Thesis (Ph.D.)--University of Wollongong, 2008. / Typescript. Includes bibliographical references: leaf 190-205.

Thesis (Ph.D.)--Ohio University, November, 2007. / Abstract only has been uploaded to OhioLINK. Includes bibliographical references (leaves 155-178)

L’angiogenèse tumorale réfère à la capacité d’une tumeur à stimuler la formation de nouveaux vaisseaux sanguins. L’induction de l’angiogenèse dépend notamment de la présence de certains récepteurs exprimés à la surface de cellules endothéliales et tumorales. Ces récepteurs sont impliqués dans la formation de nouveaux vaisseaux sanguins mais aussi dans la progression des tumeurs, l’invasion locale des tissus avoisinants et la formation de métastases. Nous nous intéressons ici essentiellement aux récepteurs de type intégrines (et surtout l’intégrine αvß3) ou neuropiline-1 (NRP1).Les intégrines sont des récepteurs transmembranaires décrits initialement parce qu’ils permettent aux cellules d’adhérer et de se déplacer sur la matrice extracellulaire (ECM) en particulier parce qu’elles se lient à la séquence tri-peptidique RGD, mais elles interviennent aussi directement et indirectement dans les échanges biochimiques entre les cellules et leur micro-environnement. NRP1 est un corécepteur du VEGF (vascular endothelial growth factor). Pour cela, NRP1 s’associe au récepteur principal VEGFR2, surexprimé dans les tumeurs et dont l’expression a été corrélée avec l’angiogenèse. Il est très important de noter que l’intégrine αvß3 et le récepteur NRP1 peuvent interagir physiquement et fonctionnellement. Notre hypothèse de travail est alors qu’en bloquant la fonction de ces 2 récepteurs nous pourrons augmenter l’efficacité des thérapies anti-angiogèniques anti-tumorales.Nous avons généré des nanoparticules de silices bifonctionnelles car elles présentent à leur surface à la fois des peptides cycliques cRGD ciblant l’intégrine αvß3 et ATWLPPR qui cible NRP1. Nous avons testé des ratio différents de peptides cRGD et ATWLPPR (100/0, 25/75, 50,75/50/25 et 0/100), et nous avons aussi optimisé le nombre total de ces ligands/NP. Nous avons analysé l’affinité des différentes molécules, leur sélectivité et activité biologique ainsi que leurs propriétés anti-angiogéniques et anti-tumorale en particulier sur des cellules endothéliales humaines (ECs) et sur des lignées de cellules tumorales.Notre étude suggère que ces nanoparticules bifonctionnelles présentent un grand potentiel si leur composition est soigneusement définie. En particulier, elles peuvent présenter des activités extrêmement variables voir opposées suivant la nature et composition de leur surface et de la concentration à laquelle les NPs sont utilisées. En effet, à « haute concentration » en NP, ce qui correspond en fait à une faible concentration en peptides, nous montrons qu’il est possible d’obtenir un effet « pro-angiogénique » lié au recrutement d’autres récepteurs de facteurs de croissance (IGF1-R/IR) qui a priori ne devaient pas intervenir dans notre système, mais semblent pouvoir être fonctionnellement liés aux intégrines et/ou NRP1 en réponse aux particules présentant les 2 peptides cRGD et ATWLPPR. Ces résultats contribuent à expliquer certains échecs thérapeutiques des agents anti-angiogéniques mais nous permettent aussi de proposer des solutions attractives pour la définitions nouveaux agents thérapeutiques. / Tumor angiogenesis refers to the ability of a tumor to stimulate new blood vessels formation. Angiogenesis strongly depends on cell surface receptors and integrin activation to promote tumor progression, local invasion and dissemination. Integrins (especially integrin αvß3) and Neuropilin-1 (NRP1), a co-receptor of VEGFR2, are over-expressed in the tumor vasculature and by tumor cells, and their expression has been correlated with tumor progression. Importantly, integrin αvß3 and NRP1 can physically and functionally interact.The use of dual targeted drugs that block the integrin αvß3 and the NRP1 receptor simultaneously is thus expected to augment the anti-angiogenic and anti-tumor activities, as compared to each “mono-therapy” separately. During my PhD studies, in collaboration with the group of chemists leaded by Pr G. Subra, we generated different batches of bifunctional cRGD/ATWLPPR peptides coated nanoparticles (NPs) targeting integrin αvß3 and NRP1 simultaneously. We introduced different ratio of cRGD and ATWLPPR peptides (100/0, 25/75, 50/50, 75/25 and 0/100), and we also increased the amount of total ligands on the surface of the silica NPs. Systematic studies including molecules' affinity, selectivity, and biological activity as well as anti-angiogenic and anti-tumoral effects were performed on primary endothelial cells (ECs), immortalized ECs and several tumor cells. NPs properties were also evaluated in vivo in a mouse tumor model. We report here that these NPs present highly variable biological activities in ECs and tumor cells depending on the peptides ratio, surface coating of the NPs and on their concentration. In particular, “elevated” concentrations of NPs, which actually correspond to usual concentrations of peptides, can activate an unexpected IGF1-R/IR-AKT signaling pathway that could lead to a counter-productive pro-angiogenic activity (agonist instead of antagonist). This could mimic the conflicting results obtained in clinical trials using Cilengitide, an RGD-presenting peptide, and thus provide new areas of investigations and new possibilities to design active nano-drugs.This work can thus participate to the general effort of our research community to design efficient targeted anti-angiogenic therapies that could be applied in particular for cancer treatment.

