Impact of engineered nanomaterials on health : considerations for benefit-risk assessment

Nanotechnology encompasses the design, characterisation, production and application of materials and systems by controlling shape and size at the nanoscale (nanometres). Nanomaterials may differ from other materials because of their relatively large specific surface area, such that surface properties become particularly important. There has been rapid growth in investment in nanotechnology by both the public and private sectors worldwide. In the EU, nanotechnology is expected to become an important strategic contributor to achieving economic gain and societal and individual benefits. At the same time there is continuing scientific uncertainty and controversy about the safety of nanomaterials. It is important to ensure that timely policy development takes this into consideration. Uncertainty about safety may lead to polarised public debate and to business unwillingness to invest further. A clear regulatory framework to address potential health and environmental impacts, within the wider context of evaluating and communicating the benefit-risk balance, must be a core part of Europe's integrated efforts for nanotechnology innovation. While a number of studies have been carried out on the effect of environmental nanoparticles, e.g. from combustion processes, on human health, there is yet no generally acceptable paradigm for safety assessment of nanomaterials in consumer and other products. Therefore, a working group was established to consider issues for the possible impact of nanomaterials on human health focussing specifically on engineered nanomaterials. This represents the first joint initiative between EASAC and the Joint Research Centre of the European Commission. The working group was given the remit to describe the state of the art of benefits and potential risks, current methods for safety assessment, and to evaluate their relevance, identify knowledge gaps in studying the safety of current nanomaterials, and recommend on priorities for nanomaterial research and the regulatory framework. This report focuses on key principles and issues, cross-referencing other sources for detailed information, rather than attempting a comprehensive account of the science. The focus is on human health although environmental effects are also discussed when directly relevant to health

Reinvention of chemotherapy: drug conjugates and nanoparticles.

PURPOSE OF REVIEW: Recent advances in nanotechnology have addressed some of the issues related to lack of selectivity and nonspecific toxicities associated with conventional chemotherapy. Nanoparticles are therapeutic carriers that can be fine tuned for specific application and for passive or active tumor targeting. RECENT FINDINGS: Although the nanoparticle field is rapidly expanding, there are to date only six nanoparticle-based drug delivery platforms and two antibody-drug conjugates that are clinically approved for cancer therapy. Here, we review the clinical data of liposomal anthracyclines, nanoparticle formulations of paclitaxel and trastuzumab emtansine. We then briefly comment on efficacy and safety issues of nanoparticles, as well as on the next-generation nanoparticles for cancer therapy. SUMMARY: The emerging development of cancer nanotechnology offers the opportunity of reinvestigating the potential of cytotoxic agents, improving tumor targeting and drug delivery, leading to better safety profile and antitumor activity. Adding specificity to nanoparticles may allow personalization of cancer therapy using chemotherapy.

Diffraction grating couplers milled in Si3N4 rib waveguides with a focused ion beam

Focused ion beam milling is a processing technology which allows flexible direct writing of nanometer scale features efficiently substituting electron beam lithography. No mask need results in ability for patterns writing even on fragile micromechanical devices. In this work we studied the abilities of the tool for fabrication of diffraction grating couplers in silicon nitride waveguides. The gratings were fabricated on a chip with extra fragile cantilevers of sub micron thickness. Optical characterization of the couplers was done using excitation of the waveguides in visible range by focused Gaussian beams of different waist sizes. Influence of Ga+ implantation on the device performance was studied.

The multi-facets of sustainable nanotechnology : lessons from a nanosafety symposium

An international symposium for nanosafety was held recently at the Nanyang Technological University in Singapore. Topics relating to understanding nanomaterial properties, tools, and infrastructure required for predicting hazardous outcomes, measuring nanomaterial exposure levels, systems approach for risk assessment and public's perception of nanotechnology were covered. The need for a multidisciplinary approach, across both natural and social sciences, for developing sustainable nanotechnology solutions was heavily emphasized. This commentary highlights the major issues discussed and the commitment of the nanosafety research community in Singapore to contribute collectively to realise the vision of sustainable nanotechnology.

