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.

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.

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.

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.

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.

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.

Glycan-Gold Nanoparticles as Multifunctional Probes for Multivalent Lectin-Carbohydrate Binding: Implications for Blocking Virus Infection and Nanoparticle Assembly.

Multivalent lectin-glycan interactions are widespread in biology and are often exploited by pathogens to bind and infect host cells. Glycoconjugates can block such interactions and thereby prevent infection. The inhibition potency strongly depends on matching the spatial arrangement between the multivalent binding partners. However, the structural details of some key lectins remain unknown and different lectins may exhibit overlapping glycan specificity. This makes it difficult to design a glycoconjugate that can potently and specifically target a particular multimeric lectin for therapeutic interventions, especially under the challenging in vivo conditions. Conventional techniques such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) can provide quantitative binding thermodynamics and kinetics. However, they cannot reveal key structural information, e.g., lectin's binding site orientation, binding mode, and interbinding site spacing, which are critical to design specific multivalent inhibitors. Herein we report that gold nanoparticles (GNPs) displaying a dense layer of simple glycans are powerful mechanistic probes for multivalent lectin-glycan interactions. They can not only quantify the GNP-glycan-lectin binding affinities via a new fluorescence quenching method, but also reveal drastically different affinity enhancing mechanisms between two closely related tetrameric lectins, DC-SIGN (simultaneous binding to one GNP) and DC-SIGNR (intercross-linking with multiple GNPs), via a combined hydrodynamic size and electron microscopy analysis. Moreover, a new term, potential of assembly formation (PAF), has been proposed to successfully predict the assembly outcomes based on the binding mode between GNP-glycans and lectins. Finally, the GNP-glycans can potently and completely inhibit DC-SIGN-mediated augmentation of Ebola virus glycoprotein-driven cell entry (with IC50 values down to 95 pM), but only partially block DC-SIGNR-mediated virus infection. Our results suggest that the ability of a glycoconjugate to simultaneously block all binding sites of a target lectin is key to robust inhibition of viral infection.

Analysis of complex, beam-sensitive materials by transmission electron microscopy and associated techniques.

We review the use of transmission electron microscopy (TEM) and associated techniques for the analysis of beam-sensitive materials and complex, multiphase systems in-situ or close to their native state. We focus on materials prone to damage by radiolysis and explain that this process cannot be eliminated or switched off, requiring TEM analysis to be done within a dose budget to achieve an optimum dose-limited resolution. We highlight the importance of determining the damage sensitivity of a particular system in terms of characteristic changes that occur on irradiation under both an electron fluence and flux by presenting results from a series of molecular crystals. We discuss the choice of electron beam accelerating voltage and detectors for optimizing resolution and outline the different strategies employed for low-dose microscopy in relation to the damage processes in operation. In particular, we discuss the use of scanning TEM (STEM) techniques for maximizing information content from high-resolution imaging and spectroscopy of minerals and molecular crystals. We suggest how this understanding can then be carried forward for in-situ analysis of samples interacting with liquids and gases, provided any electron beam-induced alteration of a specimen is controlled or used to drive a chosen reaction. Finally, we demonstrate that cryo-TEM of nanoparticle samples snap-frozen in vitreous ice can play a significant role in benchmarking dynamic processes at higher resolution. This article is part of a discussion meeting issue 'Dynamic in situ microscopy relating structure and function'.

Methyl Selenol as a Precursor in Selenite Reduction to Se/S Species by Methane-Oxidizing Bacteria.

A wide range of microorganisms have been shown to transform selenium-containing oxyanions to reduced forms of the element, particularly selenium-containing nanoparticles. Such reactions are promising for the detoxification of environmental contamination and the production of valuable selenium-containing products, such as nanoparticles for application in biotechnology. It has previously been shown that aerobic methane-oxidizing bacteria, including Methylococcus capsulatus (Bath), are able to perform the methane-driven conversion of selenite (SeO32-) to selenium-containing nanoparticles and methylated selenium species. Here, the biotransformation of selenite by Mc. capsulatus (Bath) has been studied in detail via a range of imaging, chromatographic, and spectroscopic techniques. The results indicate that the nanoparticles are produced extracellularly and have a composition distinct from that of nanoparticles previously observed from other organisms. The spectroscopic data from the methanotroph-derived nanoparticles are best accounted for by a bulk structure composed primarily of octameric rings in the form Se8 -x S x with an outer coat of cell-derived biomacromolecules. Among a range of volatile methylated selenium and selenium-sulfur species detected, methyl selenol (CH3SeH) was found only when selenite was the starting material, although selenium nanoparticles (both biogenic and chemically produced) could be transformed into other methylated selenium species. This result is consistent with methyl selenol being an intermediate in the methanotroph-mediated biotransformation of selenium to all the methylated and particulate products observed.IMPORTANCE Aerobic methane-oxidizing bacteria are ubiquitous in the environment. Two well-characterized strains, Mc. capsulatus (Bath) and Methylosinus trichosporium OB3b, representing gamma- and alphaproteobacterial methanotrophs, respectively, can convert selenite, an environmental pollutant, to volatile selenium compounds and selenium-containing particulates. Both conversions can be harnessed for the bioremediation of selenium pollution using biological or fossil methane as the feedstock, and these organisms could be used to produce selenium-containing particles for food and biotechnological applications. Using an extensive suite of techniques, we identified precursors of selenium nanoparticle formation and also found that these nanoparticles are made up of eight-membered mixed selenium and sulfur rings.

