O Câncer nas últimas décadas tem se tornado um evidente problema de saúde pública mundial. Os números de novos casos que surgem a cada ano e as altas taxas de mortalidade levam os pesquisadores a procurar formas de conter o avanço dessa doença. A principal forma de tratamento e que possui a maior incidência de cura é o uso de quimioterápicos, que são substâncias químicas utilizadas isoladas ou em combinação, com o objetivo de tratar as neoplasias malignas. Entretanto eles atuam sem especificidade, não destruindo seletivamente e exclusivamente as células tumorais o que causa graves efeitos colaterais aos pacientes. Com o intuito de aumentar a seletividade do tratamento, diminuir a toxicidade e aumentar o poder de cura o presente trabalho utiliza duas abordagens para o combate do câncer, a nanotecnologia (uso de sistemas de liberação de fármacos) e a hipertermia. Foram preparadas, caracterizadas e avaliadas quanto à atividade antitumoral in vitro nanocápsulas contendo o agente quimioterápico selol (composto semi-sintético provindo do óleo de girassol e que possui selênio na sua estrutura) e fluído magnético iônico (composto por nanopartículas magnéticas de maghemita, ?-Fe2O3. Ao total foram preparadas quatro diferentes formulações pelo método de nanoprecipitação descrito por Fessi com algumas modificações. As nanocápsulas apresentaram um tamanho de partícula máximo de 230,5 nm (± 4,5), com índice de polidispersividade < 0,267 (± 0,05) e potencial zeta que variou de -54,4 mV (± 3,4) a -28,6 mV (± 4,3). Foram realizadas análises da morfologia das nanocápsulas através de microscopia eletrônica de transmissão que confirmaram o tamanho nanométrico do sistema preparado. Todas as formulações demonstraram ser estáveis durante o tempo 3 meses quando armazenadas a temperatura de 4oC. Nos estudos celulares foram utilizadas as linhagens B16- F10 (melanoma murino) e OSCC (carcinoma epidermóide de boca humano), sendo que as mesmas mostraram diferentes comportamentos quando incubadas com as formulações em diferentes concentrações. Na linhagem B16-F10 foi observado um maior efeito de morte causado pelo selol (a viabilidade celular chegou a 52,5 % ± 8,4), já quando o campo magnético foi utilizado não foi possível observar um aumento da morte celular. No estudos com a linhagem OSCC, a mesma demonstrou resistência quando foi tratada com selol e na ausência de campo magnético, já quando o campo magnético foi utilizado a viabilidade celular chegou a 33,3% (± 0,3), indicando um forte efeito hipertêmico nesta linhagem. Mais estudos devem ser realizados para entendermos o efeito do sistema preparado perante diferentes linhagens celulares, no entanto podemos confirmar o sucesso no preparo do mesmo e a capacidade de causar morte de diferentes células neoplásicas, o que indica uma importante arma para atuar futuramente no combate do câncer. / In the latest decades, cancer has become a clear public health problem worldwide. The neoplastic diseases increase each year and high mortality rates lead researchers to develop new approaches able to contain the progress of this disease. The main treatment type which has the highest incidence of cure is based on chemotherapeutic agents used alone or in combination. However, they act without specificity and selectively destroying both tumor and normal cells causing serious side effects to patients. In order to enhance the selectivity of the treatment decreasing toxicity and increase the healing power, the present study employs two approaches to treat the cancer, nanotechnology (the use of drug delivery systems) and hyperthermia (magnetic fluid). Nanocapsules containing the chemotherapeutic agent selol (semi-synthetic compound coming from sunflower oil and that has selenium in its structure) and maghemite magnetic nanoparticles (?-Fe2O3) were prepared, characterized, and evaluated in respect with their in vitro antitumor activity. Four different formulations were prepared by the nanoprecipitation method described by Fessi et al. with some modifications. The nanocapsules presented a particle size up to 230.5 nm (±4.5) with polydispersity index of 0.267 (±0.05), and zeta potential ranged from - 54.4 mV (±3.4) to - 28.6 mV (±4.3). The transmission electron microscopy analysis of nanocapsules confirmed the nanometric size system prepared. All formulations proved to be stable during 3 months as stored at 4°C. The cell lines studied were B16-F10 (murine melanoma) and OSCC (oral squamous cell carcinoma). These cell lines showed different behavior after incubation at different formulation concentrations. For cytotoxicty study on B16-F10 cells, it was observed a strong effect caused by Selol (cell viability reached 52.5% ±8.4). On the other hand, there was no cytotoxic effect on B16-F10 cells (p > 0.05) under magnetic field application. OSCC cell line showed a resistance to treatment with selol and in the absence of AC magnetic field. However, after magnetic field activation the cell viability reached 33.3% (±0.3) indicating a strong hyperthermic effect on OSCC cells. Therefore, it has been confirmed nanocapsules containing selol and magnetic fluid are able to destroy B16-F10 or OSCC neoplastic cells indicating an important weapon for future work in the treatment against cancer.

