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.

Study on Pore Distribution and Formation Rule of Sepiolite Mineral Nanomaterials

Theoretical research and specific surface area analysis of nitrogen adsorption indicated that a lot of structural micropores exist in sepiolite minerals fibers. However, the microporous size, existing form, and the distribution relationship between microporous structures were not proved yet. In this paper, the section TEM samples of nanofibers were prepared on the basis of the metal embedding and cutting technique, and the inner structure of sepiolite nanofibers was observed by TEM. The results showed that sepiolite fibers have multiplayer structure similar to concentric circles, and many micropores with the size of about 2–5 nm are normal and parallel to the -axis. The reason for the previously mentioned phenomenon was explained by using BET analysis and X-ray diffraction analysis results.

Structure, Morphology, and Electrochemical Properties of Transition Metal Oxide, Hydroxide, and Phosphate Nanomaterials for Energy Storage

Energy storage technologies are crucial for efficient utilization of electricity. Supercapacitors and rechargeable batteries are of currently available energy storage systems. Transition metal oxides, hydroxides, and phosphates are the most intensely investigated electrode materials for supercapacitors and rechargeable batteries due to their high theoretical charge storage capacities resulted from reversible electrochemical reactions. Their insulating nature, however, causes sluggish electron transport kinetics within these electrode materials, hindering them from reaching the theoretical maximum. The conductivity of these transition metal based-electrode materials can be improved through three main approaches; nanostructuring, chemical substitution, and introducing carbon matrices. These approaches often lead to unique electrochemical properties when combined and balanced.

Ethanol-mediated solvothermal synthesis we developed is found to be highly effective for controlling size and morphology of transition metal-based electrode materials for both pseudocapacitors and batteries. The morphology and the degree of crystallinity of nickel hydroxide are systematically changed by adding various amounts glucose to the solvothermal synthesis. Nickel hydroxide produced in this manner exhibited increased pseudocapacitance, which is partially attributed to the increased surface area. Interestingly, this morphology effect on cobalt doped-nickel hydroxide is found to be more effective at low cobalt contents than at high cobalt contents in terms of improving the electrochemical performance.

Moreover, a thin layer of densely packed nickel oxide flakes on carbon paper substrate was successfully prepared via the glucose-assisted solvothermal synthesis, resulting in the improved electrode conductivity. When reduced graphene oxide was used for conductive coating on as-prepared nickel oxide electrode, the electrode conductivity was only slightly improved. This finding reveals that the influence of reduced graphene oxide coating, increasing the electrode conductivity, is not that obvious when the electrode is already highly conductive to begin with.

We were able to successfully control the interlayer spacing and reduce the particle size of layered titanium hydrogeno phosphate material using our ethanol-mediated solvothermal reaction. In layered structure, interlayer spacing is the key parameter for fast ion diffusion kinetics. The nanosized layered structure prepared via our method, however, exhibited high sodium-ion storage capacity regardless of the interlayer spacing, implying that interlayer space may not be the primary factor for sodium-ion diffusion in nanostructured materials, where many interstitials are available for sodium-ion diffusion.

Our ethanol-mediated solvothermal reaction was also effective for synthesis of NaTi2(PO4)3 nanoparticles with uniform size and morphology, well connected by a carbon nanotube network. This composite electrode exhibited high capacity, which is comparable to that in aqueous electrolyte, probably due to the uniform morphology and size where the preferable surface for sodium-ion diffusion is always available in all individual particles.

Fundamental understandings of the relationship between electrode microstructures and electrochemical properties discussed in this dissertation will be important to design high performance energy storage system applications.