In vitro detection of in vitro secondary mechanisms of genotoxicity induced by engineered nanomaterials.

BACKGROUND: It is well established that toxicological evaluation of engineered nanomaterials (NMs) is vital to ensure the health and safety of those exposed to them. Further, there is a distinct need for the development of advanced physiologically relevant in vitro techniques for NM hazard prediction due to the limited predictive power of current in vitro models and the unsustainability of conducting nano-safety evaluations in vivo. Thus, the purpose of this study was to develop alternative in vitro approaches to assess the potential of NMs to induce genotoxicity by secondary mechanisms. RESULTS: This was first undertaken by a conditioned media-based technique, whereby cell culture media was transferred from differentiated THP-1 (dTHP-1) macrophages treated with γ-Fe2O3 or Fe3O4 superparamagnetic iron oxide nanoparticles (SPIONs) to the bronchial cell line 16HBE14o-. Secondly construction and SPION treatment of a co-culture model comprising of 16HBE14o- cells and dTHP-1 macrophages. For both of these approaches no cytotoxicity was detected and chromosomal damage was evaluated by the in vitro micronucleus assay. Genotoxicity assessment was also performed using 16HBE14o- monocultures, which demonstrated only γ-Fe2O3 nanoparticles to be capable of inducing chromosomal damage. In contrast, immune cell conditioned media and dual cell co-culture SPION treatments showed both SPION types to be genotoxic to 16HBE14o- cells due to secondary genotoxicity promoted by SPION-immune cell interaction. CONCLUSIONS: The findings of the present study demonstrate that the approach of using single in vitro cell test systems precludes the ability to consider secondary genotoxic mechanisms. Consequently, the use of multi-cell type models is preferable as they better mimic the in vivo environment and thus offer the potential to enhance understanding and detection of a wider breadth of potential damage induced by NMs.

Toward Developing a Predictive Approach To Assess Electron Beam Instability during Transmission Electron Microscopy of Drug Molecules.

During drug development control of polymorphism, particle properties and impurities are critical for ensuring a good quality, reproducible, and safe medicine. A wide variety of analytical techniques are employed in demonstrating the regulators control over the drug substance and product manufacturing, storage, and supply. Transmission electron microscopy (TEM) offers the opportunity to analyze in detail pharmaceutical systems at a length scale and limit of detection not readily achieved by many traditional techniques. However, the use of TEM as a characterization tool for drug development is uncommon due to possible damage caused by the electron beam. This work outlines the development of a model, using molecular descriptors, to predict the electron beam stability of active pharmaceutical ingredients (API). For a given set of conditions and a particular imaging or analytical mode, the total number of electrons per unit area, which causes observable damage to a sample in the TEM, can be defined as the critical fluence ( CF). Here the CF of 20 poorly water-soluble APIs were measured using selected area electron diffraction. Principal component analysis was used to select the most influential molecular descriptors on CF, which were shown to be descriptors involving the degree of conjugation, the number of hydrogen bond donors and acceptors, and the number of rotatable bonds. These were used to generate several multiple linear regression models. The model that provided the best fit to the measured CF data included the ratio of the number of conjugated carbons to nonconjugated carbons, the ratio of the number of hydrogen bond donors to acceptors, and the ratio of the number of hydrogen bond acceptors to donors. Using this model, the CF of the majority of the compounds was predicted within ±2 e-/Å2. Molecules with no hydrogen bond acceptors did not fit the model accurately possibly due to the limited sample size or the influence of other parameters not included in this model, such as intermolecular bond energies. The model presented can be used to support pharmaceutical development by quickly assessing the stability of other poorly soluble drugs in TEM. Provided that the model suggests that the API is relatively stable to electron irradiation, TEM offers the prospect of determining the presence of crystalline material at low levels at length scales and limits of detection unobtainable by other techniques. This is particularly so for amorphous solid dispersions.

Dissecting Multivalent Lectin-Carbohydrate Recognition Using Polyvalent Multifunctional Glycan-Quantum Dots.