Made available in DSpace on 2016-03-04T13:55:11Z (GMT). No. of bitstreams: 2 4.pdf: 1725584 bytes, checksum: 95e2e35ced41be5b57d848564e502b68 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Fármacos/Farmanguinhos. Rio de Janeiro, RJ, Brasil. / A nanotecnologia é uma tecnologia transdisciplinar que está sendo desenvolvida e aplicada em diversas áreas, dentre as quais cabe ressaltar a da saúde, principalmente no que tange à terapêutica e ao diagnóstico. Na oncologia os tratamentos são muito prolongados, a necessidade de exames de imagem é frequente, as doses administradas dos tratamentos são muito elevadas e a toxicidade para o paciente é, muitas vezes, o fator limitante da terapia. Com os avanços da nanotecnologia, espera-se que essas deficiências sejam resolvidas ou, pelo menos, amenizadas, tendo em vista algumas características especiais destes materiais. Entretanto, ainda não se tem clara a relação entre essas características e seus efeitos toxicológicos. Por isso, é necessário entender se os requisitos regulatórios, em termos de avaliação toxicológica, para registro de um medicamento com base em nanotecnologia, são capazes de identificar os possíveis riscos advindos desta nova tecnologia. Esse trabalho teve por objetivo comparar a abordagem regulatória da EMA, FDA e ANVISA com relação à avaliação de nanomedicamentos em comparação com medicamentos convencionais. Para isso, foi analisado o perfil toxicológico do DOXIL® em relação a doxorrubicina convencional. Esse medicamento foi escolhido para ser analisado por ser o primeiro lipossoma aprovado pelo FDA, em 1995, e pela importância da doxorrubicina no tratamento oncológico. Foram analisadas as possíveis deficiências dos testes requeridos pelas agências reguladoras e quando possível foi sugerido procedimentos para sua melhoria. Ainda nesse sentido, foi destacada a importância do compartilhamento de experiência sobre a regulamentação da nanomedicina entre os países e, mais especificamente, seu potencial para impulsionar o desenvolvimento dessa área no Brasil. Pode-se concluir que os testes toxicológicos preconizados atualmente pelas agências reguladoras dos Estados Unidos da América, União Europeia e no Brasil, apesar de estarem alinhados, não são específicos para a avaliação de nanomedicamentos. Em base às informações disponíveis, não se pode garantir que os dados gerados pela bateria de testes solicitada sejam confiáveis para o estabelecimento de uma relação risco/benefício robusta para os nanomedicamentos. Além disso, foram demonstradas muitas das limitações desses testes e algumas sugestões de melhorias para a condução dos mesmos. Ainda nesse sentido, foi ressaltada a importância da caracterização bio-físico-química de cada nanomedicamento submetido às análises. Com relação à bateria de testes toxicológicos solicitada para a avaliação do DOXIL®, pode-se concluir que seu perfil de segurança não pode ser adequadamente estabelecido pelos testes realizados. Entretanto, os dados de pós-comercialização demonstraram que seu perfil de toxicidade está bem estabelecido e que manteve alinhamento com os resultados obtidos durante o seu desenvolvimento. Não se pode, entretanto, extrapolar este mesmo comportamento para outros casos, os quais deverão seguir normativas atualizadas. À luz dessas diferenças e limitações, este estudo traz à discussão os esforços para entender melhor a aplicabilidade dos requisitos atuais de avaliação toxicológica para aprovação de nanomedicamentos e visa contribuir com a ANVISA para a implementação de um programa de avaliação toxicológica robusto para garantir o desenvolvimento seguro da nanomedicina. / Nanotechnology is a transdisciplinary technology which is under developement and is being applied in various fields, among those it is worth highlighted in health, especially with regard to the therapy and diagnosis. Oncologic treatments are very long and expensive, the need for imaging studies is frequent, treatment doses are very high and toxicity is often the limiting therapy factor for patients. The advances in nanotechnology bring a promise expectation to overcome these deficiencies and even mitigate it, taking into consideration some particular characteristics of these materials. However, there is still no clear relationship between these characteristics and their toxicological effects and more studies on this field need to be done in order to validate this as an efficient therapy alternative for those patients. Therefore, it is necessary to understand if the current regulatory requirements in terms of toxicological