As células dendríticas (DCs) são células apresentadoras de antígeno suscetíveis a muitos sinais de ativação, os quais induzem diferentes padrões de ativação e resposta de linfócitos T. Neste trabalho, estudamos os efeitos de dois sinais de perigo, a febre e o LPS, sobre o fenótipo e função de DCs. A exposição de DCs ao calor não afetou a expressão de CD80 e CD86, capacidade endocítica ou produção de citocinas, características que foram afetadas pelo LPS. Entretanto, DCs expostas ao calor apresentaram uma maior atividade aloestimuladora e maior expressão de CD40. Quando DCs ativadas com LPS foram também estimuladas pelo calor, nenhuma alteração fenotípica na superfície celular foi notada, mas as DCs induziram maior produção de IFN-<font face=\"symbol\">g por linfócitos T alogenêicos e favoreceram a proliferação de linfócitos T CD8+. Esses dados indicam que a febre pode favorecer uma resposta celular, através de sua ação sobre DCs ativadas, com provável participação do CD40. Além de seu significado fisiológico, esse fenômeno pode ter aplicação em estratégias imunoterapêuticas. / Dendritic cells (DCs) are the main antigen presenting cells and susceptible to many activation signals that will induce different patterns of DC activation and of T cell responses. In this work we studied the effects of two danger signals, fever and LPS, on DC phenotype and function. Exposure of immature monocyte-derived dendritic cells to heat did not affect CD80 and CD86 expression, their endocytic ability, or their cytokine production, characteristics that were affected by LPS. However, heat-exposed DCs presented a higher allo-stimulatory activity and enhanced CD40 expression. When LPS activated DCs were also stimulated by heat, no cell surface phenotypic change was noted but these cells induced a higher IFN-<font face=\"symbol\">g secretion by allogeneic T lymphocytes and favored the proliferation of CD8+ cells. These data indicate that fever may cause a bias toward cellular responses, through its action on activated DCs, probably through CD40. Besides its physiological meaning, this phenomenon may have applications in immunotherapeutic strategies.

Orientadores: Flávio Garcia, Kleber Roberto Pirota / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin / Made available in DSpace on 2018-08-24T22:02:36Z (GMT). No. of bitstreams: 1 Dias_CarlosSatoBaraldi_D.pdf: 19717682 bytes, checksum: 212154446eec9e3ee26eee15692adfec (MD5) Previous issue date: 2014 / Resumo: Durante este trabalho, estudamos a viabilidade de uma nova classe de partículas magnéticas otimizadas para a hipertermia magnética e que denominamos VIP (Vortex Iron oxide Particle). Estas partículas são compostas de óxidos de ferro (magnetita ou maghemita) e assumem a forma de nanoanéis ou nanotubos. Tal morfologia confere as partículas um comportamento magnético peculiar, fruto de seu estado magnético denominado vórtice. Graças a este estado magnético, estas partículas possuem uma resposta para hipertermia superior, se comparadas aos SPIONs (Superparamagnetic Iron Oxide Nanoparticles) tradicionalmente usados para este fim, atendendo os pré-requisitos para aplicações biomédicas (baixas toxicidade e remanência). Os experimentos realizados no trabalho se focaram em duas linhas principais. Na primeira, avaliamos o desempenho das VIPs para hipertermia magnética em um ambiente in vitro. Neste experimento, avaliamos tanto a citotoxicidade das partículas quanto os mecanismos que promovem a morte celular. Estes resultados mostraram que as VIPs não são citotóxicas. Além disso, observamos também que o tamanho e a forma das partículas permitem que estas sejam internalizadas, promovendo um processo de hipertermia magnética muito eficiente. De fato, o experimento de hipertermia in vitro mostrou que as VIPs são capazes de alcançar um alto grau de seletividade, matando principalmente as células que internalizam as VIPs minimizando o aquecimento do meio celular e reduzindo assim o dano às células vizinhas. Na segunda linha, trabalhamos no desenvolvimento de uma VIP recoberta com material antiferromagnético (AFM), cujo desempenho para hipertermia magnética seria superior ao da própria VIP testada nos experimentos in vitro. Desenvolvemos um extenso estudo teórico