Nanocharacterization of Soft Biological Samples in Shear Mode with Quartz Tuning Fork Probes

Quartz tuning forks are extremely good resonators and their use is growing in scanning probe microscopy. Nevertheless, only a few studies on soft biological samples have been reported using these probes. In this work, we present the methodology to develop and use these nanosensors to properly work with biological samples. The working principles, fabrication and experimental setup are presented. The results in the nanocharacterization of different samples in different ambients are presented by using different working modes: amplitude modulation with and without the use of a Phase-Locked Loop (PLL) and frequency modulation. Pseudomonas aeruginosa bacteria are imaged in nitrogen using amplitude modulation. Microcontact printed antibodies are imaged in buffer using amplitude modulation with a PLL. Finally, metastatic cells are imaged in air using frequency modulation.

Intrinsic ROS-production capacity of nanoparticles

Engineered nanomaterials (ENMs) exhibit special physicochemical properties and thus are finding their way into an increasing number of industries, enabling products with improved properties. Their increased use brings a greater likelihood of exposure to the nanoparticles (NPs) that could be released during the life cycle of nano-abled products. The field of nanotoxicology has emerged as a consequence of the development of these novel materials, and it has gained ever more attention due to the urgent need to gather information on exposure to them and to understand the potential hazards they engender. However, current studies on nanotoxicity tend to focus on pristine ENMs, and they use these toxicity results to generalize risk assessments on human exposure to NPs. ENMs released into the environment can interact with their surroundings, change characteristics and exhibit toxicity effects distinct from those of pristine ENMs. Furthermore, NPs' large surface areas provide extra-large potential interfaces, thus promoting more significant interactions between NPs and other co-existing species. In such processes, other species can attach to a NP's surface and modify its surface functionality, in addition to the toxicity in normally exhibits. One particular occupational health scenario involves NPs and low-volatile organic compounds (LVOC), a common type of pollutant existing around many potential sources of NPs. LVOC can coat a NP's surface and then dominate its toxicity. One important mechanism in nanotoxicology is the creation of reactive oxygen species (ROS) on a NP's surface; LVOC can modify the production of these ROS. In summary, nanotoxicity research should not be limited to the toxicity of pristine NPs, nor use their toxicity to evaluate the health effects of exposure to environmental NPs. Instead, the interactions which NPs have with other environmental species should also be considered and researched. The potential health effects of exposure to NPs should be derived from these real world NPs with characteristics modified by the environment and their distinct toxicity. Failure to suitably address toxicity results could lead to an inappropriate treatment of nano- release, affect the environment and public health and put a blemish on the development of sustainable nanotechnologies as a whole. The main objective of this thesis is to demonstrate a process for coating NP surfaces with LVOC using a well-controlled laboratory design and, with regard to these NPs' capacity to generate ROS, explore the consequences of changing particle toxicity. The dynamic coating system developed yielded stable and replicable coating performance, simulating an important realistic scenario. Clear changes in the size distribution of airborne NPs were observed using a scanning mobility particle sizer, were confirmed using both liquid nanotracking analyses and transmission electron microscopy (TEM) imaging, and were verified thanks to the LVOC coating. Coating thicknesses corresponded to the amount of coating material used and were controlled using the parameters of the LVOC generator. The capacity of pristine silver NPs (Ag NPs) to generate ROS was reduced when they were given a passive coating of inert paraffin: this coating blocked the reactive zones on the particle surfaces. In contrast, a coating of active reduced-anthraquinone contributed to redox reactions and generated ROS itself, despite the fact that ROS generation due