Effect of II‐VI Semiconductor Nanomaterials and Electron Irradiation on Polystyrene (PS) & Poly(ethylene‐co‐vinyl acetate) (EVA)

Organic-inorganic nanocomposites combine unique properties of both the constituents in one material. Among this group of materials, clay based as well as ZnO, TiO2 nanocomposites have been found to have diverse applications. Optoelectronic devices require polymerinorganic systems to meet certain desired properties. Dielectric properties of conventional polymers like poly(ethylene-co-vinyl acetate) (EVA) and polystyrene (PS) may also be tailor tuned with the incorporation of inorganic fillers in very small amounts. Electrical conductivity and surface resistivity of polymer matrices are found to improve with inorganic nanofillers. II-VI semiconductors and their nano materials have attracted material scientists because of their unique optical properties of photoluminescence, UV photodetection and light induced conductivity. Cadmium selenide (CdSe), zinc selenide (ZnSe) and zinc oxide (ZnO) are some of the most promising members of the IIVI semiconductor family, used in light-emitting diodes, nanosensors, non-linear optical (NLO) absorption etc. EVA and PS materials were selected as the matrices in the present study because they are commercially used polymers and have not been the subject of research for opto-electronic properties with semiconductor nanomaterials

Applications for nanomaterials in critical technologies

This thesis is devoted to the development, synthesis, properties, and applications of nano materials for critical technologies, including three areas: (1) Microbial contamination of drinking water is a serious problem of global significance. About 51% of the waterborne disease outbreaks in the United States can be attributed to contaminated ground water. Development of metal oxide nanoparticles, as viricidal materials is of technological and fundamental scientific importance. Nanoparticles with high surface areas and ultra small particle sizes have dramatically enhanced efficiency and capacity of virus inactivation, which cannot be achieved by their bulk counterparts. A series of metal oxide nanoparticles, such as iron oxide nanoparticles, zinc oxide nanoparticles and iron oxide-silver nanoparticles, coated on fiber substrates was developed in this research for evaluation of their viricidal activity. We also carried out XRD, TEM, SEM, XPS, surface area measurements, and zeta potential of these nanoparticles. MS2 virus inactivation experiments showed that these metal oxide nanoparticle coated fibers were extremely powerful viricidal materials. Results from this research suggest that zinc oxide nanoparticles with diameter of 3.5 nm, showing an isoelectric point (IEP) at 9.0, were well dispersed on fiberglass. These fibers offer an increase in capacity by orders of magnitude over all other materials. Compared to iron oxide nanoparticles, zinc oxide nanoparticles didn’t show an improvement in inactivation kinetics but inactivation capacities did increase by two orders of magnitude to 99.99%. Furthermore, zinc oxide nanoparticles have higher affinity to viruses than the iron oxide nanoparticles in presence of competing ions. The advantages of zinc oxide depend on high surface charge density, small nanoparticle sizes and capabilities of generating reactive oxygen species. The research at its present stage of development appears to offer the best avenue to remove viruses from water. Without additional chemicals and energy input, this system can be implemented by both points of use (POU) and large-scale use water treatment technology, which will have a significant impact on the water purification industry. (2) A new family of aliphatic polyester lubricants has been developed for use in micro-electromechanical systems (MEMS), specifically for hard disk drives that operate at high spindle speeds (>15000rpm). Our program was initiated to address current problems with spin-off of the perfluoroether (PFPE) lubricants. The new polyester lubricant appears to alleviate spin-off problems and at the same time improves the chemical and thermal stability. This new system provides a low cost alternative to PFPE along with improved adhesion to the substrates. In addition, it displays a much lower viscosity, which may be of importance to stiction related problems. The synthetic route is readily scalable in case additional interest emerges in other areas including small motors. (3) The demand for increased signal transmission speed and device density for the next generation of multilevel integrated circuits has placed stringent demands on materials performance. Currently, integration of the ultra low-k materials in dual Damascene processing requires chemical mechanical polishing (CMP) to planarize the copper. Unfortunately, none of the commercially proposed dielectric candidates display the desired mechanical and thermal properties for successful CMP. A new polydiacetylene thermosetting polymer (DEB-TEB), which displays a low dielectric constant (low-k) of 2.7, was recently developed. This novel material appears to offer the only avenue for designing an ultra low k dielectric (1.85k), which can still display the desired modulus (7.7Gpa) and hardness (2.0Gpa) sufficient to withstand the process of CMP. We focused on further characterization of the thermal properties of spin-on poly (DEB-TEB) ultra-thin film. These include the coefficient of thermal expansion (CTE), biaxial thermal stress, and thermal conductivity. Thus the CTE is 2.0*10-5K-1 in the perpendicular direction and 8.0*10-6 K-1 in the planar direction. The low CTE provides a better match to the Si substrate which minimizes interfacial stress and greatly enhances the reliability of the microprocessors. Initial experiments with oxygen plasma etching suggest a high probability of success for achieving vertical profiles.

