Determinants of biological activity of nanomaterials in innate immunity cells: the role of aspect ratio, environmental contaminants and protein corona

As defined by the European Union, “ ’Nanomaterial’ (NM) means a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or agglomerate, where, for 50 % or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm-100 nm ” (2011/696/UE). Given their peculiar physico-chemical features, nanostructured materials are largely used in many industrial fields (e.g. cosmetics, electronics, agriculture, biomedical) and their applications have astonishingly increased in the last fifteen years. Nanostructured materials are endowed with very large specific surface area that, besides making them very useful in many industrial processes, renders them very reactive towards the biological systems and, hence, potentially endowed with significant hazard for human health. For these reasons, in recent years, many studies have been focused on the identification of toxic properties of nanostructured materials, investigating, in particular, the mechanisms behind their toxic effects as well as their determinants of toxicity. This thesis investigates two types of nanostructured TiO2 materials, TiO2 nanoparticles (NP), which are yearly produced in tonnage quantities, and TiO2 nanofibres (NF), a relatively novel nanomaterial. Moreover, several preparations of MultiWalled Carbon Nanotubes (MWCNT), another nanomaterial widely present in many products, are also investigated.- Although many in vitro and in vivo studies have characterized the toxic properties of these materials, the identification of their determinants of toxicity is still incomplete. The aim of this thesis is to identify the structural determinants of toxicity, using several in vitro models. Specific fields of investigation have been a) the role of shape and the aspect ratio in the determination of biological effects of TiO2 nanofibres of different length; b) the synergistic effect of LPS and TiO2 NP on the expression of inflammatory markers and the role played therein by TLR-4; c) the role of functionalization and agglomeration in the biological effects of MWCNT. As far as biological effects elicited by TiO2 NF are concerned, the first part of the thesis demonstrates that long TiO2 nanofibres caused frustrated phagocytosis, cytotoxicity, hemolysis, oxidative stress and epithelial barrier perturbation. All these effects were mitigated by fibre shortening through ball-milling. However, short TiO2 NF exhibited enhanced ability to activate acute pro-inflammatory effects in macrophages, an effect dependent on phagocytosis. Therefore, aspect ratio reduction mitigated toxic effects, while enhanced macrophage activation, likely rendering the NF more prone to phagocytosis. These results suggest that, under in vivo conditions, short NF will be associated with acute inflammatory reaction, but will undergo a relatively rapid clearance, while long NF, although associated with a relatively smaller acute activation of innate immunity cells, are not expected to be removed efficiently and, therefore, may be associated to chronic inflammatory responses. As far as the relationship between the effects of TiO2 NP and LPS, investigated in the second part of the thesis, are concerned, TiO2 NP markedly enhanced macrophage activation by LPS through a TLR-4-dependent intracellular pathway. The adsorption of LPS onto the surface of TiO2 NP led to the formation of a specific bio-corona, suggesting that, when bound to TiO2 NP, LPS exerts a much more powerful pro-inflammatory effect. These data suggest that the inflammatory changes observed upon exposure to TiO2 NP may be due, at least in part, to their capability to bind LPS and, possibly, other TLR agonists, thus enhancing their biological activities. Finally, the last part of the thesis demonstrates that surface functionalization of MWCNT with amino or carboxylic groups mitigates the toxic effects of MWCNT in terms of macrophage activation and capability to perturb epithelial barriers. Interestingly, surface chemistry (in particular surface charge) influenced the protein adsorption onto the MWCNT surface, allowing to the formation of different protein coronae and the tendency to form agglomerates of different size. In particular functionalization a) changed the amount and the type of proteins adsorbed to MWCNT and b) enhanced the tendency of MWCNT to form large agglomerates. These data suggest that the different biological behavior of functionalized and pristine MWCNT may be due, at least in part, to the different tendency to form large agglomerates, which is significantly influenced by their different capability to interact with proteins contained in biological fluids. All together, these data demonstrate that the interaction between physico-chemical properties of nanostructured materials and the environment (cells + biological fluids) in which these materials are present is of pivotal importance for the understanding of the biological effects of NM. In particular, bio-persistence and the capability to elicit an effective inflammatory response are attributable to the interaction between NM and macrophages. However, the interaction NM-cells is heavily influenced by the formation at the nano-bio interface of specific bio-coronae that confer a novel biological identity to the nanostructured materials, setting the basis for their specific biological activities.