assessment for registration of a drug based on nanotechnology are able to identify the possible risks arising from this new technology. This study aimed to compare the regulatory approach of the EMA, FDA and ANVISA regarding the evaluation of nanodrugs compared with conventional medicines. For this, the toxicological profile of DOXIL® compared to conventional doxorubicin were reviewed. This product was chosen to be analyzed by being the first liposome approved by FDA in 1995, and the importance of doxorubicin in cancer treatment. Possible shortcomings of the tests required by regulatory agencies and possible suggested procedures for improvement were analyzed. Also in this sense, was highlighted the importance of sharing experience on nanomedicine regulation across countries, and more specifically, its potential to boost the development of this area in Brazil. It was concluded that toxicological tests currently recommended by regulatory agencies in the United States, European Union and Brazil, although they are aligned, are not specific to the assessment of nanodrugs. Based on the available information, it cannot guarantee the reliability of the data generated through the battery of tests required for establishing a robust risk / benefit ratio for nanodrugs. Moreover, were demonstrated many of the limitations of these tests and some suggestions for improvement on the conduction of such investigations. Also in this sense, was highlighted the importance of biophysicochemical characterization of each nanomedicine subjected to analysis. It could be concluded that the DOXIL® safety profile were not adequately represented by the toxicological tests performed for regulatory approval. However, the post-marketing data sustain that its toxicity profile is well established and maintained in alignment with the results obtained during its development. Notwithstanding, it is not possible to extrapolate the same behavior for other cases, which should follow new standards. In light of these discrepancies and limitations, this study brings to discussion the efforts to understand better the applicability of current toxicological assessment requirements for nanomedicines approval and aims to contribute with ANVISA to implement a robust toxicological assessment program to guarantee the safe development of nanomedice.

Nanopartículas de ouro têm mostrado enorme potencial de aplicação em modalidades diagnósticas e terapêuticas fotoativadas. Em especial, nanoestruturas de ouro anisotrópicas ramificadas apresentam excelente desempenho atuando tanto como contrastes de imagens fotoacústicas, quanto como agentes ativos para terapias fototérmicas de câncer. Apesar dos avanços nas suas rotas de síntese, o desenvolvimento dessas nanoestruturas de forma simples e reprodutível ainda é desafiador. O presente trabalho visou o desenvolvimento de nanopartículas de ouro anisotrópicas ramificadas, ou nanoflores, que sejam fotoativas no infravermelho-próximo para a terapia e diagnóstico de câncer. Em particular, buscou-se o desenvolvimento de uma síntese simples para sua obtenção, assim como a verificação de sua atuação como agente de contraste fotoacústico e como agente ativo para hipertermia de tumores. Para tanto, desenvolveu-se uma síntese in situ que permitiu a obtenção de nanoflores monodispersas com tamanho e propriedades ópticas controláveis. Através da variação de aspectos da síntese, como a temperatura e a concentração de ouro, foi possível sintonizar a atividade óptica das partículas entre 590 e 960 nm. Sua formação foi confirmada por microscopia eletrônica de varredura, espalhamento de luz dinâmico e espectroscopia UV-visível. As partículas apresentaram boa estabilidade de suas características físico-químicas por dois meses e meio. Ainda, as nanoflores se mostraram estáveis, também, quando suspensas em meio de cultura, sob irradiação de lasers, e quando mantidas a temperatura corpórea por longos intervalos. Sua resposta fotoacústica foi caracterizada, apresentando sinais significativos e permitindo a obtenção de imagens claras de sua localização, mesmo em baixas concentrações. Testes realizados em cultura de células mostraram que as nanoflores foram eficazes na hipertermia de uma linhagem de hepatocarcinoma de rato (HTC), ao mesmo tempo que não apresentaram sinais de toxicidade a uma linhagem de fibroblastos de camundongos (FC3H). Esses resultados revelam uma possibilidade simples de fabricação de nanoestruturas de ouro anisotrópicas ramificadas, que podem servir como uma plataforma promissora para o diagnóstico e terapia do