Titanium dioxide based nanomaterials for photocatalytic water treatment

Water treatment using photocatalysis has gained extensive attention in recent years. Photocatalysis is promising technology from green chemistry point of view. The most widely studied and used photocatalyst for decomposition of pollutants in water under ultraviolet irradiation is TiO2 because it is not toxic, relatively cheap and highly active in various reactions. Within this thesis unmodified and modified TiO2 materials (powders and thin films) were prepared. Physico-chemical properties of photocatalytic materials were characterized with UV-visible spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectrometry (XPS), inductively coupled plasma optical emission spectroscopy (ICP-OES), ellipsometry, time-of-flight secondary ion mass spectrometry (ToF-SIMS), Raman spectroscopy, goniometry, diffuse reflectance measurements, thermogravimetric analysis (TGA) and nitrogen adsorption/desorption. Photocatalytic activity of prepared samples in aqueous environment was tested using model compounds such as phenol, formic acid and metazachlor. Also purification of real pulp and paper wastewater effluent was studied. Concentration of chosen pollutants was measured with high pressure liquid chromatography (HPLC). Mineralization and oxidation of organic contaminants were monitored with total organic carbon (TOC) and chemical oxygen demand (COD) analysis. Titanium dioxide powders prepared via sol-gel method and doped with dysprosium and praseodymium were photocatalytically active for decomposition of metazachlor. The highest degradation rate of metazachlor was observed when Pr-TiO2 treated at 450ºC (8h) was used. The photocatalytic LED-based treatment of wastewater effluent from plywood mill using commercially available TiO2 was demonstrated to be promising post-treatment method (72% of COD and 60% of TOC was decreased after 60 min of irradiation). The TiO2 coatings prepared by atomic layer deposition technique on aluminium foam were photocatalytically active for degradation of formic and phenol, however suppression of activity was observed. Photocatalytic activity of TiO2/SiO2 films doped with gold bipyramid-like nanoparticles was about two times higher than reference, which was not the case when gold nanospheres were used.

Manufactured Nanomaterials: is there a correlation between toxicological effects and the physicochemical properties?

Toxicological information on nanomaterials (NMs) is of major importance for safety assessment, since they are already used in many consumer products and promise cutting-edge applications in the future. While the number of different NMs increases exponentially, new strategies for risk assessment are needed to cope with the safety issues, keeping pace with innovation. However, recent studies have suggested that even subtle differences in the physicochemical properties of NMs that are closely related may define different nano-bio interactions, thereby determining their toxic potential. Further research in this field is necessary to allow straightforward grouping strategies leading time-effective risk assessment to enable the safe use of the emerging NMs. In this presentation the case study of the in vitro toxicity testing of a set of multi-walled carbon nanotubes (MWCNTs) in two human cell lines from the respiratory tract will be described. Those MWCNT have been previously characterized in detail, and differ in thickness, length, aspect ratio and morphology. This comprehensive toxicological investigation undertaken in parallel with physicochemical characterization in the cellular moiety showed that the same NM did not display a consistent effect in different cell types, and that, within the same class of NM, different toxic effects could be observed. The correlation of the cytotoxic and genotoxic effects characterized in the two cell lines with their physicochemical properties will be presented and the relevance of considering the NMs properties in the biological context will be discussed. Overall, this case study suggests that nanotoxicity of closely related MWCNTs depends not only on their primary physicochemical properties, or combinations of these properties, but also on the cellular system, and its context. Challenges posed to toxicologists, risk assessors and regulators when addressing the safety assessment of NMs will be highlighted.