Multivalent protein-carbohydrate interactions initiate the first contacts between virus/bacteria and target cells, which ultimately lead to infection. Understanding the structures and binding modes involved is vital to the design of specific, potent multivalent inhibitors. However, the lack of structural information on such flexible, complex, and multimeric cell surface membrane proteins has often hampered such endeavors. Herein, we report that quantum dots (QDs) displayed with a dense array of mono-/disaccharides are powerful probes for multivalent protein-glycan interactions. Using a pair of closely related tetrameric lectins, DC-SIGN and DC-SIGNR, which bind to the HIV and Ebola virus glycoproteins (EBOV-GP) to augment viral entry and infect target cells, we show that such QDs efficiently dissect the different DC-SIGN/R-glycan binding modes (tetra-/di-/monovalent) through a combination of multimodal readouts: Förster resonance energy transfer (FRET), hydrodynamic size measurement, and transmission electron microscopy imaging. We also report a new QD-FRET method for quantifying QD-DC-SIGN/R binding affinity, revealing that DC-SIGN binds to the QD >100-fold tighter than does DC-SIGNR. This result is consistent with DC-SIGN's higher trans-infection efficiency of some HIV strains over DC-SIGNR. Finally, we show that the QDs potently inhibit DC-SIGN-mediated enhancement of EBOV-GP-driven transduction of target cells with IC50 values down to 0.7 nM, matching well to their DC-SIGN binding constant (apparent Kd = 0.6 nM) measured by FRET. These results suggest that the glycan-QDs are powerful multifunctional probes for dissecting multivalent protein-ligand recognition and predicting glyconanoparticle inhibition of virus infection at the cellular level.

Nanoparticle vesicle encoding for imaging and tracking cell populations.

For phenotypic behavior to be understood in the context of cell lineage and local environment, properties of individual cells must be measured relative to population-wide traits. However, the inability to accurately identify, track and measure thousands of single cells via high-throughput microscopy has impeded dynamic studies of cell populations. We demonstrate unique labeling of cells, driven by the heterogeneous random uptake of fluorescent nanoparticles of different emission colors. By sequentially exposing a cell population to different particles, we generated a large number of unique digital codes, which corresponded to the cell-specific number of nanoparticle-loaded vesicles and were visible within a given fluorescence channel. When three colors are used, the assay can self-generate over 17,000 individual codes identifiable using a typical fluorescence microscope. The color-codes provided immediate visualization of cell identity and allowed us to track human cells with a success rate of 78% across image frames separated by 8 h.

Spatially orthogonal chemical functionalization of a hierarchical pore network for catalytic cascade reactions.

The chemical functionality within porous architectures dictates their performance as heterogeneous catalysts; however, synthetic routes to control the spatial distribution of individual functions within porous solids are limited. Here we report the fabrication of spatially orthogonal bifunctional porous catalysts, through the stepwise template removal and chemical functionalization of an interconnected silica framework. Selective removal of polystyrene nanosphere templates from a lyotropic liquid crystal-templated silica sol-gel matrix, followed by extraction of the liquid crystal template, affords a hierarchical macroporous-mesoporous architecture. Decoupling of the individual template extractions allows independent functionalization of macropore and mesopore networks on the basis of chemical and/or size specificity. Spatial compartmentalization of, and directed molecular transport between, chemical functionalities affords control over the reaction sequence in catalytic cascades; herein illustrated by the Pd/Pt-catalysed oxidation of cinnamyl alcohol to cinnamic acid. We anticipate that our methodology will prompt further design of multifunctional materials comprising spatially compartmentalized functions.

Microbial transformations of selenite by methane-oxidizing bacteria.

Methane-oxidizing bacteria are well known for their role in the global methane cycle and their potential for microbial transformation of wide range of hydrocarbon and chlorinated hydrocarbon pollution. Recently, it has also emerged that methane-oxidizing bacteria interact with inorganic pollutants in the environment. Here, we report what we believe to be the first study of the interaction of pure strains of methane-oxidizing bacteria with selenite. Results indicate that the commonly used laboratory model strains of methane-oxidizing bacteria, Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b, are both able to reduce the toxic selenite (SeO32-) but not selenate (SeO42-) to red spherical nanoparticulate elemental selenium (Se0), which was characterized via energy-dispersive X-ray spectroscopy (EDXS), X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS). The cultures also produced volatile selenium-containing species, which suggests that both strains may have an additional activity that can transform either Se0 or selenite into volatile methylated forms of selenium. Transmission electron microscopy (TEM) measurements and experiments with the cell fractions cytoplasm, cell wall and cell membrane show that the nanoparticles are formed mainly on the cell wall. Collectively, these results are promising for the use of methane-oxidizing bacteria for bioremediation or suggest possible uses in the production of selenium nanoparticles for biotechnology.

Biomagnetic Recovery and Bioaccumulation of Selenium Granules in Magnetotactic Bacteria.