to oxidation by Ag NPs themselves was quenched. Further objectives of this thesis included development of ROS methodology and the analysis of ROS case studies. Since the capacity of NPs to create ROS is an important effect in nanotoxicity, we attempted to refine and standardize the use of 2'7-dichlorodihydrofluorescin (DCFH) as a chemical tailored for the characterization of NPs' capacity for ROS generation. Previous studies had reported a wide variety of results, which were due to a number of insufficiently well controlled factors. We therefore cross-compared chemicals and concentrations, explored ways of dispersing NP samples in liquid solutions, identified sources of contradictions in the literature and investigated ways of reducing artificial results. The most robust results were obtained by sonicating an optimal sample of NPs in a DCFH-HRP solution made of 5,M DCFH and 0.5 unit/ml horseradish peroxidase (HRP). Our findings explained how the major reasons for previously conflicting results were the different experimental approaches used and the potential artifacts appearing when using high sample concentrations. Applying our advanced DCFH protocol with other physicochemical characterizations and biological analyses, we conducted several case studies, characterizing aerosols and NP samples. Exposure to aged brake wear dust engenders a risk of potential deleterious health effects in occupational scenarios. We performed microscopy and elemental analyses, as well as ROS measurements, with acellular and cellular DCFH assays. TEM images revealed samples to be heterogeneous mixtures with few particles in the nano-scale. Metallic and non-metallic elements were identified, primarily iron, carbon and oxygen. Moderate amounts of ROS were detected in the cell-free fluorescent tests; however, exposed cells were not dramatically activated. In addition to their highly aged state due to oxidation, the reason aged brake wear samples caused less oxidative stress than fresh brake wear samples may be because of their larger size and thus smaller relative reactive surface area. Other case studies involving welding fumes and differently charged NPs confirmed the performance of our DCFH assay and found ROS generation linked to varying characteristics, especially the surface functionality of the samples. Les nanomatériaux manufacturés (ENM) présentent des propriétés physico-chimiques particulières et ont donc trouvés des applications dans un nombre croissant de secteurs, permettant de réaliser des produits ayant des propriétés améliorées. Leur utilisation accrue engendre un plus grand risque pour les êtres humains d'être exposés à des nanoparticules (NP) qui sont libérées au long de leur cycle de vie. En conséquence, la nanotoxicologie a émergé et gagné de plus en plus d'attention dû à la nécessité de recueillir les renseignements nécessaires sur l'exposition et les risques associés à ces nouveaux matériaux. Cependant, les études actuelles sur la nanotoxicité ont tendance à se concentrer sur les ENM et utiliser ces résultats toxicologiques pour généraliser l'évaluation des risques sur l'exposition humaine aux NP. Les ENM libérés dans l'environnement peuvent interagir avec l'environnement, changeant leurs caractéristiques, et montrer des effets de toxicité distincts par rapport aux ENM originaux. Par ailleurs, la grande surface des NP fournit une grande interface avec l'extérieur, favorisant les interactions entre les NP et les autres espèces présentes. Dans ce processus, d'autres espèces peuvent s'attacher à la surface des NP et modifier leur fonctionnalité de surface ainsi que leur toxicité. Un scénario d'exposition professionnel particulier implique à la fois des NP et des composés organiques peu volatils (LVOC), un type commun de polluant associé à de nombreuses sources de NP. Les LVOC peuvent se déposer sur la surface des NP et donc dominer la toxicité globale de la particule. Un mécanisme important en nanotoxicologie est la création d'espèces réactives d'oxygène (ROS) sur la surface des particules, et les LVOC peuvent modifier cette production de ROS. En résumé, la recherche en nanotoxicité ne devrait pas être limitée à la toxicité des ENM originaux, ni utiliser leur toxicité pour évaluer les effets sur la santé de l'exposition aux NP de l'environnement; mais les interactions que les NP ont avec d'autres espèces environnementales doivent être envisagées et étudiées. Les effets possibles sur la santé de l'exposition aux