Os grandes desafios da medicina contemporânea destacam a necessidade de uma intensa pesquisa para desenvolver novos tratamentos para muitas doenças crônicas, tais como as reumáticas, que sejam efetivos, seguros e com qualidade. Uma das novas ferramentas para o desenvolvimento de novos medicamentos é a nanotecnologia, a qual nos últimos anos tem aumentado a sua aplicação na área farmacêutica contemplando um crescente otimismo acerca do seu potencial uso para obter melhores oportunidades de diagnóstico e de terapias mais eficazes. No presente trabalho foram encapsulados dois antiinflamatórios em sistemas nanoparticulados, nanoesferas de acido poli-láctico co-glicólico (PLGA), a técnica utilizada permitiu a encapsulação de compostos hidrofílicos e hidrofóbicos na mesma nanopartícula polimérica, diclofenaco de sódio (DS) e dexametasona (DX), respectivamente, obtendo nanopartículas com potencial para o tratamento de doenças inflamatórias crônicas. Para o desenvolvimento das nanoesferas se utilizou a técnica de emulsão/evaporação do solvente. As nanoesferas foram caraterizadas por microscopia eletrônica de varredura (SEM), microscopia eletrônica de transmissão (TEM), medição do pH, medição do tamanho de partícula, potencial zeta e polidisversividade e espectroscopia vibracional de infravermelho (IR). A eficiência de encapsulação (EE) dos fármacos (diclofenaco de sódio e dexametasona) nas nanoesferas foi realizada pelas técnicas analíticas, cromatografia líquida de alta eficiência (HPLC) e eletroforese capilar (CE), previamente validadas. Na melhor formulação foi obtida encapsulação de 51,4 ± 5,5 % para o diclofenaco e 66,9 ± 8,4 para a dexametasona. / The great challenges of contemporary medicine emphasize the need for intensive research to develop new treatments for many chronic diseases, such as the rheumatic, to be effective, safe and of good quality. One of the new tools for the development of new drugs is the nanotechnology, which in recent years has increased its application in the pharmaceutical area contemplating a growing optimism about its potential use to get better opportunities for diagnosis and more effective therapies. In the present work were encapsulated two anti-inflammatories in nanoparticulate systems, nanospheres poly-lactic co-glycolic acid (PLGA), the technique used allows the encapsulation of hydrophilic and hydrophobic compounds in the same polymer nanoparticle, diclofenac sodium (DS) and dexamethasone (DX), respectively, obtaining nanoparticles with potential for the treatment of chronic inflammatory diseases. For the development of nanospheres the technique used was emulsion / solvent evaporation. The nanospheres were characterized by zeta potential infrared, particle size, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and pH. The encapsulation of the drug (diclofenac sodium and dexamethasone) in the nanospheres was performed by previously validated analytical techniques high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). In the best formulation was achieved encapsulation 51.4 ± 5.5% for diclofenac and 66.9 ± 8.4 for dexamethasone

DNA vaccines offer unique promise as a means of generating immunity against infectious and malignant disease. Unfortunately a number of obstacles, including rapid degradation of naked plasmid DNA (pDNA), poor cellular uptake by antigen presenting cells (APCs) and subsequent low levels of gene expression have limited the ability of DNA vaccines to raise sufficient immune responses towards the target antigen. This thesis is focused on investigating the immunostimulatory potential of liposomal nanoparticulate (LN) formulations of pDNA (stabilized plasmid lipid particles; SPLP) and cytosine-guanine oligodeoxynucleotides (CpG-ODN; LN CpG-ODN), and examining their ability to act together as a non-viral DNA vaccine in attempt to address the shortcomings of current DNA vaccine approaches. One focus of this thesis concerns investigating the immunostimulatory activity of LN formulations of CpG-ODN and pDNA. It is shown that despite dramatic differences in pharmacokinetics and biodistribution of LN CpG-ODN following intravenous (i.v.) and subcutaneous (s.c.) administration the resultant immune response is very similar, which is concluded to be due to the intrinsic ability of APCs to sequester LN CpG- ODN. In addition, it is demonstrated that lipid encapsulation dramatically enhances the immunostimulatory potential of pDNA and it is observed that SPLP maintains immunostimulatory activity in Toll-like receptor 9 (TLR9) knock-out mice. Together theses findings highlight the need for DNA-based therapies to consider both TLR9-dependent and -independent immunostimulatory activities of pDNA when constructing non-viral vectors. Furthermore, a new role for SPLP as a non-viral gene delivery vehicle for the generation of a systemically administered genetic vaccine in the presence of LN CpG-ODN is introduced. The ability of vaccination with SPLP to act prophylactically, to protect mice from tumour challenge, and therapeutically, in a novel vaccination strategy where the antigen is expressed at the tumour site as a result of SPLP-mediated transfection, is explored, demonstrating that in the presence of LN CpG-ODN SPLP possesses potential as a non-viral delivery system for DNA-based cancer vaccines. In summary, this work represents a substantial advance in the understanding of the immunostimulatory potential of both SPLP and LN CpG-ODN and provides insight into their ability to work together as a non-viral DNA vaccine. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Mixed metal oxides are ionic compounds containing at least two metal ions within an oxide structure. The literature contains a plethora of examples of mixed metal oxides on the bulk scale, which have been well characterised, however, mixed metal oxides on the nanoscale are far less well understood. The work presented here investigates the Bi2O3-CeO2-Nb2O5 mixed oxide system and characterises the resulting nanoparticles and crystal structures. Although the parent oxides are well known and much work has previously been done in analysing their crystal structures, combinations of these oxides have not been well characterised, especially on the nanoscale. Using high resolution electron microscopy (HRTEM), powder X-ray diffraction (PXRD), electron dispersive X-ray spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS) as analytical tools, the structures of the nanoparticles in this system have been explored. As each of the parent oxides possess useful properties, which have been utilised in industrial applications such as electrolyte components in solid oxide fuel cells and as catalysts in a range of chemical reactions, it was hypothesised that if all three metal ions could be contained in one particle they could show novel and interesting characteristics. It was proposed that due to the more relaxed crystal structure in nanoparticles, the solid solubility of the metal ions should be increased, and a solid solution of ions would form. This work presents results showing the synthesis of binary and ternary oxides in the nano-form within the Bi2O3-CeO2-Nb2O5 system, including quantitative analysis of these particles. Secondly, and most importantly, it presents the first successful synthesis of quaternary oxide nanoparticles containing bismuth, cerium and niobium using the low temperature resin-gel method. Finally, the work attempts to explain how and why the ions are ordered in a given arrangement, with bismuth showing a preference for surface site occupation, as shown by XPS data, and describes some preliminary computational results which corroborate the experimental data.

Driven by exciting new research and applications, top-down and bottom-up fabrication techniques are producing ever more intricate, reproducible, plasmonic nano-architectures with gaps and junctions approaching the single nanometre and atomic scales. Such atomic-sized features promote the intersection of physics, chemistry and biology in plasmonics. Consequently, understanding light-matter interactions in such closely spaced, electromagnetically coupled, metallic nanosystems is of vital importance to a tremendous variety of current and future nanophotonic technologies. This thesis describes the first dynamically controlled, optically broadband, experimental investigations of light-driven plasmonic coupling between two metal nanostructures with sub-nanometre separation. A new experimental apparatus and nanosystem alignment technique was developed to enable the required sub-nanometre inter-nanoparticle geometry to be created and probed. Two conducting atomic force microscopy tips with nanoparticle functionalised apices are brought into nanoscale `tip-to-tip' axial alignment with dynamically-controlled spacing and ultra-wide optical access. Resonant electrical parametric mixing, created by oscillating the electromechanically coupled tips, is utilised to extract an electronic signal due to nanoscale changes in inter-tip position. Experimental results match theory confirming the viability of the technique. By functionalising the tip apices, this unique multi-functional observation platform allows the plasmonic response of nanoparticle dimers with sub-nanometre separations to be characterised. By simultaneously capturing both the electrical and optical properties of tip-mounted gold nanoparticles with controllable sub-nanometre separation, the first evidence for the quantum regime of optically driven tunnelling plasmonics is revealed in unprecedented detail. It is demonstrated that quantum mechanical effects are critically important at approximately the 0.3 nm scale where spatially non-local tunnelling plasmonics controls the optical response. All observed phenomena are in good agreement with a recently developed quantum-corrected model of plasmonic systems. The findings imply that tunnelling establishes a quantum limit for plasmonic field enhancement and confinement. Additionally, the work suggests the highly enhanced local density of photonic states in nanoscale cavities could enable coherent plasmon-exciton coupling. This thesis prompts new experimental and theoretical investigations into quantum-domain plasmonic systems, and impacts the future of nanoplasmonic device engineering, nanoscale photochemistry and plasmon-mediated electron tunnelling.