fundamental, baseado em simulações micromagnéticas, que permitiram prever muitos dos fenômenos que seriam observados experimentalmente. No entanto os experimentos para a síntese da VIP@AFM não foram completamente finalizados, impossibilitando maiores conclusões sobre o sistema simulado. Sendo assim, acreditamos que os resultados alcançados no projeto promoveram o desenvolvimento de partículas magnéticas otimizadas para hipertermia magnética. Tanto por mostrar sua viabilidade, quando testados in vitro, quanto por promover o conceito da VIP@AFM como um próximo passo para o aprimoramento desta classe de partículas / Abstract: In this work, we studied the feasibility of a new class of magnetic particles named as Vortex Iron oxide Particle or VIP and specifically designed for magnetic hyperthermia. Those iron oxide (magnetite or magnetite) particles have a ring shape morphology that grants them a very specific magnetic configuration know as vortex state. This magnetic configuration would grant a superior hyperthermia response when compared to the traditional Superparamagnetic Iron Oxide Particle (SPION) without compromising biomedical requirements as low cytotoxicity and lack of magnetic remanence. The experiments presented on this work explored two main paths. The first one evaluated the VIP performance in vitro. We were able to assess both the cytotoxicity of the particle as to observe the killing mechanism. The results showed that the VIPs are not cytotoxic and that the size and shape of the particle may promote the internalization of those particles, resulting on a very efficient magnetic hyperthermia. With the in vivo experiment, we notice a high degree of selectivity, thanks to the cell internalization, that preserved the surrounding cells. In the second research path, we further developed the concept of a VIP, by creating a core-shell structure where the VIP would be coated by an antiferromagnetic (AFM) material and improving the magnetic hyperthermia response. However, the experiments for the development of the VIP@AFM were not completed. Although we were able to complete a study based on micromagnetic simulations, the experiments intending to fabricate the VIP@AFM were not fully successful, preventing any further conclusion about this system. In any case, we believe that the results achieved on this project represent an important contribution to the development of magnetic hyperthermia specific particles / Doutorado / Física / Doutor em Ciências

Cette thèse présente la conception de nano-objets magnétiques dendronisés permettant à la fois le diagnostic par imagerie par IRM et la thérapie par hyperthermie magnétique (HM). La validation in vitro et in vivo des propriétés de ces nano-objets et la démonstration de leur efficacité pour le ciblage spécifique de tumeurs sont rapportées. Un état de l’art sur la synthèse des NPs d’oxydes métalliques, leur fonctionnalisation ainsi que leurs propriétés en IRM et en HM est présenté. L’influence de la méthode de fonctionnalisation de NPs d’oxyde de fer puis l’influence de l’architecture de la molécule greffée sur la stabilité colloïdale et la relaxivité ont été étudiées. Des NPs de différentes tailles, morphologies et composition ont ensuite été synthétisées et fonctionnalisées par une molécule dendron et leurs les propriétés en IRM et en HM ont été étudiées. Le ciblage spécifique de la mélanine au sein de mélanome a été montré. / This thesis presents the design of dendronized magnetic nano- objects for both diagnostic imaging by MRI and magnetic hyperthermia therapy (HM). In vitro and in vivo validation of these nano-objects properties and the demonstrattion of their effectiveness for specific targeting of tumors are reported. A state of the art on the synthesis of metal oxide NPs, their functionalization and their properties in MRI and HM is presented. The influence of the NPs iron oxide functionalization method and the influence of the molecule architecture on the colloidal stability and relaxivity were studied. NPs of different sizes, morphologies and composition were then synthesized and functionalized with a dendron molecule and the MRI and HM properties were investigated. The specific targeting of melanin in melanoma was demonstrated.