NP devraient être dérivés de ces NP aux caractéristiques modifiées et à la toxicité distincte. L'utilisation de résultats de toxicité inappropriés peut conduire à une mauvaise prise en charge de l'exposition aux NP, de détériorer l'environnement et la santé publique et d'entraver le développement durable des industries de la nanotechnologie dans leur ensemble. L'objectif principal de cette thèse est de démontrer le processus de déposition des LVOC sur la surface des NP en utilisant un environnement de laboratoire bien contrôlé et d'explorer les conséquences du changement de toxicité des particules sur leur capacité à générer des ROS. Le système de déposition dynamique développé a abouti à des performances de revêtement stables et reproductibles, en simulant des scénarios réalistes importants. Des changements clairs dans la distribution de taille des NP en suspension ont été observés par spectrométrie de mobilité électrique des particules, confirmé à la fois par la méthode dite liquid nanotracking analysis et par microscopie électronique à transmission (MET), et a été vérifié comme provenant du revêtement par LVOC. La correspondance entre l'épaisseur de revêtement et la quantité de matériau de revêtement disponible a été démontré et a pu être contrôlé par les paramètres du générateur de LVOC. La génération de ROS dû aux NP d'argent (Ag NP) a été diminuée par un revêtement passif de paraffine inerte bloquant les zones réactives à la surface des particules. Au contraire, le revêtement actif d'anthraquinone réduit a contribué aux réactions redox et a généré des ROS, même lorsque la production de ROS par oxydation des Ag NP avec l'oxygène a été désactivé. Les objectifs associés comprennent le développement de la méthodologie et des études de cas spécifique aux ROS. Etant donné que la capacité des NP à générer des ROS contribue grandement à la nanotoxicité, nous avons tenté de définir un standard pour l'utilisation de 27- dichlorodihydrofluorescine (DCFH) adapté pour caractériser la génération de ROS par les NP. Des etudes antérieures ont rapporté une grande variété de résultats différents, ce qui était dû à un contrôle insuffisant des plusieurs facteurs. Nous avons donc comparé les produits chimiques et les concentrations utilisés, exploré les moyens de dispersion des échantillons HP en solution liquide, investigué les sources de conflits identifiées dans les littératures et étudié les moyens de réduire les résultats artificiels. De très bon résultats ont été obtenus par sonication d'une quantité optimale d'échantillons de NP en solution dans du DCFH-HRP, fait de 5 nM de DCFH et de 0,5 unité/ml de Peroxydase de raifort (HRP). Notre étude a démontré que les principales raisons causant les conflits entre les études précédemment conduites dans la littérature étaient dues aux différentes approches expérimentales et à des artefacts potentiels dus à des concentrations élevées de NP dans les échantillons. Utilisant notre protocole DCFH avancé avec d'autres caractérisations physico-chimiques et analyses biologiques, nous avons mené plusieurs études de cas, caractérisant les échantillons d'aérosols et les NP. La vielle poussière de frein en particulier présente un risque élevé d'exposition dans les scénarios professionnels, avec des effets potentiels néfastes sur la santé. Nous avons effectué des analyses d'éléments et de microscopie ainsi que la mesure de ROS avec DCFH cellulaire et acellulaire. Les résultats de MET ont révélé que les échantillons se présentent sous la forme de mélanges de particules hétérogènes, desquels une faible proportion se trouve dans l'échelle nano. Des éléments métalliques et non métalliques ont été identifiés, principalement du fer, du carbone et de l'oxygène. Une quantité modérée de ROS a été détectée dans le test fluorescent acellulaire; cependant les cellules exposées n'ont pas été très fortement activées. La raison pour laquelle les échantillons de vielle poussière de frein causent un stress oxydatif inférieur par rapport à la poussière de frein nouvelle peut-être à cause de leur plus grande taille engendrant une surface réactive proportionnellement plus petite, ainsi que leur état d'oxydation avancé diminuant la réactivité. D'autres études de cas sur les fumées de soudage et sur des NP différemment chargées ont confirmé la performance de notre test DCFH et ont trouvé que la génération de ROS est liée à certaines caractéristiques, notamment la fonctionnalité de surface des échantillons.