Direct ethanol fuel cells have been shown to be a good alternative to internal combustion engines in order to reduce the CO2 emissions. In this study, Pd and Pd-based nanocatalysts were deposited on various supports (carbon black, graphene, SnO2, CeO2, TiO2, TiO2 nanotubes and SnO2/TiO2 nanotubes) and their effects on the catalytic properties of the deposited metal for ethanol oxidation in alkaline media are studied. These modifications to the catalytic systems have shown to cause an increase in the reaction rate at the surface of the catalyst and to reduce the overpotential of the ethanol oxidation reaction. Two different promotion mechanisms have been identified. Firstly, the supply of OH- ions at the metal-support interface facilitates the oxidation of adsorbed molecules on neighbouring Pd sites. Secondly, an increase in electron density of Pd nanoparticles with increasing support reducibility modifies the adsorption strength of ethanol and its oxidation intermediates.

Methylmercury (MeHg) is a ubiquitous contaminant and potent neurotoxicant with no completely effective therapy, although selenium antagonises MeHg toxicity. Furthermore, nanoparticles are promising as a novel drug delivery system. We researched the potential of selenium nanoparticles (SeNPs) in antagonising MeHg neurotoxicity compared to selenomethionine (SeMet) using primary astrocyte cell cultures and examining outcomes related to oxidative stress. We found that SeNPs were more toxic than SeMet. Increasing SeNPs significantly decreased MeHg cellular uptake and MeHg significantly decreased uptake of SeNPs at the highest concentration. Finally, SeNPs alone produced significantly higher reactive oxidative species and altered the ratio of reduced-to-oxidised glutathione, but MeHg, SeMet, and co-exposures did not. There were no significant effects on glutathione peroxidase or reductase activity. This suggests that SeNPs are more toxic than MeHg in cerebellar astrocytes and that they may not be suitable as a therapy at the doses and formulation used in this research.

In this study, crystalline poly(N-isopropylacrylamide-co-acrylic acid) (PNIPAm-co-AAc) nanoparticle network in organic solvents was obtained by self assembling precursor particles in acetone/epichlorohydrin mixture at room temperature followed by inter-sphere crosslinking at ~98 °C. The crystals thus formed can endure solvent exchanges or large distortions under a temporary compressing force with the reoccurrence of crystalline structures. In acetone, the crystals were stable, independent of temperature, while in water crystals could change their colors upon heating or changing pH values. By passing a focused white light beam through the crystals, different colors were displayed at different observation angles, indicating typical Bragg diffraction. Shear moduli of the gel nanoparticle crystals were measured in the linear stress-yield ranges for the same gel crystals in both acetone and water. Syntheses of particles of different sizes and the relationship between particle size and the color of the gel nanoparticle networks at a constant solid content were also presented. Temperature- and pH- sensitive crystalline PNIPAm-co-AAc hydrogel was prepared using osmosis crosslinking method. Not only the typical Bragg diffraction phenomenon was observed for the hydrogel but also apparent temperature- and pH- sensitive properties were performed. The phase behavior of PNIPAm nanoparticles dispersed in water was also investigated using a thermodynamic perturbation theory combined with lightscattering and spectrometer measurements. It was shown how the volume transition of PNIPAM particles affected the interaction potential and determined a novel phase diagram that had not been observed in conventional colloids. Because both particle size and attractive potential depended on temperature, PNIPAM aqueous dispersion exhibited phase transitions at a fixed particle number density by either increasing or decreasing temperature. The phase transition of PNIPAm-co-AAc colloids was also studied. The results from the comparison between pure PNIPAm and charged PNIPAm colloids showed that the introducing of carboxyl (-COOH) group not only contributed to the synthesis of three-dimensional nanoparticle network but also effectively increased the crystallization temperature and concentration range. The phase transitions at both low and high temperatures were observed from the turbidity change by using UV-Vis spectrometer. Centrifugal vibration method was used to make crystalline PNIPAm-co-AAc dispersion at high concentration (8%). The turbidity test proved the formation of iridescent pattern.