L'hyperthermie magnétique est une technique de traitement de cancers en plein essor. Cette conversion d'une énergie électromagnétique en énergie thermique par des nanoparticules soumises à une excitation magnétique oscillant à haute fréquence est étudiée depuis près de deux décennies mais elle est encore mal décrite pour des systèmes complexes.Le travail présenté ici s'attache à compléter la connaissance de ce phénomène en reliant l'état de dispersion de nanoparticules à leurs propriétés d'hyperthermie.Tout d'abord sont présentés la caractérisation et le contrôle de dispersions dans des milieux complexes tels qu'un liquide ionique ou une matrice polymère.En effet, l'obtention d'une dispersion colloïdalement stable dans un liquide ionique soulève de nombreux défis. Une étude approfondie du rôle de la charge de surface des particules, contrôlée à l'aide du pH dans un liquide ionique protique, a permis de mieux comprendre le rôle du solvant et la nature des interactions dans ce type de milieu.En outre, disperser finement des nanoparticules hydrophiles dans une matrice hydrophobe telle qu'une matrice siliconée est également une tâche délicate et une méthode nouvelle d'évaporation d'émulsion ferrofluide-dans-matrice polymère est présentée ici, ainsi que l'étude des dispersions ainsi obtenues.Enfin est exposée une étude du pouvoir chauffant de nanoparticules dans des états de dispersion variés, faisant notamment intervenir une méthode de mesure développée dans le cadre de cette thèse. Ces résultats soulignent l'influence de l'agrégation des particules qui provoque une baisse significative de leur échauffement ainsi que le rôle clef de la compacité des agrégats formés. / Magnetic hyperthermia is a promising therapeutic technique against cancer. It consists in turning electromagnetic energy into heat thanks to nanoparticles that are excited by a radiofrequency oscillating magnetic field. Although this phenomenon has been studied for more than two decades, it remains poorly described. This work aims at filling the gap of knowledge about magnetic hyperthermia through the study of the correlations between the dispersion state and the heating efficiency of the particles. The characterisation and the control of dispersions in complex media such as ionic liquids or a polymer matrix is dealt with in the first part. Obtaining a colloidally stable dispersion in an ionic liquid proves indeed a challenging task. The particles surface charge can be controlled in a protic ionic liquid by tuning the pH. A thorough study of the influence of this parameter allowed a better insight into the role of the solvent and the nature of the interactions. Furthermore, the formation of a fine dispersion of hydrophilic nanoparticles in a hydrophobic silicon matrix is also a ticklish task. A new method is presented here, that consists in the evaporation of a ferrofluid-in-polymer matrix emulsion. The dispersion state of the resulting materials is then characterised.The heating efficiency of particles showing different dispersion states is studied in the last part. It includes results obtained with a new measurement method developed in the scope of this thesis. These results highlight that the particles aggregation causes a decrease of their heating properties. They also evidence the key role of the aggregates compactness in this respect.

Introduction. L’efficacité de la chimiothérapie intracavitaire dépend de la pénétration du produit au sein du péritoine (CHIP) ou de la plèvre. L’hyperthermie et l’hyperpression peuvent augmenter cette pénétration. Ce travail étudie leur effet intrapéritonéal, puis établit la méthode optimale pour les délivrer. L’étude de la faisabilité et de la tolérance d’une hyperpression intrapleurale est essentielle pour transposer ces bénéfices à la cavité thoracique. Méthodes. Quatre groupes de porcs ont reçu une CHIP ouverte avec de l’oxaliplatine à une concentration constante (150 mg/l) pendant 30 minutes en normothermie ou hyperthermie (42-43°C) ; et en pression atmosphérique ou hyperpression (25 cmH2O). Deux groupes ont reçu une procédure fermée en hyperthermie et hyperpression ou forte hyperpression (40 cmH2O). L’absorption systémique et tissulaire d’oxaliplatine a été étudiée. La tolérance d’une perfusion pleurale a été étudiée chez 21 porcs avec ou sans résection associée, avec ou sans chimiothérapie (cisplatine + gemcitabine), à divers niveaux de pression de 15 à 25 cmH2O. Résultats. L’hyperthermie augmente les concentrations de platine dans les surfaces viscérales (p=0.0014), alors que l’hyperpression l’augmente dans les surfaces viscérales et pariétales (respectivement p= 0.0058 et p= 0.0044). L’association des deux facteurs permet d’obtenir les concentrations les plus importantes dans le péritoine viscéral (p= 0.00001) et pariétal (p= 0.0003). Les concentrations obtenues lors des procédures fermées sont inférieures à celles obtenues en ouvert, même lorsque la pression atteint 40 cmH2O. Une chimiothérapie intrapleurale à 20 cmH2O sans résection associée est le niveau maximal toléré durant 60 minutes. Conclusion. Au cours d’une CHIP, l’hyperthermie augmente la pénétration d’oxaliplatine dans le péritoine viscéral, alors que l’hyperpression est efficace dans le péritoine viscéral et pariétal. Leur association est synergique et la procédure ouverte semble la meilleure pour la délivrer. Une chimiothérapie intrapleurale est faisable à 20 cmH2O dans ce modèle. / Introduction. In order to achieve a good effect, chemotherapy drugs need to penetrate into the peritoneal (HIPEC) or pleural tissue. Hyperthermia and high-pressure may enhance this penetration. The aim of this study was to evaluate their peritoneal effect and to establish the best technique to it. A feasibility study of an intrapleural high-pressure was an essential step to export these effects to the thoracic space. Methods. Four groups of pigs underwent an open HIPEC with a constant concentration (150 mg/l) of oxaliplatin during 30 minutes either in normothermia, or in hyperthermia (42-43°C); and either with atmospheric pressure or with high-pressure (25 cmH2O). Two more groups underwent a closed procedure with hyperthermia and either high-pressure or very high-pressure (40 cmH2O). The systemic and tissue absorption of oxaliplatin were studied. The haemodynamic and respiratory tolerance of a pleural infusion was also tested in 21 pigs with and without associated resection; with and without chemotherapy infusion (cisplatin + gemcitabin) and at various levels of pressure (from 15 to 25 cmH2O). Results. Hyperthermia enhances the concentrations of platinum in visceral surfaces (p=0.0014), whereas high-pressure enhances it both in visceral and in parietal surfaces (p= 0.0058 and p= 0.0044, respectively). Their association obtains the highest concentrations both in the visceral (p= 0.00001) and the parietal peritoneum (p= 0.0003). The concentrations obtained during closed procedure are lower than those achieved with the open technique, even with 40 cmH2O of pressure. A 60-minutes intrapleural chemotherapy perfusion with 20 cmH2O of pressure without any lung resection was the maximal tolerated level. Conclusion. During HIPEC, hyperthermia improves the penetration of oxaliplatin in the visceral peritoneum, whereas high-pressure is effective in both peritoneal surfaces. Their association is synergic and the open technique seems to be the best one to deliver it. An intrapleural chemotherapy with a 20 cmH2O pressure is feasible in this model.

TRAIL (TNF-related apoptosis inducing ligand) est une protéine du système immunitaire appartenant à la famille du TNF (Tumor necrosis factor). L'intérêt de TRAIL en thérapie anti-cancéreuse réside dans sa capacité à induire la mort par apoptose des cellules tumorales, sans exercer de toxicité envers les cellules saines. Le principal frein à l’utilisation de TRAIL est la survenue courante de résistances dans les tumeurs, limitant ainsi son efficacité. Mon travail de thèse a consisté à étudier l’intérêt de l’hyperthermie (ou choc thermique) en tant qu’adjuvant à TRAIL et a décrire ses capacités à contourner les mécanismes de résistance.Dans un premier temps, l’activité et la portée de cette combinaison a été évaluée dans de nombreuses et diverses lignées cellulaires cancéreuses sensibles ou résistantes à TRAIL. Un choc thermique (1h 42°C) permet de sensibiliser efficacement les lignées devenues résistantes à TRAIL et cette association s’est avérée efficace dans toutes les lignées testées.Dans un deuxième temps, mon travail s’est focalisé sur les mécanismes induits par l’hyperthermie, responsables de la sensibilisation et de l’apoptose des cellules. Les analyses des complexes initiateurs de la mort (DISC - Death-inducing signaling complex) ont révélé de nombreuses disparités suivant les conditions thermiques. Les différences majeures impliquent avant tout l’absence de FLIP dans le DISC, celui-ci étant l’inhibiteur principal de la voie, ainsi qu’un retard de la formation du complexe en condition d’hyperthermie. Ceci est associé à l’activation des caspases initiatrices d’une meilleure qualité, une fois la température revenue à la normale.L’absence de FLIP est expliquée par son inactivation due à l’agrégation de cette protéine suite à l’augmentation de la température. Ce phénomène est indépendant d’une quelconque modification post-traductionnelle connue, mais peut être inhibé par la présence de glycérol qui stabilise les protéines dénaturées.L’ensemble de ce travail met en lumière l’intérêt de la combinaison de TRAIL avec une hyperthermie et présente un point de vue nouveau sur