câncer. / Gold nanoparticles have shown enormous potential of application in photodiagnostic and in phototherapeutic procedures. Notably, branched anisotropic gold nanostructures present distinguished performance acting as contrast agents of photoacoustic images and as active agents for photothermal therapies for cancer. Despite advances in their synthesis routes, the growth of these nanostructures in a simple and reproducible way is still challenging. The present study was aimed at developing branched anisotropic gold nanoparticles, coined nanoflowers, that are photoactive in the near-infrared for therapy and diagnosis of cancer. In particular, we sought to develop a simple synthesis route, as well as to verify its application for both, as photoacoustic contrast agents and as active agents for tumor hyperthermia. An in situ synthesis was developed which allowed the development of monodisperse nanoflowers with controllable size and optical properties. Through variations of certain aspects of this procedure, such as temperature and gold ions concentration, it was possible to tune the optical activity of the particles between 590 and 960 nm. The nanostructure morphology was confirmed by scanning electron microscopy, dynamic light scattering and UV-visible spectroscopy. The particles exhibited consistent physicochemical characteristics and good stability for two and a half months. Furthermore, the nanoflowers were also stable when suspended in cell culture medium, under laser irradiation and when maintained at body temperature for long intervals. Its photoacoustic response was characterized, presenting significant responses and generating clear images of its location, even at low concentrations. In vitro tests revealed that these nanoflowers were effective therapeutic agents for photothermal therapy of a rat hepatocarcinoma (HTC) lineage, while showing no signs of toxicity to mouse fibroblast (FC3H) cell line. These results reveal a simple procedure of synthesizing branched anisotropic gold nanostructures, which can serve as a promising platform for cancer diagnosis and therapy.

Nanotubos de Carbono (Carbon Nanotubes - CNTs) são nanoestruturas de carbono na forma de tubos cilíndricos que apresentam excelentes propriedades ópticas, elétricas, térmicas e mecânicas. A imobilização dos CNTs em conjunto com polieletrólitos condutores e naturais na forma de filmes ultrafinos, utilizando a técnica automontagem, apresenta uma nova alternativa para o desenvolvimento de nanocompósitos aplicados em dispositivos sensores e biossensores. Neste trabalho de mestrado foram construídos e estudados filmes automontados contendo CNTs funcionalizados com grupos ácidos carboxílicos e imobilizados com os polieletrólitos polianilina (PANI) e quitosana (QUIT). Também foram fabricados para comparação filmes de PANI e poliestireno sulfonado (PSS). O crescimento dos filmes de PANI/CNT e PANI/PSS revelou um crescimento linear com o número de bicamadas, conforme mostraram as medidas de espectroscopia UV-Vis e voltametria cíclica (CV), enquanto que para o filme de QUIT/CNT, as medidas de microbalança de cristal de quartzo (QCM) mostraram um crescimento exponencial. Análises de espectroscopias Raman e infravermelho com transformada de Fourier (FTIR) indicaram alterações nas bandas características dos filmes, comparadas com seus materiais precursores, indicando interação entre os componentes nas multicamadas dos filmes. Análises morfológicas obtidas por AFM mostraram um aumento da rugosidade com o aumento do número de bicamadas. O mapeamento Raman revelou que os filmes contendo CNTs apresentaram maior heterogeneidade química na superfície do que aos filmes somente com polieletrólitos. Foi observada uma queda na resistividade dos filmes, com o aumento do número de bicamadas, sendo esta, mais acentuada com a presença dos CNTs. Os eletrodos contendo os filmes automontados apresentaram alta estabilidade eletroquímica, a presença dos CNTs aumentou a intensidade das correntes de pico e tornou os sistemas mais reversíveis. Os eletrodos que se mostraram mais sensíveis à detecção de peróxido de hidrogênio foram selecionados para imobilização da enzima Glicose Oxidase (GOx). Foram investigados os seguintes sistemas biossensores: (PANI/CNT)7, (PANI/PSS)1 e (QUIT/CNT)5 apresentando na mesma ordem: valores de sensibilidades de 190 nA/(mmol/L), 36 nA/(mmol/L) e 220 nA/(mmol/L); Limite de detecção de 2,2 µmol/L, 67,5 µmol/L e 8,5 µmol/L, e valores deconstante de Michealis-Menten(\'K IND.M\'POT.APP\' ) de 2,2 µmol/L, 67,5 µmol/L e 8,5 µmol/L. Os resultados