Les films nanocomposites sont des revêtements composés de nanoparticules enrobées dans une matrice solide d’un matériau différent. L’intérêt de ces matériaux réside dans leur capacité à exploiter les caractéristiques inédites des nano-objets qu’ils contiennent tout en bénéficiant des propriétés de résistance mécanique et chimique de la matrice. Ces composites disposent de propriétés très prometteuses pour un grand nombre d’applications comme le photovoltaïque ou la photocatalyse. Plusieurs procédés de synthèse existants permettent de produire des matériaux nanocomposites par des méthodes physiques ou chimiques (co-pulvérisation, sol-gel,…). Cependant, aucun n’est assez flexible pour envisager la synthèse d’une large gamme de nanocomposites par le même procédé. Ceci est un frein au développement à l’échelle industrielle de ce type de matériaux. Le premier objectif de la thèse est de développer un procédé original de synthèse de films nanocomposites. Ce procédé présente un caractère universel en ce qu’il permet un choix a priori illimité dans la nature des nanoparticules et celle de la matrice. Le procédé développé combine un jet de nanoparticules sous vide formé par une lentille aérodynamique à un dispositif de pulvérisation magnétron qui permet de déposer la matrice. Le jet de nanoparticules permet de coupler toute source de nanoparticules à la pulvérisation. Les nanoparticules peuvent être soit synthétisées in situ en phase gazeuse, soit synthétisées préalablement en voie liquide. Une grande variété de nanoparticules peut donc être utilisée. La pulvérisation magnétron permet par ailleurs de disposer d’une très large gamme de matériaux pour la matrice (métaux, céramique, polymère). Dans le cadre de cette thèse, deux types de sources de nanoparticules ont été utilisés. Le premier est un réacteur de pyrolyse laser et le second un générateur d’aérosol. Le réacteur de pyrolyse laser permet une synthèse in-situ des nanoparticules en phase gazeuse alors que le générateur d’aérosol permet d’utiliser une suspension de nanoparticules préalablement synthétisées. Afin d’éprouver la robustesse du procédé de co-dépôt, deux types de matériaux nanocomposites ont été développés. Le premier matériau étudié est composé de nanoparticules d’or sphériques de 35 nm de diamètre, synthétisées préalablement par voix liquide, dans une matrice de silice. Le but ici est de bénéficier des propriétés optiques uniques des nanoparticules d’or dans un film résistant mécaniquement et chimiquement. Les caractérisations réalisées sur ces matériaux ont permis d’optimiser la concentration en nanoparticules d’or dans les films de manière à garder des propriétés mécaniques et chimiques compatibles avec les applications tout en gardant des propriétés optiques satisfaisantes. Le second type de matériaux étudiés est composé de nanoparticules semi-conductrices synthétisées in situ par pyrolyse laser et d’une matrice métallique. La synthèse de ce matériau permet de démontrer la flexibilité du procédé de co-dépôt à synthétiser une large gamme de films nanocomposites. Enfin, la robustesse du procédé ayant été démontrée, la conception d’un pilote industriel a été entreprise. Le but final étant de disposer d’une machine répondant aux exigences industrielles dans l’optique d’un transfert technologique. / The nanocomposite films are coatings of nanoparticles embedded in a solid matrix of a different material. The advantage of these materials is their ability to exploit the unique properties of nano-objects while benefiting of the mechanical and chemical resistance properties of the matrix. These composites have very promising properties for many applications such as photovoltaics and photocatalysis. Several existing synthetic methods can produce nanocomposite materials by physical or chemical methods (co-sputtering, sol-gel, ...). However, none is flexible enough to consider the synthesis of a wide range of nanocomposites by the same method. This is an obstacle to the development on an industrial scale of this type of material. The first objective of the thesis is to develop an original synthesis process of nanocomposite films. This method is universal in which it presents no limit in the choice of nanoparticles and matrix. The developed method combines vacuum nanoparticle jets formed by an aerodynamic lens with a magnetron sputtering device for depositing the matrix. The nanoparticle jets can be coupled with any source of nanoparticles. Nanoparticles may be synthesized in situ in the gas phase or beforehand solution synthesis. A wide variety of nanoparticles can be used. Magnetron sputtering also enables to have a very wide range of materials for the matrix (metal, ceramic, polymer). During this thesis, two types of nanoparticles