Growth and magnetic characterization of Co nanoparticles obtained by femtosecond pulsed laser deposition.

We present a detailed study on the morphology and magnetic properties of Co nanostructures deposited onto oxidized Si substrates by femtosecond pulsed laser deposition. Generally, Co disks of nanometric dimensions are obtained just above the ablation threshold, with a size distribution characterized by an increasingly larger number of disks as their size diminishes, and with a maximum disk size that depends on the laser power density. In Au/Co/Au structures, in-plane magnetic anisotropy is observed in all cases, with no indication of superparamagnetism regardless of the amount of material or the laser power density. Magnetic force microscopy observations show coexistence of single-domain and vortex states for the magnetic domain structure of the disks. Superconducting quantum interference device magnetometry and x-ray magnetic circular dichroism measurements point to saturation magnetization values lower than the bulk, probably due to partial oxidation of the Co resulting from incomplete coverage by the Au capping layer.

Identification of new transformation products during enzymatic treatment of tetracycline and erythromycin antibiotics at laboratory scale by an on-line turbulent flow liquid-chromatography coupled to a high resolution mass spectrometer LTQ-Orbitrap

This work describes the formation of transformation products (TPs) by the enzymatic degradation at laboratory scale of two highly consumed antibiotics: tetracycline (Tc) and erythromycin (ERY). The analysis of the samples was carried out by a fast and simple method based on the novel configuration of the on-line turbulent flow system coupled to a hybrid linear ion trap – high resolution mass spectrometer. The method was optimized and validated for the complete analysis of ERY, Tc and their transformation products within 10 min without any other sample manipulation. Furthermore, the applicability of the on-line procedure was evaluated for 25 additional antibiotics, covering a wide range of chemical classes in different environmental waters with satisfactory quality parameters. Degradation rates obtained for Tc by laccase enzyme and ERY by EreB esterase enzyme without the presence of mediators were ∼78% and ∼50%, respectively. Concerning the identification of TPs, three suspected compounds for Tc and five of ERY have been proposed. In the case of Tc, the tentative molecular formulas with errors mass within 2 ppm have been based on the hypothesis of dehydroxylation, (bi)demethylation and oxidation of the rings A and C as major reactions. In contrast, the major TP detected for ERY has been identified as the “dehydration ERY-A”, with the same molecular formula of its parent compound. In addition, the evaluation of the antibiotic activity of the samples along the enzymatic treatments showed a decrease around 100% in both cases

Surface Studies of Thiolate Adsorbates on Some Metals

The results and discussions in this thesis are based on my studies about selfassembled thiol layers on gold, platinum, silver and copper surfaces. These kinds of layers are two-dimensional, one molecule thick and covalently organized at the surface. They are an easy way to modify surface properties. Self-assembly is today an intensive research field because of the promise it holds for producing new technology at nanoscale, the scale of atoms and molecules. These kinds of films have applications for example, in the fields of physics, biology, engineering, chemistry and computer science. Compared to the extensive literature concerning self-assembled monolayers (SAMs) on gold, little is known about the structure and properties of thiolbased SAMs on other metals. In this thesis I have focused on thiol layers on gold, platinum, silver and copper substrates. These studies can be regarded as a basic study of SAMs. Nevertheless, an understanding of the physical and chemical nature of SAMs allows the correlation between atomic structure and macroscopic properties. The results can be used as a starting point for many practical applications. X-ray photoelectron spectroscopy (XPS) and synchrotron radiation excited high resolution photoelectron spectroscopy (HR-XPS) together with time-offlight secondary ion mass spectrometry (ToF-SIMS) were applied to investigate thin organic films formed by the spontaneous adsorption of molecules on metal surfaces. Photoelectron spectroscopy was the main method used in these studies. In photoelectron spectroscopy, the sample is irradiated with photons and emitted photoelectrons are energy-analyzed. The obtained spectra give information about the atomic composition of the surface and about the chemical state of the detected elements. It is widely used in the study of thin layers and is a very powerful tool for this purpose. Some XPS results were complemented with ToF-SIMS measurements. It provides information on the chemical composition and molecular structure of the samples. Thiol (1-Dodecanethiol, CH3(CH2)11SH) solution was used to create SAMs on metal substrates. Uniform layers were formed on most of the studied metal surfaces. On platinum, surface aligned molecules were also detected in investigations by XPS and ToF-SIMS. The influence of radiation on the layer structure was studied, leading to the conclusion that parts of the hydrocarbon chains break off due to radiation and the rest of the layer is deformed. The results obtained showed differences depending on the substrate material. The influence of oxygen on layer formation was also studied. Thiol molecules were found to replace some of the oxygen from the metal surfaces.