indicam que a utilização dos CNTs é bastante promissora para fabricação de dispositivos biossensores para aplicação em Nanomedicina, e além disso, a escolha dos componentes para formação dos nanocompósitos exerce grande influência no desempenho do dispositivo. / Carbon Nanotubes (CNTs) are cylindrical carbon nanostructures exhibiting excellent electrical, thermal, optical and mechanical properties. The immobilization of CNTs in nanostructured thin films in conjunction with polymers using the Layer-by-Layer (LbL) technique provides a new alternative for development of nanocomposites to be used as sensors and biosensors. In this study we report the fabrication of LbL films containing CNTs functionalized with carboxylic acid immobilized in conjunction with polyaniline (PANI) or chitosan (QUIT). Films comprising PANI and sulfonated polystyrene (PSS) were also produced for comparison. Film growth was monitored by UV-Vis spectroscopy, cyclic voltammetry (CV) and quartz crystal microbalance (QCM) and the results showed a linear increase with the number of bilayers in PANI films, and an exponential growth for QUIT/CNT films. FTIR and Raman analyses revealed changes in the bands of nanocomposites compared to their precursor materials, indicating the interactions between the components in the multilayers. Morphological analysis of the films obtained by atomic force microscopy (AFM) showed that the roughness of the films increased with the number of bilayers. Raman mapping showed that the presence of CNTs generated a high heterogeneity in film surface in comparison to a films formed only by polyelectrolytes. Electrical resistivity of the films decrease upon increasing the number of bilayers, especially for CNTs-containing films. Furthermore, electrodes containing LbL films exhibited high electrochemical stability, in which the presence of CNTs increased the intensity of response signal. The electrodes that exhibited best performance toward hydrogen peroxide detection were employed for immobilization of glucose oxidase (GOx) and used as glucose biosensors. The systems (PANI/CNT)7, (PANI/PSS) 1 and (QUIT/CNT)5 exhibited a sensitivity of 190 nA/(mmol/L), 36 nA/(mmol/L) and 220 nA/(mmol/L), respectively. The detection limit was estimated at 2.2 mmol/L, 67.5 mmol/L and 8,5 mmol/L, whereas the Michaelis-Menten constant ( \'K IND.M\'POT.APP\') values was found to be 2.2 mmol/L, 67.5 mmol/L and 8.5 mmol/L, respectively, to the three systems employed. The results indicated that the use of CNTs in Layer-by-layer thin films is promising for use as biosensors. Furhtermore, we showed that the choice of the polyelectrolyte is a crucial parameter to tailor specific, high performance sensors.

Essa tese de doutoramento utiliza a auto-organização dos filmes finos layer-by-layer (LbL), auto-organização por alcanotióis mistas (SAMmix) e monocamada de Langmuir no desenvolvimento de dispositivos e novas metodologias para aplicações em nanomedicina. Foram desenvolvidos e aplicados biossensores utilizando as técnicas de LbL e SAM. Dentre os biossensores construídos está o sensor para óxido nítrico (NO•), que é de grande importância no sistema fisiológico. O sensor foi construído por meio da modificação de ultramicroeletrodos de fibra de carbono pela técnica LbL. A caracterização do sensor foi realizada por voltametrias e espectroscopias de impedância eletroquímica. Os resultados revelaram que a difusão de NO• é dependente do número de bicamadas empregadas e da disposição das moléculas no filme. O sensor com arquitetura CF-(PAMAM/NiTsPc), fibra de carbono (CF), ftalocianina de níquel tetrasulfonada (NiTsPc) e dendrímero poliamidoamina (PAMAM), apresentou o melhor sinal analítico. Além disso, foi analisada a detecção de NO• com interferentes como nitrito, nitrato, peróxido de hidrogênio, ácido ascórbico, dopamina, epinefrina e a norepinefrina. Os resultados mostraram alta seletividade devido à utilização do dendrímero PAMAM. O segundo biossensor utilizou a enzima acetilcolinesterase imobilizada em monocamadas auto-organizadas mistas (SAMmix) de alcanotióis. A detecção eletroquímica mostrou-se altamente sensível, uma vez que não há o uso do glutaraldeído como agente reticulante. Com essa plataforma foi possível desenvolver um biossensor de acetilcolina estável e robusto, sendo calculado o valor de Km app = 0,46x10-3 mol L-1, limite de detecção LD=3,32x10-10 mol L-1 e limite de quantificação LQ=1,11x10-9 mol L-1, valores inferiores aos encontrados na literatura, ressaltando a eficiencia da nova plataforma. Seguindo a mesma idéia de