les mécanismes expliquant son efficacité. / The TNF-family member TRAIL (TNF-related apoptosis inducing ligand) is a cytokine involved in the immune anti-tumour surveillance. TRAIL is a promising agent currently under investigation for its anti-cancer properties with limited side effects on healthy cells. However, the use of TRAIL in oncology has been limited due to its lack of efficiency, mainly associated with cell resistance to apoptosis. The aim of this project was to study the interest of hyperthermia (or heat shock - HS) as an adjuvant for TRAIL therapy and the mechanisms involved in this sensitization.We have first evaluated the significance of this combination in a large variety of cancer cell lines known to be sensitive or resistant to TRAIL. We could demonstrate that hyperthermia was able to efficiently sensitize resistant cancer cells to TRAIl-induced apoptosis in almost every cell lines tested.We next, focused our work on the molecular mechanisms responsible for the sensitization, during hyperthermia. Analyses of the DISC (Death-Inducing Signaling Complex) revealed a lack of recruitment of FLIP in the DISC, the main inhibitor of the extrinsic pathway, and a delay in the formation of the complex under hyperthermic conditions. Inhibition of FLIP recruitment was associated with enhanced initiator caspases activation when cells were reincubated at 37°C after the HS.The absence of FLIP within the TRAIL DISC was due to its aggregation during HS and was independent of post-translational modifications. Inhibition of FLIP aggregation by glycerol, which stabilizes denaturerated proteins, restored FLIP recruitment within the TRAIL DISC and consequently inhibited TRAIL-induced cell death. Taken together, these results highlight the interest of combining TRAIL with hyperthermia and highlight new mechanisms explaining its efficiency.

Orientador: Marcelo Knobel / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin / Made available in DSpace on 2018-08-30T18:13:51Z (GMT). No. of bitstreams: 1 OrozcoHenao_JuanManuel_M.pdf: 7687296 bytes, checksum: d9aecf2d118aed0c43ab747d0dcff34c (MD5) Previous issue date: 2016 / Resumo: Nanopartículas de magnetita com diâmetros entre 5nm e 19nm sintetizadas mediante um método de decomposição térmica são apresentadas. A caracterização estrutural é feita usando diferentes técnicas experimentais como a microscopia eletrônica de transmissão (TEM), difração de raios-X e espalhamento de raios-X a baixos ângulos (SAXS) de onde são obtidos os tamanhos e a forma das nanopartículas. As propriedades magnéticas e de magneto-hipertermia das nanopartículas são estudadas para diferentes parâmetros de produção como concentração dos surfactantes, temperaturas de refluxo e atmostfera de crescimento. A dependência com a temperatura das propriedades magnéticas são analisadas dentro do marco do modelo usual do superparamagnetismo e o modelo de interação superparamagnética (ISP), de onde os parâmetros magnéticos dependentes do tamanho como anisotropia magnética (1.06x10^4 J/m^3 até 9.91x10^4 J/m^3), momento magnético por partícula (2618?B até 11500?B), temperatura de bloqueio (18K até mais de 300K) e energia de ineração dipolar magnética (0.55x10^-21 J até 5.5x10^-21 J) são inferidos. Os resultados de magneto-hipertermia foram obtidos mediante a medição da resposta térmica das nanopartículas de magnetita suspendidas em tolueno. Valores da taxa de absorção específica (SAR) são calculados experimental e teoricamente utilizando a teoria de resposta linear para um sistema superparamagnético não interagente. Valores de SAR entre 3.0W/g e 40.3W/g e a sua dependência com a frequência e o campo aplicado são apresentados. Como resultado interessante, a resposta de magneto-hipertermia para as nanopartículas de 19nm preparadas na presença de oxigênio e mais de 10 vezes maior do que nanopartículas similares mas obtidas na ausência da atmosfera de oxigênio. Também é destacada a possibilidade de prever a resposta de magneto-hipertermia num sistema de nanopartículas magnéticas mediante a obtenção dos parâmetros de caracterização magnetica e estrutural / Abstract: Magnetite nanoparticles with diameters between 5nm and 19nm synthesized by means of a thermal decomposition method are presented. Structural characterization is made by different experimental techniques such as transmission electron microscopy (TEM), X-ray diffraction and Small Angle X-ray Scattering (SAXS) from where nanoparticles size and shape are obtained. Magnetic and magneto-hyperthermia properties of the nanoparticles are studied for different production parameters, such as surfactant concentrations, refluxing temperature and growth atmosphere. Temperature dependence of the magnetic properties are analyzed in the framework of the standard superparamagnetism model and