternative to invasive animal experimentation. The aim of this study was to determine the genotoxicity of non-functionalised (neutral), amine- and carboxyl-functionalised FLG upon both human-transformed type-I (TT1) alveolar epithelial cell monocultures, as well as co-cultures of TT1 and differentiated THP-1 monocytes (d.THP-1 (macrophages)). RESULTS: In monocultures, TT1 and d.THP-1 macrophages showed a statistically significant (p < 0.05) cytotoxic response with each ENM following 24-h exposures. Monoculture genotoxicity measured by the in vitro cytokinesis blocked micronucleus (CBMN) assay revealed significant (p < 0.05) micronuclei induction at 8 µg/ml for amine- and carboxyl-FLG. Transmission electron microscopy (TEM) revealed ENMs were internalised by TT1 cells within membrane-bound vesicles. In the co-cultures, ENMs induced genotoxicity in the absence of cytotoxic effects. Co-cultures pre-exposed to 1.5 mM N-acetylcysteine (NAC), showed baseline levels of micronuclei induction, indicating that the genotoxicity observed was driven by oxidative stress. CONCLUSIONS: Therefore, FLG genotoxicity when examined in monocultures, results in primary-indirect DNA damage; whereas co-cultured cells reveal secondary mechanisms of DNA damage.

In Vitro Primary-Indirect Genotoxicity in Bronchial Epithelial Cells Promoted by Industrially Relevant Few-Layer Graphene.

Few-layer graphene (FLG) has garnered much interest owing to applications in hydrogen storage and reinforced nanocomposites. Consequently, these engineered nanomaterials (ENMs) are in high demand, increasing occupational exposure. This investigation seeks to assess the inhalation hazard of industrially relevant FLG engineered with: (i) no surface functional groups (neutral), (ii) amine, and (iii) carboxyl group functionalization. A monoculture of human lung epithelial (16HBE14o- ) cells is exposed to each material for 24-h, followed by cytotoxicity and genotoxicity evaluation using relative population doubling (RPD) and the cytokinesis-blocked micronucleus (CBMN) assay, respectively. Neutral-FLG induces the greatest (two-fold) significant increase (p < 0.05) in micronuclei, whereas carboxyl-FLG does not induce significant (p < 0.05) genotoxicity. These findings correlate to significant (p < 0.05) concentration-dependent increases in interleukin (IL)-8, depletion of intracellular glutathione (rGSH) and a depletion in mitochondrial ATP production. Uptake of FLG is evaluated by transmission electron microscopy, whereby FLG particles are observed within membrane-bound vesicles in the form of large agglomerates (>1 µm diameter). The findings of the present study have demonstrated the capability of neutral-FLG and amine-FLG to induce genotoxicity in 16HBE14o- cells through primary indirect mechanisms, suggesting a possible role for carboxyl groups in scavenging radicals produced via oxidative stress.

Detoxification, Active Uptake, and Intracellular Accumulation of Chromium Species by a Methane-Oxidizing Bacterium.

Despite the wide-ranging proscription of hexavalent chromium, chromium(VI) remains among the major polluting heavy metals worldwide. Aerobic methane-oxidizing bacteria are widespread environmental microorganisms that can perform diverse reactions using methane as the feedstock. The methanotroph Methylococcus capsulatus Bath, like many other microorganisms, detoxifies chromium(VI) by reduction to chromium(III). Here, the interaction of chromium species with M. capsulatus Bath was examined in detail by using a range of techniques. Cell fractionation and high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) indicated that externally provided chromium(VI) underwent reduction and was then taken up into the cytoplasmic and membranous fractions of the cells. This was confirmed by X-ray photoelectron spectroscopy (XPS) of intact cultures that indicated negligible chromium on the surfaces of or outside the cells. Distribution of chromium and other elements within intact and sectioned cells, as observed via transmission electron microscopy (TEM) combined with energy-dispersive X-ray spectroscopy (EDX) and electron energy loss spectroscopy (EELS), was consistent with the cytoplasm/membrane location of the chromium(III), possibly as chromium phosphate. The cells could also take up chromium(III) directly from the medium in a metabolism-dependent fashion and accumulate it. These results indicate a novel pattern of interaction with chromium species distinct from that observed previously with other microorganisms. They also suggest that M. capsulatus and similar methanotrophs may contribute directly to chromium(VI) reduction and accumulation in mixed communities of microorganisms that are able to perform methane-driven remediation of chromium(VI).IMPORTANCEM. capsulatus Bath is a well-characterized aerobic methane-oxidizing bacterium that has become a model system for biotechnological development of methanotrophs to perform useful reactions for environmental cleanup and for making valuable chemicals and biological products using methane gas. Interest in such technology has increased recently owing to increasing availability of low-cost methane from fossil and biological sources. Here, it is demonstrated that this versatile methanotroph can reduce the toxic contaminating heavy metal chromium(VI) to the less toxic form chromium(III) while accumulating the chromium(III) within the cells. This is expected to diminish the bioavailability of the chromium and make it less likely to be reoxidized to chromium(VI). Thus, M. capsulatus has the capacity to perform methane-driven remediation of chromium-contaminated water and other materials and to accumulate the chromium in the low-toxicity chromium(III) form within the cells.