auto-organização, foram realizados estudos de nanotoxicidade utilizando modelos de membrana a partir de filmes de Langmuir. O principal objetivo foi elucidar a ação dos nanotubos de carbono (SWCNT), PAMAM e do nanocomplexo entre os dois materiais (SWCNT-PAMAM) nas membranas celulares, a nível molecular, usando um sistema modelo de membrana. A penetração de SWCNT e dos nanocomplexos em monocamadas lipídicas foi estudada utilizando microscopia de ângulo de Brewster (BAM) simultaneamente com cinética de absorção e pressão de superfície. Os resultados confirmaram a interação entre os nanomateriais e a membrana, indicando que a presença dos nanomateriais afeta o empacotamento dos lipídios. Foram realizados ainda estudos de citotoxicidade dos mesmos nanomateriais em sistemas celulares in vitro. Os resultados de citometria, proliferação celular, morfologia e inibição de adesão apresentaram-se evidenciaram que a combinação entre SWCNT e PAMAM, proporciona um maior índice de toxicidade em relação ao SWCNT, um comportamento diferente do que relatado nos componentes individuais. A toxicidade de nanocomplexos de SWCNT-PAMAM e de seus componentes individuais podem estar fortemente ligados ao tipo de material e como estes estão disponíveis no meio de cultura. Os estudos contidos nessa tese mostram a versatilidade dos filmes finos em sistemas auto-organizados e biomiméticos, e podem ser relevantes para o avanço de pesquisas sobre interação de nanomateriais e biossistemas. / In this thesis we employed the concept of self-organization, including the layer-by-layer (LbL) technique, alkanethiols self-assembled monolayers (SAMmix) and Langmuir monolayers, to develop new methods for materials and devices manipulation for application in nanomedicine. Two different types of biosensors were developed. The first one was based on the LbL technique to detect nitric oxide (NO•), which is of great importance in the medicine. The second biosensor was based on SAM monolayers supporting acetylcholinesterase for pesticide monitoring. The NO• was constructed by modified carbon fiber (CF) assembled with nickel phtalocyanine tetrasulfonade (NiTsPc) and polyamidoamine dendrimer (PAMAM) in the form of ultramicroelectrodes (UMEs) by the LbL technique. The sensor was characterized using differential pulse voltammetry and electrochemical impedance spectroscopy. The results showed that NO• diffusion is dependent on the number of bilayers employed and the arrangement of molecules in the film. The sensor architecture with CF-(PAMAM/NiTsPc) presented the best analytical signal. In addition, we analyzed the detection of interfering with NO• as nitrite, nitrate, hydrogen peroxide, ascorbic acid, dopamine, epinephrine and norepinephrine. The results showed high selectivity due to the use of PAMAM dendrimer as selective layer. The second biosensor used the enzyme acetylcholinesterase immobilized on SAMmix. The electrochemical detection of carbaryl was highly sensitive, since there is no use of glutaraldehyde as crosslinking agent. Using acetylcholine as a probe, Kmapp value was determined at 0.46x10-3 mol L-1, with detection limit of 3.32x10-10 mol L-1 and quantification limit of 1.11x10-9 mol L-1, values lower than those found in the literature, highlighting the efficiency of the new platform. Langmuir films made of lipids were employed as cell membrane models, in order to investigate the interactions between single-wall carbon nanotubes (SWCNT), PAMAM and their nanocomplex (SWCNT-PAMAM) at the molecular level. The interation of SWCNT and nanocomplexes in lipid monolayers was studies using Brewster angle microscopy (BAM) in conjunction with absorption kinetics and surface pressure. The results confirmed the interaction between nanomaterials and the membrane, indicating that the presence of nanomaterials affects the packing of the lipids. Cytotoxicity studies were also employed to investigate the interaction of nanomaterials in in vitro cell systems. The results of flow cytometry, cell proliferation, morphology and inhibition of adhesion revealed the toxicological aspects of the materials, demonstrating a higher toxicity to the nanocomplex, compared to SWCNT, differently of the individual components. The toxicity of SWCNT nanocomplex and its individual components can be related to the type of material and how these materials are available in the culture medium. The studies in this thesis show the versatility of self-assembly thin films on biomimetic systems and may be relevant to the advance of research on the interaction of nanomaterials and biosystems.

