The thesis has been focused on testing of micromethod of DNA isolation using magnetic particles from thermic-managed food products in a quality suitable for polymerase chain reaction (PCR). Currant jams were selected for the analysis. These were homogenized using plastic copist and stomacher in lysis buffer with cetyltrimethylammonium bromide (CTAB). The effect of chloroform-octanol and isopropanol in the preparation of homogenates was tested. Homogenates were used for DNA isolation by magnetic particles. Rough fraction of DNA was purified by binding on the magnetic particles after centrifugation of the CTAB complexes with proteins, polyphenols and polysaccharides. Two types of magnetic particles were tested: microparticles of poly(hydroxyethylmethacrylate-co-glycidylmethacrylate) - P(HEMA-co-GMA) and nanoparticles of iron oxides covered by poly(L-lysine) - PLL. Isolated DNA was analyzed spectrophotometrically - it was assessed its concentration and contamination by polyphenols and proteins. After that, amplification of the DNA was tested in PCR. Primers specific for plant ribosomal DNA were used. PCR products of expected length 700 bp were detected by agarose gel electrophoresis. It was shown that DNA isolated from currant jams using magnetic particles was in PCR-ready quality.

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In this study, the magnetically-induced vibrations of functionalized magnetic particle suspensions were probed for the development of a novel optical spectroscopy technique. Through this work (1) the frequency-dependence of the faraday rotation in ferrofluids and (2) the extension of this system to elucidating particle size and conformation as an alternative immunossay to costly and labor/time intensive Western Blotting and ELISA has been shown. With its sensitivity and specificity, this method has proven to be a promising multi-functional tool in biosensing, diagnostic, and therapeutic nanomedicine efforts. Due to its ubiquitous nature in all optically-transparent materials, the farady rotation, or circular birefringence, was developed as a robust and sensitive nanoscale biomolecule characterization technique through Brownian relaxation studies of particle suspensions. Current efforts have shown the applicability of this phenomenon in solid, pure liquid, and colloidal samples as well as simultaneous advancements of magnetic nanoparticle research in the magnetometric and magneto-optical regimes. By merging these two fields, a clinically relevant spectroscopy (fd-FRS, Frequency Domain Faraday Rotation Spectroscopy) was developed based on a newly revised model stemming from Debye relazation theory. Through this work, an optical bench with a variable permeability core electromagnet and a frequency-domain lock-in amplifier setup (DC to 20 kHz) have been used to distinguish between Fe3O4-core nanoparticles with functionalization layers of PEG4/PEG8 polymer with future applications involving the Anti-BSA/BSA antibody/antigen couple. Particle concentrations down to 500 nM (magnetic nanoparticles) and 0.01 Volume % (magnetic beads) were studied with diameters ranging from 200 nm to 1μm. currently, the characteristic peak corresponding to the out-of-phase relazation of the suspended particles has been elusive, despite a wide particle size distribution and the use of a balanced photodetector. Future work will involved highly monodisperse samples, faster scan times, and thermal characterization applications of fs-FRS.