An undergraduate level experiment on the synthesis of Au nanoparticles and their size-dependent optical and catalytic properties

The synthesis of gold nanoparticles (Au NPs) 15, 26, and 34 nm in diameter, followed by the investigation of their size-dependent optical and catalytic properties, is described herein as an undergraduate level experiment. The proposed experiment covers concepts on the synthesis, stabilization, and characterization of Au NPs, their size-dependent optical and catalytic properties at the nanoscale, chemical kinetics, and the role of a catalyst. The experiment should be performed by groups of two or three students in three lab sessions of 3 h each and organized as follows: i) synthesis of Au NPs of different sizes and investigation of their optical properties; ii) evaluation of their catalytic activity; and iii) data analysis and discussion. We believe that this activity enables students to integrate these multidisciplinary concepts in a single experiment as well as to become introduced/familiarized with an active research field and current literature in the areas of nanoparticle synthesis and catalysis.

CARBOXYLATION OF SILVER NANOPARTICLES FOR THE IMMOBILIZATION OF β-GALACTOSIDASE AND ITS EFFICACY IN GALACTO-OLIGOSACCHARIDES PRODUCTION

The present study investigated the carboxylation of silver nanoparticles (AgNPs) by 1:3 nitric acid-sulfuric acid mixtures for immobilizing Aspergillus oryzae β-galactosidase. Carboxylated AgNPs retained 93% enzyme upon immobilization and the enzyme did not leach out appreciably from the modified nanosupport in the presence of 100 mmol L-1 NaCl. Atomic force micrograph revealed the binding of β-galactosidase on the modified AgNPs. The optimal pH for soluble and carboxylated AgNPs adsorbed β-galactosidase (IβG) was observed at pH 4.5 while the optimal operating temperature was broadened from 50 ºC to 60 ºC for IβG. Michaelis constant, Km was increased two and a half fold for IβG while Vmax decreases slightly as compared to soluble enzyme. β-galactosidase immobilized on surface functionalized AgNPs retained 70% biocatalytic activity even at 4% galactose concentration as compared to enzyme in solution. Our study showed that IβG produces greater amount of galacto-oligosaccharides at higher temperatures (50 ºC and 60 ºC) from 0.1 mol L-1 lactose solution at pH 4.5 as compared to previous reports.

NANOPARTICLES OF TUNGSTEN AS LOW-COST MONOMETALLIC CATALYST FOR SELECTIVE HYDROGENATION OF 3-HEXYNE

Low-cost tungsten monometallic catalysts containing variable amounts of metal (4.5, 7.1 and 8.5%W) were prepared by impregnating alumina with ammonium metatungstate as an inexpensive precursor. The catalysts were characterized using ICP, XPS, XRD, TPR and hydrogen chemisorption. These techniques revealed mainly WO3-Al2O3 (W6+) species on the surface. The effects of the content of W nanoparticles and reaction temperature on activity and selectivity for the partial hydrogenation of 3-hexyne, a non-terminal alkyne, were assessed under moderate conditions of temperature and pressure. The monometallic catalysts prepared were found to be active and stereoselective for the production of (Z )-3-hexene, had the following order: 7.1WN/A > 8.5 WN/A ≥ 4.5 WN/A. Additionally, the performance of the synthesized xWN/A catalysts exhibited high sensitivity to temperature variation. In all cases, the maximum 3-hexyne total conversion and selectivity was achieved at 323 K. The performance of the catalysts was considered to be a consequence of two phenomena: a) the electronic effects, related to the high charge of W (+6), causing an intensive dipole moment in the hydrogen molecule (van der Waals forces) and leading to heterolytic bond rupture; the H+ and H- species generated approach a 3-hexyne adsorbate molecule and cause heterolytic rupture of the C≡C bond into C- = C+; and b) steric effects related to the high concentration of WO3 on 8.5WN/A that block the Al2O3 support. Catalyst deactivation was detected, starting at about 50 min of reaction time. Electrodeficient W6+ species are responsible for the formation of green oil at the surface level, blocking pores and active sites of the catalyst, particularly at low reaction temperatures (293 and 303 K). The resulting best catalyst, 7.1WN/A, has low fabrication cost and high selectivity for (Z )-3-hexene (94%) at 323 K. This selectivity is comparable to that of the classical and more expensive industrial Lindlar catalyst (5 wt% Pd). The alumina supported tungsten catalysts are low-cost potential replacements for the Lindlar industrial catalyst. These catalysts could also be used for preparing bimetallic W-Pd catalysts for selective hydrogenation of terminal and non-terminal alkynes.