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Aminoglycosides are a group of broad-spectrum antibiotics that, under neutral pH conditions, carry a positive charge. The net cationic charge arises from the high number of amino groups in the core structure of aminoglycosides. Previous studies performed have shown that negatively charged citrate ligand-capped gold nanoparticles (AuNPs) can interact with various biomolecules such as aminoglycosides. AuNPs bound to biomolecules have been used in conjugation with various assaying techniques to detect and study compounds in vitro and in vivo. AuNPs also have strong light scattering properties that can be used with a wide variety of imaging and assaying techniques. Our laboratory has previously performed experiments on the aminoglycoside antibiotic ribostamycin sulfate. During this experiment, the concentration dependent rod-like assembly of ribostamycin sulfate was characterized. This experiment used three analytical techniques in conjunction with AuNPs: (1) dynamic light scattering (DLS), (2) UV-Vis absorption spectroscopy, and (3) dark field optical microscope imaging (DFM). This suite of techniques was used to analyze mixtures of ribostamycin sulfate at different concentration with different sized AuNPs. The primary objective of this research was to determine if the techniques used to characterize the self-assembly of ribostamycin sulfate could be generalized and applied to other aminoglycoside antibiotics. The secondary objective of this research was to determine if other aminoglycoside antibiotics formed rod-like assemblies. This study demonstrated that AuNPs can be used to detect self-assembled oligomers for different aminoglycoside antibiotics. In addition, this study also revealed that not all aminoglycoside antibiotics will self assemble into rod-like oligomers similar to ribostamycin. It was observed that the aminoglycoside antibiotic amikacin self assembled into rod-like aggregates similar to ribostamycin sulfate but the aminoglycoside antibiotics neomycin sulfate and streptomycin sulfate did not.

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Systemic administration of adenovirus type 5 (Ad5) vectors for the treatment of cancer is limited by poor circulation kinetics and inefficient uptake from the bloodstream into tumours. This study reports a novel method for linkage of highly-PEGylated gold nanoparticles (AuPEG) to Ad5 by a single reduction cleavable bond. The resulting ‘dandelion’ structure provides very effective steric shielding with only minimal and reversible modification of the Ad5 capsid. This ablates in vitro cell infection, improves protection against the binding of antibodies, and enhances in vivo circulation kinetics. Focused ultrasound is a promising technology for the non-invasive, targeted treatment of cancer. In the context of drug delivery, cavitational energy generated upon exposure of ultrasound contrast agents to focused ultrasound can be used as a powerful stimulus to move therapeutics over distances of hundreds of microns away from blood vessels. In addition to providing a platform for effective stealthing, conjugation of AuPEG to Ad5 also increases the effective density of Ad5. This increase in density imparts a second major advantage on the strategy, observed for the first time in the present study: denser particles are transported significantly farther by cavitation-induced microstreaming than identically-sized particles of lower density. Specifically, in in vitro tests using a tumour-mimicking flow-channel phantom model and in in vivo experiments using tumour bearing mice, Ad5–AuPEG was delivered farther from vessels in response to ultrasound induced cavitation than either naked Ad5 or polymer-coated Ad5. The enhancements in stealthing and improvements in response to ultrasound provided by this strategy enabled up to 12% (S.D. 0.97) of the injected dose to be deposited in the tumour, compared to just 0.12% (S.D. 0.05) for Ad5 without ultrasound (p < 0.001). Consequently, in a survival study, mice treated with Ad5–AuPEG with focussed ultrasound had the slowest tumour growth and longest survival rate when compared to mice treated with Ad5 alone, Ad5–AuPEG alone, or Ad5 with focussed ultrasound. These results provide compelling evidence that the combination of focussed ultrasound with density-augmented stealthed Ad5 results in improved delivery to tumours and therapeutic efficacy. This combination of ultrasound with particle modification for optimal cavitation-mediated delivery has the potential to be applied to a broad range of anti-cancer nano-medicines and therapeutics to augment their bio-availability for improved cancer treatment.