sources were used. The first one is a laser pyrolysis reactor and the second is an aerosol generator. The laser pyrolysis reactor enables in-situ gas phase synthesis of the nanoparticles while the aerosol generator use a suspension of previously synthesized nanoparticles. To test the robustness of the co-deposition process, two types of nanocomposite materials have been developed. The first material is composed of 35 nm spherical gold nanoparticles, chemically synthesized, in a silica matrix. The goal here is to benefit from the unique optical properties of gold nanoparticles in a film mechanically and chemically resistant. The characterizations carried out on these materials have optimized the gold nanoparticle concentration in the films to keep the mechanical and chemical properties compatible with applications while maintaining satisfactory optical properties. The second type of materials studied is composed of semiconductor nanoparticles in situ synthesized by laser pyrolysis and a metal matrix. The synthesis of this material demonstrates the flexibility of the co-deposition method to synthesize a wide variety of nanocomposite films. Finally, the design of an industrial pilot was undertaken. The final goal is to have a pilot-scale setup that meets industry requirements in the context of a technology transfer.

The infiltration of nanoparticle electrocatalysts into solid oxide fuel cell (SOFC) electrodes has been proven to produce a high density of electrochemically active sites, and reduce charge transfer polarization losses in SOFC electrodes. This is crucial for intermediate temperature operation, as these losses increase greatly at lower temperatures. Nickel-yttria stabilized zirconia (Ni-YSZ) cermets are low-cost, and exhibit excellent stability, but their main disadvantage stems from nickel coarsening and performance loss over their operational lifetimes. Infiltration of electrocatalyst nanoparticles has been shown to mitigate nickel coarsening and the consequent anode degradation. In this work, the effects of these infiltrants have been observed in a standard Ni-YSZ electrode. In addition to nickel, mixed ionic and electronic conducting (MIEC) phases have been infiltrated into Ni-YSZ scaffolds and their performance characterized using electrochemical impedance spectroscopy (EIS). Cross-sectional microscopy of fractured cells has been used to compare electrode microstructure and particle statistics. A model has been proposed to explain the origin of anode performance enhancement from nanoscale electrocatalysts.

The aim of my PhD research was to develop and establish techniques for surface functionalization of nanoparticles, which can be employed to study the dynamics, function and activity of recombinantly expressed as well as endogenous proteins inside living cells. A prerequisite to achieve this goal was the ability to bio-functionalize nanoparticles with proteins in the cytoplasm of living cells. The HaloTag technology was utilized for generic site-specific targeting of nanoparticles to proteins. Fast and efficient targeting of nanoparticles to proteins was then achieved by using an engineered clickHTL exhibiting fast reactivity towards the HaloTag-enzyme. Application of this approach to track individual proteins in the outer membrane of mitochondria revealed that the physicochemical properties of the nanoparticles biased the mobility of the targeted proteins. To circumvent this, a model nanoparticle was systematically engineered in order to identify physicochemical properties that are important for tracking intracellular membrane proteins without affecting their diffusion dynamics. Nanoparticles exhibiting stealth properties were finally obtained upon densely coating the nanoparticle surface with PEG2k. These particles were mono-functionalization with clickHTL, to ensure labeling in a 1:1 stoichiometry, and could be successfully used for unbiased tracking of individual membrane proteins. Beyond the observation of proteins, generic approaches that allow intracellular manipulation and probing of protein activities are desired. To this end, 500 nm superparamagnetic nanoparticles were used as mobile nanoscopic hotspots self-assebled into active signaling platforms. Inside living cells, precise and accurate manipulation of endogeneous Rac1 activity was possible at different subcellular locations and over extended time periods. These experiments demonstrated that Rac1 signaling is dependent on the subcellular-context by spatial isolation of distinct signaling pathways. Furthermore, these MNPs provided well defined platforms for selective spectroscopy in order to quantify bait-prey protein interactions in the cytoplasm as was demonstrated by the interaction of cdc42 and N-WASP.