the interacting superparamagnetic model (ISP), from where size dependent magnetic parameters for each sample such as anisotropy (1.06×10^4 J/m^3 to 9.91x10^4 J/m^3) magnetic moment per particle (2618?B to 11500?B), blocking temperature (18K to above 300K) and magnetic dipolar interaction energy on dried nanoparticle samples (0.55 × 10^?21 J to 5.5 × 10^?21 J) are inferred. Magneto-hyperthermia results are obtained by measuring the thermal response of magnetite nanoparticles dissolved in toluene. Specific absorption rate (SAR) values are theoretically and experimentally calculated by means of a linear response theory approach of a non-interacting superparamagnetic system. SAR values between 5.8W/g and 40.3W/g are reported; interestingly, the magneto-hyperthermia response for 19nm nanoparticles prepared in presence of an oxygen atmosphere is more than 10 times larger than similar particles obtained in absence of oxygen atmosphere. Also it is important to highlight the possibility to obtain the magneto-hyperthermia behavior of a magnetic nanoparticles system by knowing a priori its structural and magnetic characterization parameters / Mestrado / Física / Mestre em Física / 1247647/2013 / CAPES

Estudos com tratamento hipertérmico de tumores utilizando nanopartículas metálicas têm sido realizados durante as últimas décadas e mostram resultados bons quanto à remissão de tumores, por vezes chegando à cura completa. O mesmo acontece em relação aos tratamentos baseados em ação fotodinâmica de fotossensibilizadores. Tratamentos aliando a terapia hipertérmica com nanopartículas de ouro e a terapia fotodinâmica com diversos fotossensibilizadores tem efeito sinérgico e apresenta excelente potencial terapêutico, em que pese serem necessários mais estudos para que uma nova terapia conjunta possa ser implementada. A proposta deste trabalho foi investigar esse efeito sinérgico utilizando nanobastões de ouro complexados com fotossensibilizadores. Após a síntese dos nanobastões pelo método de seeding, a eficácia do tratamento fotodinâmico e da terapia hipertérmica, separadamente, foi investigada. A metodologia do recobrimento dos nanobastões por fotossensibilizador, em um primeiro momento, não logrou êxito com a porfirina, porém com a ftalocianina tetracarboxilada se mostrou mais eficaz. A taxa de fotodegradação da ftalocianina em solução foi investigada como parâmetro para a eficiência em geração de oxigênio singlete. Após centrifugação e lavagem das nanopartículas, no entanto, evidenciou-se por espectrofotometria que o fotossensibilizador não permaneceu aderido aos nanobastões. Em um segundo momento, optamos por recobrir os nanobastões por porfirinas tetrassulfonadas, com ou sem grupamentos metil-glucamina. Após o processo de recobrimento, essas ftalocianinas formaram complexos iônicos com o CTAB que recobre os nanobastões. Os complexos nanobastões-ftalocianinas foram analisados por microscopia eletrônica de transmissão e as taxas de geração de oxigênio singlete e de radical hidroxil foram investigadas. Além disso, foram utilizadas para testes in vivo e in vitro com células de melanoma melanótico (B16F10) ou amelanótico (B16G4F). As células tumorais em cultura ou os tumores em camundongos C57BL6 foram irradiados com luz em 635 nm e os tumores foram observados por 15 dias após o tratamento. Houve evidente aumento na geração de oxigênio singlete por ambos fotossensibilizadores, e maior geração de radicais livres por parte do fotossensibilizador metilglucaminado. O oposto ocorre com o fotossensibilizador sem metilglucamina. Houve, também, moderada citotoxicidade no escuro quando células foram incubadas com nanopartículas recobertas por ftalocianinas ou não. Quando ativados pela luz, os complexos ftalocianinas-nanobastões desencadearam um aumento de 5ºC no meio de cultura das células, e a morte celular observada foi extensa (91% para a linhagem B16G4F e 95% para a linhagem B16F10). Tanto os resultados in vitro quanto os in vivo indicam que as propriedades das ftalocianinas testadas são melhoradas significativamente quando elas estão complexadas aos nanobastões. Este é um estudo pioneiro por utilizar duas porfirinas tetrassulfonadas específicas e por utilizar o mesmo comprimento de onda para a ativação dos fotossensibilizadores e nanobastões. / Studies with hyperthermic tumor ablation using metallic nanoparticles have been performed on the last decades, and show promising results on tumor remission, sometimes achieving the complete cancer elimination. The same occurs regarding on treatments based on photodynamic activity of photosensitizer compounds. Studies indicate that those therapeutic interventions - hyperthermic therapy using gold nanorods and photodynamic activity with many photosensitizers - together can present a synergistic effect, and offer