Dans cette thèse, l’auto-assemblage en molécules colloïdales de virus en forme de filament, les bactériophages M13, est étudié. Comme première approche, l’affinité de la streptavidine pour la biotine ou un Strep-tag est utilisée et quantitativement comparée. Pour ce faire, des virus modifiés génétiquement, M13-AS, présentant des Strep-tag et des virus M13C7C chimiquement bioconjugués par de la biotine ont réagi via leur extrémité proximale avec des nanoparticules fonctionnalisées par de la streptavidine. Il en résulte la formation de molécules colloïdales en étoile, dont la valence ou nombre de virus par structure, peut être simplement contrôlée par l’excès molaire initial. Cependant, la stabilité de ces molécules colloïdales est limitée par la libération progressive et la dégradation de la streptavidine. Nous avons alors développé une seconde approche basée sur l’affinité soufre-métal, qui s’est avérée à la fois pratique expérimentalement et fiable. Grâce aux groupements disulfures présents sur les cystéines de la protéine P3, des nanoparticules métalliques peuvent se lier à l’extrémité des virus. Le caractère générique de cette méthode est vérifié en faisant varier la nature du métal des nanoparticules ainsi que la souche des virus, dont la sauvage. La valence des structures formées est déterminée en fonction de plusieurs paramètres, dont l’excès molaire initial, la taille des nanoparticules et la force ionique. Un modèle rendant compte des résultats expérimentaux a été élaboré, dont les principales variables sont la surface des nanoparticules et le diamètre effectif électrostatique des virus. Cette approche est étendue à la réalisation de diblocs colloïdaux hétéro bifonctionnels, utilisant les virus comme briques constitutives. Comme preuve de concept, des diblocs bicolores à base de virus sont obtenus par auto-assemblage et leur dynamique est étudiée à l’échelle du bloc élémentaire en microscopie optique de fluorescence. Ainsi, nous avons montré dans cette thèse la réalisation par auto-assemblage d’une nouvelle génération de molécules colloïdales, dont l’auto-organisation peut conduire à la formation de superstructures hiérarchiques hybrides de complexité croissante, potentiellement utiles en sciences des matériaux. / In this thesis, the self-assembly of rod-like viral particles, specifically the M13 bacteriophages, into colloidal molecules is studied. As the first method, the affinity of streptavidin to biotin or Strep-tag is used and quantitatively compared. In this case, both biologically engineered M13-AS displaying Strep-tags and chemically biotinylated M13C7C viruses have reacted with streptavidin activated nanoparticles via their functionalized proximal ends. This results in star-like colloidal molecules, whose valency – or number of viruses par structure – can be solely controlled by tuning the initial molar excess. However, the stability of these colloidal molecules is limited by streptavidin release and degradation. Thus, we develop the second method based on the sulfur—metal interactions, which is more convenient and reliable. Thanks to the exposed disulfide groups located at p3 proteins, metallic nanoparticles are able to bind to proximal ends of the M13 virus. The generic feature of this method is verified by using different metals and two virus strains including wt-M13. Afterwards, the control of the valency is explored by varying the initial molar excess, the nanoparticle size and the ionic strength. A quantitative model is built correspondingly, using the surface area of Au nanobead and the effective electrostatic diameter of the virus as variables, which accounts for the assembly of colloidal molecules with desired valencies. This method is further applied to assemble heterobifunctional diblocks by using filamentous viruses as building units. As a proof-of-concept experiment, bicolored diblocks are produced and tracked by each block simultaneously. Overall, we demonstrate the synthesis of a new generation of hybrid colloidal molecules, whose self-organization could serve as a promising means to create novel hierarchical biologic/inorganic superstructures that may find applications in materials science.

A incorporação de nanopartículas metálicas em tecidos tumorais tem sido estudada em Radioterapia devido ao aumento de dose que pode ser obtido no volume alvo do tratamento. Estudos indicam que nanopartículas de ouro (AuNP) estão entre as de maior viabilidade biológica para essas aplicações, devido ao baixo potencial tóxico. Além disso, estudos mostram que AuNP de alguns nanômetros até alguns micrômetros podem permear vasos sanguíneos que alimentam tumores, permitindo sua incorporação nas células tumorais. Desta forma, este trabalho visou estudar os fatores de aumento de dose obtidos em Radioterapia com AuNP incorporadas ao tecido tumoral utilizando feixes de ortovoltagem, de braquiterapia e de teleterapia. Este trabalho utilizou de uma metodologia computacional, através de simulação Monte Carlo com o código PENELOPE. Foram simulados feixes clínicos de 50, 80, 150 e 250 kVp, Ir-192 e 6 MV, e um modelo de célula tumoral com AuNPs incorporadas com diferentes concentrações de ouro. O modelo de células utilizado possui 13 µm de diâmetro externo máximo e 2 µm de diâmetro no núcleo. Dois modelos de incorporação