Investigation and development of techniques for the characterisation of the synthetic/biological interface

The purpose of this study is to increase our knowledge of the nature of the surface properties of polymeric materials and improve our understanding of how these factors influence the deposition of proteins to form a reactive biological/synthetic interface. A number of surface analytical techniques were identified as being of potential benefit to this investigation and included in a multidisciplinary research program. Cell adhesion in culture was the primary biological sensor of surface properties, and it showed that the cell response to different materials can be modified by adhesion promoting protein layers: cell adhesion is a protein-mediated event. A range of surface rugosity can be produced on polystyrene, and the results presented here show that surface rugosity does not play a major role in determining a material's cell adhesiveness. Contact angle measurements showed that surface energy (specifically the polar fraction) is important in promoting cell spreading on surfaces. The immunogold labelling technique indicated that there were small, but noticeable differences, between the distribution of proteins on a range of surfaces. This study has shown that surface analysis techniques have different sensitivities in terms of detection limits and depth probed, and these are important in determining the usefulness of the information obtained. The techniques provide information on differing aspects of the biological/synthetic interface, and the consequence of this is that a range of techniques is needed in any full study of such a complex field as the biomaterials area.

Synthetic analogues of protein-lipid complexes

Hypercoiling poly(styrene-alt-maleic anhydride) (PSMA) is known to undergo conformational transition in response to environmental stimuli. The association of PSMA with lipid 2-dilauryl-sn-glycero-3-phosphocholine (DLPC) produces polymer-lipid complex analogues to lipoprotein assemblies found in lung surfactant. These complexes represent a new bio-mimetic delivery vehicle with applications in the cosmetic and pharmaceutical industries. The primary aim of this study was to develop a better understanding of PSMA-DLPC association by using physical and spectroscopic techniques. Ternary phase diagrams were constructed to examine the effects of various factors, such as molecular weight, pH and temperature on PSMA-DLPC association. 31P-NMR spectroscopy was used to investigate the polymorphic changes of DLPC upon associating with PSMA. The Langmuir Trough technique and surface tension measurement were used to explore the association behaviour of PSMA both at the interface and in the bulk of solution, as well as its interaction with DLPC membranes. The ultimate aim of this study was to investigate the potential use of PSMA-DLPC complexes to improve the bioavailability and therapeutic efficacy of a range of drugs. Typical compounds of ophthalmic interest range from new drugs such as Pirenzepine, which has attracted clinical interest for the control of myopia progression, to the well-established family of non-steroid anti-inflammatory drugs. These drugs have widely differing structures, sizes, solubility profiles and pH-sensitivities. In order to understand the ways in which these characteristics influence incorporation and release behaviour, the marker molecules Rhodamine B and Oil Red O were chosen. PSMA-DLPC complexes, incorporated with marker molecules and Pirenzepine, were encapsulated in hydrogels of the types used for soft contact lenses. Release studies were conducted to examine if this smart drug delivery system can retain such compounds and deliver them at a slow rate over a prolonged period of time.

Microstructural analysis of surface and interface zones in concrete

Several of OPC paste and concrete specimens, with different mix proportions, were cast against CPF and impermeable formwork (IF) and the profiles of pore structure, microhardness and scratch hardness of the cover zone were established. The chloride ingress and the depth of carbonation of the surface zone of concrete cast against CPF and IF were investigated. The main mechanisms controlling the ECR processes and the factors affecting such treatment were critically reviewed. Subsequently, as a means of restoring passivation of steel embedded in carbonated concrete, such HCP specimens were subjected to ECR. The influence of ECR on the chemistry of the pore solution and the microstructure of the surface and the steel/cement past interface zones were also studied. The main findings of this investigation were as follows: (a) The thickness of the microstructure gradient of cover concrete is significantly decreased with increasing period of water curing but is relatively unaffected by curing temperature, w/e ratio and the use of cement replacement materials. (b) The scratch hardness technique was shown to be potentially useful for characterising the microstructure and microhardness gradients of the surface zone. (c) A relationship between the microstructure gradient and mass transport properties of the surface zone was established. (d) The use of CPF resulted in a significant reduction in porosity of both the cement paste matrix and the aggregate/cement paste transition zone, and a marked improvement in the resistance of the surface zone to carbonation and the ingress of chloride ions. (e) The ECR treatment resulted in a marked densification of the pore structure and in changes to the pore solution chemistry and the cement phases of near-surface and steel/cement paste transition zones. This effect was more pronounced with current density, period of treatment and particularly with the use of sodium phosphate as an electrolyte.