UNLABELLED: Using microorganisms to remove waste and/or neutralize pollutants from contaminated water is attracting much attention due to the environmentally friendly nature of this methodology. However, cell recovery remains a bottleneck and a considerable challenge for the development of this process. Magnetotactic bacteria are a unique group of organisms that can be manipulated by an external magnetic field due to the presence of biogenic magnetite crystals formed within their cells. In this study, we demonstrated an account of accumulation and precipitation of amorphous elemental selenium nanoparticles within magnetotactic bacteria alongside and independent of magnetite crystal biomineralization when grown in a medium containing selenium oxyanion (SeO3 (2-)). Quantitative analysis shows that magnetotactic bacteria accumulate the largest amount of target molecules (Se) per cell compared with any other previously reported nonferrous metal/metalloid. For example, 2.4 and 174 times more Se is accumulated than Te taken up into cells and Cd(2+) adsorbed onto the cell surface, respectively. Crucially, the bacteria with high levels of Se accumulation were successfully recovered with an external magnetic field. The biomagnetic recovery and the effective accumulation of target elements demonstrate the potential for application in bioremediation of polluted water. IMPORTANCE: The development of a technique for effective environmental water remediation is urgently required across the globe. A biological remediation process of waste removal and/or neutralization of pollutant from contaminated water using microorganisms has great potential, but cell recovery remains a bottleneck. Magnetotactic bacteria synthesize magnetic particles within their cells, which can be recovered by a magnetic field. Herein, we report an example of accumulation and precipitation of amorphous elemental selenium nanoparticles within magnetotactic bacteria independent of magnetic particle synthesis. The cells were able to accumulate the largest amount of Se compared to other foreign elements. More importantly, the Se-accumulating bacteria were successfully recovered with an external magnetic field. We believe magnetotactic bacteria confer unique advantages of biomagnetic cell recovery and of Se accumulation, providing a new and effective methodology for bioremediation of polluted water.

Genotoxic capacity of Cd/Se semiconductor quantum dots with differing surface chemistries.

Quantum dots (QD) have unique electronic and optical properties promoting biotechnological advances. However, our understanding of the toxicological structure-activity relationships remains limited. This study aimed to determine the biological impact of varying nanomaterial surface chemistry by assessing the interaction of QD with either a negative (carboxyl), neutral (hexadecylamine; HDA) or positive (amine) polymer coating with human lymphoblastoid TK6 cells. Following QD physico-chemical characterisation, cellular uptake was quantified by optical and electron microscopy. Cytotoxicity was evaluated and genotoxicity was characterised using the micronucleus assay (gross chromosomal damage) and the HPRT forward mutation assay (point mutagenicity). Cellular damage mechanisms were also explored, focusing on oxidative stress and mitochondrial damage. Cell uptake, cytotoxicity and genotoxicity were found to be dependent on QD surface chemistry. Carboxyl-QD demonstrated the smallest agglomerate size and greatest cellular uptake, which correlated with a dose dependent increase in cytotoxicity and genotoxicity. Amine-QD induced minimal cellular damage, while HDA-QD promoted substantial induction of cell death and genotoxicity. However, HDA-QD were not internalised by the cells and the damage they caused was most likely due to free cadmium release caused by QD dissolution. Oxidative stress and induced mitochondrial reactive oxygen species were only partially associated with cytotoxicity and genotoxicity induced by the QD, hence were not the only mechanisms of importance. Colloidal stability, nanoparticle (NP) surface chemistry, cellular uptake levels and the intrinsic characteristics of the NPs are therefore critical parameters impacting genotoxicity induced by QD.

Quantifying the cellular uptake of semiconductor quantum dot nanoparticles by analytical electron microscopy.

Semiconductor quantum dot nanoparticles are in demand as optical biomarkers yet the cellular uptake process is not fully understood; quantification of numbers and the fate of internalized particles are still to be achieved. We have focussed on the characterization of cellular uptake of quantum dots using a combination of analytical electron microscopies because of the spatial resolution available to examine uptake at the nanoparticle level, using both imaging to locate particles and spectroscopy to confirm identity. In this study, commercially available quantum dots, CdSe/ZnS core/shell particles coated in peptides to target cellular uptake by endocytosis, have been investigated in terms of the agglomeration state in typical cell culture media, the traverse of particle agglomerates across U-2 OS cell membranes during endocytosis, the merging of endosomal vesicles during incubation of cells and in the correlation of imaging flow cytometry and transmission electron microscopy to measure the final nanoparticle dose internalized by the U-2 OS cells. We show that a combination of analytical transmission electron microscopy and serial block face scanning electron microscopy can provide a comprehensive description of the internalization of an initial exposure dose of nanoparticles by an endocytically active cell population and how the internalized, membrane bound nanoparticle load is processed by the cells. We present a stochastic model of an endosome merging process and show that this provides a data-driven modelling framework for the prediction of cellular uptake of engineered nanoparticles in general.