de AuNPs foram implementados: modelo homogêneo e modelo heterogêneo. No modelo homogêneo, as AuNP foram distribuídas homogeneamente no núcleo e as células foram irradiadas nas diferentes energias estudadas para avaliar o fator de aumento de dose (DEF) em função da concentração de ouro na célula e da energia do feixe. No modelo heterogêneo, aglomerados de AuNPs foram simulados individualmente dentro da célula. Neste modelo foram utilizados somente os espetros de radiação que apresentaram os melhores desempenhos no modelo homogêneo. Foram avaliadas a fluência de partículas ejetadas nas AuNPs, o DEF, as distribuições de doses e os perfis de dose com aglomerados de 50 a 220 nm na célula. Os resultados obtidos para o modelo homogêneo mostram que os feixes de baixa energia são os que proporcionam maior DEF para uma mesma concentração de AuNP. Os maiores DEFs obtidos foram de 2,80; 2,99; 1,62 e 1,61, para os feixes de 50 kVp, 80 kVp, 150 kVp, 250 kVp, respectivamente, sendo a maior incerteza de 1,9% para o feixe de 250 kVp. Através dos resultados obtidos com o modelo heterogêneo foi possível concluir que os elétrons ejetados possuem maior influência no aumento local da dose. Os perfis de dose, extraídos das distribuições de doses, para os aglomerados simulados permitiram obter os alcances das isodoses de 50, 20 e 10% da dose no entorno das AuNPs. Através desses perfis de dose pode-se concluir que o aumento de dose é local, da ordem de alguns micrômetros, dependendo do tamanho das nanopartículas e da energia do feixe primário. Para o feixe de 50 kVp, o DEF encontrado para uma incorporação heterogênea de seis aglomerados de AuNPs, correspondendo a um modelo clínico real, foi de 1,79, com incerteza de 0,4%. Com base nos resultados obtidos pode-se concluir que as energias de ortovoltagem proporcionam maior fator de aumento de dose que feixes de megavoltagem utilizados em teleterapia convencional. Além disso, o reforço local de dose pode proporcionar um fator de radiossensibilização celular se as AuNPs forem incorporadas no núcleo das células, nas redondezas do DNA, proporcionando um maior potencial de controle tumoral. / The incorporation of metal nanoparticles into tumor tissues has been studied in radiation therapy given of the dose enhancement that can be obtained in the target volume of the treatment. Studies indicate that gold nanoparticles (AuNP) are among the highest biologically viable for such applications, due to their low toxic potential. In addition, studies show that AuNP from a few nanometers to a few micrometers can permeate blood vessels that feed tumors, allowing their incorporation into tumor cells. Hence, this study´s goal was to study the dose enhancement factors obtained in radiation therapy with AuNP incorporated in the tumor using orthovoltage, brachytherapy and teletherapy beams. This work used a computational methodology, through Monte Carlo simulation with the PENELOPE package. Clinical beams of 50, 80, 150 and 250 kVp, Ir-192 and 6 MV were simulated with a tumor cell model with incorporated AuNPs. The cell model has maximum outer diameter of 13 m and 2 m of nucleus diameter. Two models of AuNP incorporation were implemented: homogeneous model and heterogeneous model. In the homogeneous model the AuNP were distributed homogeneously in the nucleus and the cells were irradiated in the different beams studied to evaluate the dose enhancement factors (DEF) as a function of concentration of gold in the cell and radiation beam. In the heterogeneous model, clusters of AuNPs were simulated individually within the cell. In this model, the radiation spectra used was selected among those that presented the best performances in the homogeneous model. The fluence of particles ejected from the AuNPs, the DEFs, the dose distributions and dose profiles for clusters of 50 to 220 nm in the cell were evaluated. The results obtained for the homogeneous model show that lower energy beams provide the highest DEFs for the same concentration of AuNP. The highest DEFs obtained were 2.80; 2.99; 1.62 and 1.61, for the beams of 50 kVp, 80 kVp, 150 kVp, 250 kVp, respectively, with a maximun uncertainty of 1.9% for the 250 kVp beam. Through the results obtained with the heterogeneous model it was possible to conclude that the electrons ejected from he AuNPs have the major influence on the local dose enhancement. The dose profiles extracted from the dose distributions for the simulated clusters allowed the evaluation of the ranges for the 50, 20 and 10% isodoses in the surroundings of the AuNPs. Through these dose profiles, it can be concluded that the dose increase is local, in the order of a few micrometers, depending on the size of the nanoparticles and the energy of the primary beam. For the 50 kVp beam, the DEF found for a heterogeneous incorporation of six clusters of AuNPs, corresponding to an actual clinical model, was 1.79, with uncertainty of 0.4%. Based on the results obtained it can be concluded that kilovoltage energies provide a higher dose enhancement factor than megavoltage beams used in teletherapy. In addition, local dose enhancement may provide a cellular radiosensitization factor if the nanoparticles are incorporated in the nucleus of the cells, in the vicinity of the DNA, providing an enhanced potential for tumor control.