Using iron super-paramagnetic and ferromagnetic nanoparticles composed of Fe3O4 molecules, scientists analyze the effectiveness and practicality of this new treatment theory, hyperthermia. The problems of magnetic particle density, isothermal barriers/cellular cooling thresholds, and nanoparticle specific targeting are addressed in this review. Iron magnetic nanoparticles were chosen due to their relatively low biological reactivates and lack of subsequent cellular toxicity. However, there are significant heating problems associated with these magnetic nanoparticles due to their relative size and short thermal time constants or thermal half-lives. Effectively, these aforementioned issues create a phenomenon where cancerous cells, surrounded by unheated healthy tissue, exhibit properties similar to those of an isothermal barrier. As a result, target cells experience limited gross heating, which is localized to the area directly surrounding the active magnetic nanoparticle within the cytoplasm. The effects of isothermal barriers and HSP up regulation on particle-based hypothermia are profound and prevent therapeutic temperatures from being achieved in single cell heating limiting the applications for Fe3O4 magnetic nanoparticle hyperthermia applications. It has been shown that reaching a certain magnetic nanoparticle density within the cell can result in a larger heating capacity, though this effect is also dependent on the particle dispersion pattern within cytoplasm. It has yet to be concluded whether ferromagnetic particles or super-paramagnetic particles are superior or more practical for hyperthermic treatments as they each have distinct benefits, and further study is needed. Finally, the popular targeting mechanism associated with magnetic nanoparticle research, monoclonal antibodies, require that they have an organic coating (such as starch) as a means of both providing an organic binding point and as camouflage for avoiding host filtration pathways. Forgoing this organic coating could lead to increased particle density within the cell and the adoption of a more specific targeting mechanism such as virus like particles (VLPs) altered to target HSP’s could lead to an increase in yield. Furthermore the up regulation of HSPs in response to therapeutic temperature is problematic for the therapies practically.

Considérée comme l’avenir de la pratique médicale, la nanomédecine est l’application des nanotechnologies aux soins de santé. Plus qu’un nouveau domaine d’application technologique, la nanomédecine est porteuse d’un nouveau paradigme biomédical qui promeut une conception technoscientifique de la santé. Ce nouveau paradigme regroupe sous le préfixe nano l’ensemble des grandes tendances actuelles de la recherche en santé : la médecine prédictive, la médecine personnalisée et la médecine régénératrice. Centré sur le développement d’innovations visant au contrôle technique des éléments et des processus biologiques fondamentaux, ce nouveau paradigme se développe largement grâce au soutien des gouvernements et aux promesses économiques qu’il soulève. Il se construit à la croisée du scientifique, du politique et de l’économique. Interroger la nanomédecine revient alors à examiner plus largement la forme et les conditions du sens des innovations biomédicales et à soulever de la sorte les implications de la « technoscientifisation » des soins de santé.L’objectif de cette thèse est ainsi de rendre compte de la spécificité et des enjeux sociaux, culturels et politico-économiques caractéristiques du modèle biomédical technoscientifique porté par la nanomédecine à partir de sa conceptualisation sous la forme d’un idéaltype : la nanosanté. Si la nanomédecine renvoie de manière générale aux applications techniques de la nanotechnologie au domaine biomédical, la nanosanté renvoie aux diverses dimensions sociologiques constitutives de ces technologies et à leurs effets sur la santé et la société. Notre modèle de la nanosanté s’organise autour de trois dimensions : la transversalité, l’amélioration et la globalisation. Compte tenu de sa nature synthétique, ce modèle tridimensionnel permet d’aborder de front plusieurs questionnements cruciaux soulevés par le développement de la nanomédecine. Il permet d’éclairer le rapport contemporain à la santé et ses implications sur l’identité ; de mettre en lumière la centralité des technosciences dans la conception du progrès médical et social ; de mieux saisir les nouvelles formes globales de pouvoir sur la vie et les nouvelles formes d’inégalité et d’exploitation caractéristiques d’une société qui accorde une valeur grandissante à l’adaptabilité technique de l’humain et à l’économisation de la santé et du corps ; mais aussi de mieux comprendre le sens et les répercussions de l’engagement scientifique, politique et économique dans les innovations moléculaires et cellulaires. / Nanomedicine – the application of nanotechnology to medicine – is seen as the medicine of the future. Thus, nanomedicine is not just a new biomedical field. It carries a new biomedical paradigm promoting a technoscientific conception of healthcare. This new paradigm grows from and brings together the three current tendencies of healthcare research: predictive medicine, personalized medicine and regenerative medicine. Its focus is on