Ligand diffusion on protein surface observed in molecular dynamics simulation

The process of binding of small ligands to dihydrofolate reductase protein has been investigated using all-atom molecular dynamics simulations. The existence of a mechanism that facilitates the search of the binding site by the ligand is demonstrated. The mechanism consists of ligand diffusing on the protein’s surface. It has been discussed in the literature before, but has not been explicitly confirmed for realistic molecular systems. The strength of this nonspecific binding is roughly estimated and found to be essential for the binding kinetics.

Wear resistance of surface treated metal-working tools

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Receptor activity-modifying protein-dependent effects of mutations in the calcitonin receptor-like receptor:implications for adrenomedullin and calcitonin gene-related peptide pharmacology

Background and Purpose Receptor activity-modifying proteins (RAMPs) define the pharmacology of the calcitonin receptor-like receptor (CLR). The interactions of the different RAMPs with this class B GPCR yield high-affinity calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors. However, the mechanism for this is unclear. Experimental Approach Guided by receptor models, we mutated residues in the N-terminal helix of CLR, RAMP2 and RAMP3 hypothesized to be involved in peptide interactions. These were assayed for cAMP production with AM, AM2 and CGRP together with their cell surface expression. Binding studies were also conducted for selected mutants. Key Results An important domain for peptide interactions on CLR from I32 to I52 was defined. Although I41 was universally important for binding and receptor function, the role of other residues depended on both ligand and RAMP. Peptide binding to CLR/RAMP3 involved a more restricted range of residues than that to CLR/RAMP1 or CLR/RAMP2. E101 of RAMP2 had a major role in AM interactions, and F111/W84 of RAMP2/3 was important with each peptide. Conclusions and Implications RAMP-dependent effects of CLR mutations suggest that the different RAMPs control accessibility of peptides to binding residues situated on the CLR N-terminus. RAMP3 appears to alter the role of specific residues at the CLR-RAMP interface compared with RAMP1 and RAMP2. © 2013 The Authors. British Journal of Pharmacology published by John Wiley &. Sons Ltd on behalf of The British Pharmacological Society.

An ultra-sensitive localised surface plasmon resonance fibre device for environmental sensing based upon a structured bi-metal coating

We demonstrate a bi-metal coating (platinum and gold or silver) localised surface plasmon resonance fibre device that produces an index spectral sensitivity of over 11,000 nm/RIU, yielding an index resolution of 5×10-6in the aqueous index regime, consisting of a structured multi-layered thin film on D-shaped fibre. © 2014 SPIE.

Nanoindentation measurement of surface residual stresses in particle-reinforced metal matrix composites

The surface residual stresses in SiC particle-reinforced Al matrix composites are measured using a recently developed nanoindentation technique. The tensile biaxial residual stress in Al is found to increase with the particle concentration. The stress magnitudes are in reasonable agreement with those from numerical modeling.

Impedance analysis of Al2O3/H-terminated diamond metal-oxide-semiconductor structures

Impedance spectroscopy (IS) analysis is carried out to investigate the electrical properties of the metal-oxide-semiconductor (MOS) structure fabricated on hydrogen-terminated single crystal diamond. The low-temperature atomic layer deposition Al2O3 is employed as the insulator in the MOS structure. By numerically analysing the impedance of the MOS structure at various biases, the equivalent circuit of the diamond MOS structure is derived, which is composed of two parallel capacitive and resistance pairs, in series connection with both resistance and inductance. The two capacitive components are resulted from the insulator, the hydrogenated-diamond surface, and their interface. The physical parameters such as the insulator capacitance are obtained, circumventing the series resistance and inductance effect. By comparing the IS and capacitance-voltage measurements, the frequency dispersion of the capacitance-voltage characteristic is discussed.