a great therapeutic potential, although more investigation needs to be performed before such approach could be implemented. We proposed to investigate the effect of the attachment of photosensitizers onto the surface of gold nanorods (well-characterized devices for hyperthermia generation). After nanorods synthesis through a seed-mediated method, the PDT and hyperthermia\'s efficacy was assessed separately. The method used for covering the gold nanorods with photosensitizers did not permit, in a first approach, the attachment of porphyrins onto the nanoparticles surface, but the attachment of tetrasulfonated phthalocyanines was more successful. The phthalocyanine\'s degradation rate was assessed as an indirect parameter of singlet oxygen generation. After centrifuging and washing the nanoparticles, we saw that the photosensitizers do not keep attached to the nanorods. On a second approach, we chose to recover the nanorods with two zinc phthalocyanines - with or without methyl-glucamine groups. After the recovering process, the phthalocyanines formed ionic complexes with the CTAB that is previously recovering the nanoparticles. The nanorod-phthalocyanines complexes were analyzed by TEM, and their singlet oxygen and hydroxyl radical generation yield were assessed. Furthermore, they were tested in vitro in melanoticB16F10 and amelanotic B16G4F melanoma cells, and in vivo. The tumor cells (in vitro) and the tumor tissue (in vivo) with nanoparticles were irradiated with laser (at 635 nm), and the tumor growth in mice was observed for 15 days after the laser irradiation. It is evident the increase in the singlet oxygen generation, and higher HPF activation for the glucaminated Pc, but the inverse for the other photosensitizer. It seems like there is a type I to type II switch on the action mechanism of the latter Pc. A mild cytoxocity was observed with the nanorods conjugated with photosensitizer in the dark, but when they are activated by light (and taken into account a 5ºC rise in the temperature because of the surface plasmon resonance from the gold nanorods), the cell killing is intense (91% for B16G4F cell line, and 95% for B16F10 cell line). Both in vitro and in vivo results indicate that the photodynamic properties of the phthalocyanines tested are enhanced when they are attached onto the nanorods surface. This is a novel study because we used two tetrasulfonated phthalocyanines and because we used the same wavelength to activate both the nanorods and the photosensitizers.

Magnetic fluid hyperthermia is a potential therapy for achieving interstitial hyperthermia and is currently under clinical trials. This approach is based on the instillation of magnetic nanoparticles at the tumour site, which dissipate heat when exposed to an alternating magnetic field. This procedure leads to a local increase of temperature and induction of tumour death or regression. Nanoparticles of metallic iron are potential heating agents for this therapy, but rely on the presence of a protecting coat that avoids reactions with their environment. In this work, iron nanospheres and iron nanowires with a graphite coat are explored for this purpose. From these two nanostructures, the nanospheres are shown to have a greater potential in terms of heat dissipation. The graphite shell is further investigated as an interface for conjugation with other molecules of relevance such as drugs and fluorescent probes. The effect of acidic treatments on the magnetic and surface properties of the nanospheres is systematically studied and a suitable method to generate carboxylic functionalities on the nanoparticle surface alongside with a good preservation of the magnetic properties is developed. These carboxylic groups are shown to work as a bridge for conjugation with a model molecule, methylamine, as well as with a fluorescent dye, allowing the detection of the nanoparticles in cells by means of optical methods. The carboxylic functionalities are further explored for the conjugation with the anti-cancer drug cisplatin, where the amount of drug loaded per particle is found to be dependent on the density of free carboxylic groups. The release of the drug in physiological salt solutions is time and temperature dependent, making them particularly interesting for multi-modal anti-cancer therapies, where concomitant hyperthermia and chemotherapy could be achieved. Their potential for such therapies is shown in vitro by inducing hyperthermia in cell suspensions containing these nanoparticles. These results are finally translated to a three dimensional cell culture model where the in vitro growth of tumour spheroids is inhibited. The developed nanostructures have a great potential for therapeutic approaches based on the synergistic effects of hyperthermia and chemotherapy.