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.

Simplified preparation of human arterial sections for PCR analysis of Chlamydia pneumoniae and human DNA

AIMS: To investigate multiple techniques for the preparation of solid tissue for polymerase chain reaction (PCR) analysis, and to identify the most simple techniques for routine use in the laboratory. METHODS: Techniques for the preparation of arterial tissue samples including homogenisation, ultrafiltration, and treatments involving proteinase K, Gene Clean, lectin, and Fe3+ specific chelators were evaluated using the PCR to amplify both Chlamydia pneumoniae and human DNA. RESULTS: Treatment with either Gene-Clean or lectin and the Fe3+ specific chelator deferoxamine mesylate removed PCR inhibitors from tissue homogenates. Homogenisation followed by GeneClean treatment resulted in the amplification of C pneumoniae DNA from within a section of atherosclerotic carotid artery, implying that C pneumoniae elementary bodies had been disrupted. In eight further clinical samples from patients not known to have C pneumoniae infection, human DNA was amplified and no cross contamination was observed between samples. These samples contained no evidence of C pneumoniae by PCR. CONCLUSIONS: A simple preparation of solid tissue for PCR analysis, involving homogenisation followed by GeneClean treatment has been developed, and is effective for the amplification of both C pneumoniae and human DNA.

The preparation of carbon fibres: a study of the physics and chemistry of the preparation of carbon fibres from acrylic precursors

High strength, high modulus carbon fibres are becoming increasingly important as high performance engineering materials. This thesis describes how they may be prepared by heat treatment from filaments spun from polyacrylonitrile and its copolymers. The chemistry of the first stages of heat treatment is very important in controlling the mechanical properties of the carbonised product. A cyclisation reaction has been found to be responsible for the relatively high thermal stability of pyrolysed polyacrylonitrile, but without oxidation the fibres degrade and fuse. An initial oxidation stage is, therefore, essential to the preparation of fibre of high orientation. The cyclised product of pyrolysis is probably a poly 1,4 dihydropiridine and oxidation converts this to aromatic structures, and cyclised structures containing carbonyl and other oxygenated groups. Oxidation is found to assist the carbon fibre preparation process, by producing a product which condenses at an earlier stage of heat treatment, before fusion can occur. Carbon fibre strength and modulus are dependent upon producing a highly oriented crystal structure. While oxidation of the polymer stabilises the fibre so as to prevent disorientation, further large increases in orientation, with a commensurate improvement in strength and modulus, can be obtained by stretching at temperatures above 1,700 °C. This process is analogous to the way fibre orientation is increased by the stretching of the precursor. A lamellar graphite structure can be created in high temperature fibre, by carefully controlling the degree of oxidation. This type of graphite can produce very high values of Young's modulus. More often, however, graphite fibre has a fibrillar fine structure, which is explicable in terms of continuous graphite ribbons. A ribbon model is the most satisfactory representation of the structure of carbon fibre, as it explains the mechanism of the development of long range order and the variation of Young's modulus with crystalline preferred orientation.

Polymer film formulations for the preparation of enteric pharmaceutical capsules

Objectives. Standard pharmaceutical capsules are designed to dissolve in the acidic environment of the stomach releasing the encapsulated contents for absorption. When release is required further along the gastrointestinal tract capsules can be coated with acid insoluble polymers to enable passage through the stomach and dissolution in the intestine. This paper describes formulations that have the potential to be used to produce two-piece hard capsules for post-gastric delivery without the requirement of an exterior coat. Methods. The formulation uses three polysaccharides: sodium alginate, hypromellose and gellan gum to provide acid insolubility and the ability to form capsules using standard industrial equipment. Key findings. The rheological profile, on cooling, of the base material, water content and thickness of the films were shown to be comparable with those of commercial capsules. The capsules remained intact for 2 h in 100 mm HCl at pH 1.2, and within 5 min of being removed from the acid and submerged in phosphate-buffered saline at pH 6.8 were ruptured. Conclusions. Selected formulations from this study have potential for use as delayed release capsules.

Preparation of particle-stabilized emulsions using membrane emulsification

There is currently significant interest in particle-stabilized emulsions for a variety of applications and as precursors to other materials such as micro-capsules or colloidosomes. A prerequisite for many applications is the ability to produce stable droplets with a well-controlled size. The preparation of oil-in-water (o/w) emulsions stabilized by silica colloids has been demonstrated here using membrane ulsification techniques. Emulsions were produced using both a cross-flow membrane device and a rotating membrane reactor. Under the correct conditions, highly stable emulsions with very narrow droplet size distributions can be produced. Investigations into the effects of changing the cross-flow shear rate at a fixed droplet production rate illustrate the fine control over mean droplet size that is possible with these emulsification techniques. Evidence for the importance of particle adsorption kinetics onto growing droplets prior to detachment from the membrane surface was obtained by varying the droplet production rate under fixed shear conditions. The presence of a critical surface coverage by the stabilizing particles to prevent droplet coalescence was clearly seen. Comparison with samples produced using conventional high-shear homogenization highlights the improved control over size distribution available from these membrane techniques.

Preparation of 9-substituted pyridine-stretched adenines and hypoxanthines

9-Methylsulfanyl pyridine-stretched adenine and hypoxanthine derivatives have been prepared via regioselective reaction of a 5-aminoimidazole with 2-(bis-methylsulfanylmethylene)malononitrile [(NC)C=C(SMe) ]. The 9-methylsulfanyl substituent can be replaced by sequential oxidation and substitution by nucleophiles including amines.

The preparation of a common technique for selecting counter flow and crossflow cooling towers

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Evaluation of new processes for the preparation of iron by gas/solid phase systems

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A highly emissive inorganic hexamolybdenum cluster complex as a handy precursor for the preparation of new luminescent materials

The synthesis and characterisation of a new, highly luminescent inorganic cluster complex, (Bu4N)2[Mo6I 8(NO3)6], are described. The complex possesses labile nitrato ligands and is therefore a useful precursor for the design of new luminescent materials. To exemplify this, functionalised polystyrene beads have been utilised as "polymeric ligands" to immobilise the molybdenum cluster complex. This journal is © the Partner Organisations 2014.

Preparation of a microporous silicon oximide gel from the reaction of tris(dimethylamino)silylamine with formamide and its pyrolytic conversion into a silicon oxynitride based glass

A high-surface-area silicon oximide-based gel [SiOC(H)=NSi]m[Si2N-C(H)=O]n[SiN(H)-C(H)=O]p[SiOC(H)=NH]q[SiNH]r[SiNH2]s[SiNMe2]t was prepared via a formamide-based aminolysis of tris(dimethylamino)silylamine, (Me2N)3SiNH2. The structure of the gel and the mechanism of formation are elucidated. Pyrolysis of the gel at 1000 °C under N2 flow gave an amorphous microporous oxynitride-based glass with a BET surface area of 195 m2 g−1. © The Royal Society of Chemistry 2005.