Highly sensitive, localized surface plasmon resonance fiber device for environmental sensing, based upon a structured bi-metal array of nano-wires

We demonstrate a bi-metal coated (platinum and gold or silver), localized surface plasmon resonance fiber sensor with an index sensitivity exceeding 11,900 nm/RIU, yielding an index resolution of 2 × 10-5 in the aqueous index regime. This is one of the highest index sensitivities achieved with an optical fiber sensor. The coatings consist of arrays of bi-metal nano-wires (typically 36 nm in radius and 20 μm in length), supported by a silicon dioxide thin film on a thin substrate of germanium, the nano-wires being perpendicular to the longitudinal axis of the D-shaped fiber.

Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins

Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine.

The correlation of structure and electronic properties near the surface of transition metal oxides

The strong couplings between different degrees of freedom are believed to be responsible for novel and complex phenomena discovered in transition metal oxides (TMOs). The physical complexity is directly responsible for their tunability. Creating surfaces/interfaces add an additional ' man-made' twist, approaching the quantum phenomena of correlated materials. ^ The dissertation focused on the structural and electronic properties in proximity of surface of three prototype TMO compounds by using three complementary techniques: scanning tunneling microscopy, angle-resolved photoelectron spectroscopy and low energy electron diffraction, particularly emphasized the effects of broken symmetry and imperfections like defects on the coupling between charge and lattice degrees of freedom. ^ Ca1.5Sr0.5RuO4 is a layered ruthenate with square lattice and at the boundary of magnetic/orbital instability in Ca2-xSrxRuO4. That the substitution of Sr 2+ with Ca2+ causing RuO6 rotation narrows the dxy band width and changes the Fermi surface topology. Particularly, the γ(dxy) Fermi surface sheet exhibited hole-like in Ca1.5Sr0.5RuO4 in contrast to electron-like in Sr2RuO4, showing a strong charge-lattice coupling. ^ Na0.75CoO2 is a layered cobaltite with triangular lattice exhibiting extraordinary thermoelectric properties. The well-ordered CoO2-terminated surface with random Na distribution was observed. However, lattice constants of the surface are smaller than that in bulk. The surface density of states (DOS) showed strong temperature dependence. Especially, an unusual shift of the minimum DOS occurs below 230 K, clearly indicating a local charging effect on the surface. ^ Cd2Re2O7 is the first known pyrochlore oxide superconductor (Tc ∼ 1K). It exhibited an unusual second-order phase transition occurring at TS1 = 200 K and a controversial first-order transition at TS2 = 120 K. While bulk properties display large anomalies at TS1 but rather subtle and sample-dependent changes at TS2, the surface DOS near the EF show no change at T s1 but a substantial increase below TS2---a complete reversal as the signature for the transitions. We argued that crystal imperfections, mainly defects, which were considerably enhanced at the surface, resulted in the transition at TS2. ^

Data compilation on the biological response to ocean acidification: environmental and experimental context of data sets and related literature

The exponential growth of studies on the biological response to ocean acidification over the last few decades has generated a large amount of data. To facilitate data comparison, a data compilation hosted at the data publisher PANGAEA was initiated in 2008 and is updated on a regular basis (doi:10.1594/PANGAEA.149999). By January 2015, a total of 581 data sets (over 4 000 000 data points) from 539 papers had been archived. Here we present the developments of this data compilation five years since its first description by Nisumaa et al. (2010). Most of study sites from which data archived are still in the Northern Hemisphere and the number of archived data from studies from the Southern Hemisphere and polar oceans are still relatively low. Data from 60 studies that investigated the response of a mix of organisms or natural communities were all added after 2010, indicating a welcomed shift from the study of individual organisms to communities and ecosystems. The initial imbalance of considerably more data archived on calcification and primary production than on other processes has improved. There is also a clear tendency towards more data archived from multifactorial studies after 2010. For easier and more effective access to ocean acidification data, the ocean acidification community is strongly encouraged to contribute to the data archiving effort, and help develop standard vocabularies describing the variables and define best practices for archiving ocean acidification data.