Development of hydroxyapatite-based nanomaterials to enhance biological response of osteoblast cells for clinical application

Purpose: To develop a novel chitosan/gelatin-hydroxyapatite (CGHaP) microspheres for evaluating the biological response of pre-osteoblast cells. Methods: The microsphere was prepared by water-in-oil emulsion method. Cell proliferation was studied using AlamarBlue colorimetric assay and DAPI staining while alkaline phosphatase assay was carried out by colorimetric assay method. Chitosan microspheres as well as chitosan-hydroxyapatite microspheres was prepared and tested for biological response from MC3T3-E1 cell line. Results: The results showed that CGHaP promotes MC3T3-E1 cell proliferation and spread on the surface of microspheres. The cells were clustered with more actin filaments and well-linked with neighbouring cells or adjacent cells when cultured in CGHaP microspheres whereas fewer cells were spread on chitosan (CH) microspheres. CGHaP microspheres significantly (p < 0.05) promoted cell attachment, proliferation and extracellular matrix mineralization. CGHaP microspheres presented significantly (p < 0.02) higher calcium deposition (0.5 ng) than CH microspheres (0.28 ng). Specifically, CGHaP microspheres exhibited high ALP activity (8 units; 2-fold) compared to CH with 3 units, after 7 days of incubation. The results suggest that CGHaP possesses a great ability to facilitate bone ingrowth formation and possibility of good osteointegration in vivo. Conclusion: The nanomaterial enhances the proliferation of pre-osteoblast cells in tissue engineering microspheres. The outcome of this study may have a major impact on the development of novel nanomaterials for bone tissue engineering.

Growth of boehmite nanofibers by assembling nanoparticles with surfactant micelles

It is known that boehmite (AlOOH) nanofibers formed in the presence of nonionic poly(ethylene oxide) (PEO) surfactant at 373 K. A novel approach is proposed in this study for the growth of the boehmite nanofibers: when fresh aluminum hydrate precipitate was added at regular interval to initial mixture of boehmite and PEO surfactant at 373 K, the nanofibers grow from 40 to 50 nm long to over 100 nm. It is believed that the surfactant micelles play an important role in the nanofiber growth: directing the assembly of aluminum hydrate particles through hydrogen bonding with the hydroxyls on the surface of aluminum hydrate particles. Meanwhile a gradual improvement in the crystallinity of the fibers during growth is observed and attributed to the Ostwald ripening process. This approach allows us to precisely control the size and morphology of boehmite nanofibers using soft chemical methods and could be useful for low temperature, aqueous syntheses of other oxide nanomaterials with tailorable structural specificity such as size, dimension and morphology.

Infrared and infrared emission spectroscopy of gallium oxide α-GaO(OH) nanostructures

Infrared spectroscopy has been used to study nano to micro sized gallium oxyhydroxide α-GaO(OH), prepared using a low temperature hydrothermal route. Rod-like α-GaO(OH) crystals with average length of ~2.5 μm and width of 1.5 μm were prepared when the initial molar ratio of Ga to OH was 1:3. β-Ga2O3 nano and micro-rods were prepared through the calcination of α-GaO(OH) The initial morphology of α-GaO(OH) is retained in the β-Ga2O3 nanorods. The combination of infrared and infrared emission spectroscopy complimented with dynamic thermal analysis were used to characterise the α-GaO(OH) nanotubes and the formation of β-Ga2O3 nanorods. Bands at around 2903 and 2836 cm-1 are assigned to the -OH stretching vibration of α-GaO(OH) nanorods. Infrared bands at around 952 and 1026 cm-1 are assigned to the Ga-OH deformation modes of α-GaO(OH). A significant number of bands are observed in the 620 to 725 cm-1 region and are assigned to GaO stretching vibrations.

Frontiers in Materials Science and Technology

This special issue aims to provide up-to-date knowledge and the latest scientific concepts and technological developments in the processing, characterization, testing, mechanics, modeling and applications of a broad range of advanced materials. The many contributors, from Denmark, Germany, UK, Iran, Saudi Arabia, Malaysia, Japan, the People’s Republic of China, Singapore, Taiwan, USA, New Zealand and Australia, present a wide range of topics including: nanomaterials, thin films and coatings, metals and alloys, composite materials, materials processing and characterization, biomaterials and biomechanics, and computational materials science and simulation. The work will therefore be of great interest to a broad spectrum of researchers and technologists.