the technical control of the molecular mechanisms underlying the biological development of the body. The growing of this new biomedical paradigm is largely the result of government supports and economic potentials. It is both a scientific and a politico-economic construction. In that sense, analysing nanomedicine means analysing the form and the conditions of the current biomedical progress. In other words, nanomedicine helps us to grasp and understand the issues and implications of the ‘‘technoscientifization’’ of healthcare. This thesis aims to highlight the socio-cultural nature of the technoscientific model of healthcare promoted by the nanomedicine. To do so, I propose the construction of an ideal-type of this technoscientific model, which I call nanohealth. If nanomedicine refers to the different technological applications of nanotechnology to medicine, nanohealth refers to the different sociological dimensions and impacts of these applications on health and society. The nanohealth ideal-type is constructed around three dimensions: transversality, enhancement and globalization. The synthetic nature of this tridimensional ideal-type helps us to tackle the crucial issues surrounding the development of the nanomedicine. It helps us to understand the meaning and impacts of the scientific, political and economic engagement in nanomedicine; to highlight the centrality of technoscience in the cultural conception of medical and social progress; to grasp the new forms of power upon life and identity, and the new forms of inequality and exploitation, which are characteristics of a society focusing on the technical adaptability of human being and the economization of health and body.

Pour pallier le problème d’efficacité de la plupart des médicaments disponibles sur le marché aujourd’hui, lié à des manques de spécificité et de solubilité, notamment dans le cadre du traitement du cancer, la nanomédecine, via les nanoparticules présente une alternative de grande importance. Dans ce domaine, les nanoparticules de silice ont récemment attiré une énorme attention de la part des scientifiques. Cependant, des problèmes d’élimination liés à la solidité du matériau entravent aujourd’hui sa traduction clinique. Afin d’élucider cette problématique, nous présentons, dans cette thèse, l’utilisation de nanoparticules de silice hybrides dont l’une est mésoporeuse et l’autre sous forme de nanocapsule dépourvue de porosité. Les particules qui sont sphériques ont été préparées en incorporant un groupement imine dans leur charpente afin de les rendre sensibles au pH bas, sachant que les tissus cancéreux présentent une certaine acidité par comparaison aux tissus sains. Les matériaux préparés se montrent particulièrement sensibles aux milieux acides similaires aux conditions dans les milieux cancéreux. Dans le même temps, ces particules exposent une bonne stabilité en milieu à pH neutre similaire aux conditions physiologiques. Des études in vitro réalisées avec la particule mésoporeuse sur une lignée de cellule cancéreuse issue du sein humain démontrent une bonne et rapide internalisation. De plus, lorsque le matériau est chargé avec un médicament hydrophobe très puissant utilisé dans le traitement du cancer du sein, le système en résultant indique une efficacité de grande ampleur en tuant une forte majorité des cellules cancéreuses, contrairement au système basé sur la particule non cassable et au médicament isolé. Parallèlement, les nanocapsules chargées avec un autre agent anticancéreux se montrent particulièrement cytotoxiques vis-à-vis de cellules cancéreuses très communes et qui l’internalisent de manière très rapide. / To overcome the limitations of most of the drugs avaible nowadays on the market due to their lack of solubility and specifity in cancer treatment for instance, nanomedicine plays an emerging role as an alternative. In that field, nanoparticles are endowed with several advantages, leading them to be highly considered for drug delivery systems preparation. In this respect, silica nanoparticles have recently a great deal of attention from the scientists. Nevertheless, some issues related to the in vivo elimination of silica materials represent the main obstacle impeding their clinical translation. To elucidate this problematic, we report, in this thesis, the use of breakable hybrid organosilica nanoparticles where one is mesoporous and the other one consists in a nanocapsule without porosity. Such materials have been prepared by incorporating an imine-based linker in the particles framework in order to make them pH-responsive. The advantage of the pH sensitivity relies on the fact that cancerous media present certain acidity as compared to those healthy. The particles exhibit a high pH sensitivity where, at low pH, they fully break down, while a good stability is observed in physiological conditions. Furthermore, in vitro studies performed with a drug delivery system based on the mesoporous particle and a highly hydrophobic drug show a remarkable efficiency towards a cancer cell line from human breast, which moreover, rapidly internalises the material. The nanocapsule loaded with a hydrophilic drug also demonstrates a fast internalisation towards a commonly used cancer line which does not resist to the system and thus dies by a very high rate.