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[pt] As nanopartículas magnéticas (NPMs) apresentam grande potencial em diversas aplicações tecnológicas e vem ganhando destaque na área da biomedicina devido suas propriedades superparamagnéticas. Para este trabalho foram sintetizadas nanopartículas de óxido de ferro (Fe3O4) pelo método de coprecipitação e recobertas com Pluronic F-127 (PL F-127), o qual se demonstrou, a partir de estudos anteriores, um surfactante com ótima estabilidade coloidal e alto grau de biocompatibilidade, sendo tais características relevantes para as aplicações na área da biomedicina. O método de produção se mostrou eficiente para produção de uma grande quantidade de amostra e baixo grau de oxidação, o que mantém a integridade dos resultados e do material para diversas análises no decorrer de longos períodos. A espectroscopia Raman e a difração de elétrons apontam para a composição majoritária de magnetita cristalina das amostras. As imagens obtidas através de microscopia de transmissão (MET) mostraram que o diâmetro médio das NPMs não é afetado pela concentração PL F-127 e está de acordo com os tamanhos obtidos pelas técnicas magnéticas. O MET também mostrou partículas monodispersas com formato esféricas. As técnicas de microscopia magnética de varredura (MMV), magnetômetro de amostra vibrante e de efeito Hall revelaram que o comportamento das NPMs é superparamagnético em temperatura ambiente e que a funcionalização não interferiu significativamente na magnetização de saturação. / [en] Magnetic nanoparticles (MNPs) have great potential in several technological applications and are gaining prominence in the biomedical area due to their superparamagnetic properties. For this work, iron oxide (Fe3O4) nanoparticles were synthesized by the coprecipitation method and coated with Pluronic F-127 (PL F127), which was demonstrated, from previus studies, a surfactante with excellent coloidal stability and high degree of biocompatibility, such characteristics being relevant for applications in the área of biomedicine. The production method proved to be efficient for producing a large amount of sample and a low degree of oxidation, which maintains the integrity of the results and material for several analyzes over long periods. Raman spectroscopy and eléctron diffraction indicate the samples are pure and crystalline magnetite. The images obtained through transmission eléctron microscopy (TEM) showed that the mean diameter of MNPs is not affected by the PL F-127 concentration and is in agreement with the sizes obtained by magnetic techniques. TEM also showed monodisperse particles with a spherical shape. Scanning magnetic microscopy (MMV), vibrating sample magnetometer and Hall effect techniques revealed that the behavior of NPMs is superparamagnetic at ambient temperature and that the functionalization did not significantly interfere in the saturation magnetization.

Doctor of Philosophy / Department of Anatomy and Physiology / Deryl L. Troyer / Many cell types were known to have migratory properties towards tumors and different research groups have shown reliable results regarding cells as delivery vehicles of therapeutics for targeted cancer treatment. Present report discusses proof of concept for 1. Cell mediated delivery of Magnetic nanoparticles (MNPs) and targeted Magnetic hyperthermia (MHT) as a cancer treatment by using in vivo mouse cancer models, 2. Cells surface engineering with chimeric proteins for targeted cancer treatment by using in vitro models. 1. Tumor homing cells can carry MNPs specifically to the tumor site and tumor burden will decrease after alternating magnetic field (AMF) exposure. To test this hypothesis, first we loaded Fe/Fe3O4 bi-magnetic NPs into neural progenitor cells (NPCs), which were previously shown to migrate towards melanoma tumors. We observed that NPCs loaded with MNPs travel to subcutaneous melanoma tumors. After alternating magnetic field (AMF) exposure, the targeted delivery of MNPs by the NPCs resulted in a mild decrease in tumor size (Chapter-2). Monocytes/macrophages (Mo/Ma) are known to infiltrate tumor sites, and also have phagocytic activity which can increase their uptake of MNPs. To test Mo/Ma-mediated MHT we transplanted Mo/Ma loaded with MNPs into a mouse model of pancreatic peritoneal carcinomatosis. We observed that MNP-loaded Mo/Ma infiltrated pancreatic tumors and, after AMF treatment, significantly prolonged the lives of mice bearing disseminated intraperitoneal pancreatic tumors (Chapter-3). 2. Targeted cancer treatment could be achieved by engineering tumor homing cell surfaces with tumor proteases cleavable, cancer cell specific recombinant therapeutic proteins. To test this, Urokinase and Calpain (tumor specific proteases) cleavable; prostate cancer cell (CaP) specific (CaP1 targeting peptide); apoptosis inducible (Caspase3 V266ED3)- rCasp3V266ED3 chimeric protein was designed in silico. Hypothesized membrane anchored chimeric protein (rCasp3V266ED3, rMcherry red) plasmids were constructed. Membrane anchoring and activity of designed proteins were analyzed in RAW264.7 Mo/Ma and HEK293 cells in vitro. Further, Urokinase (uPA) mediated cleavage and release of rCasp3V266ED3 from engineered cells was tested (Chapter-4). Animal models for cancer therapy are invaluable for preclinical testing of potential cancer treatments. Final chapter of present report shows evidence for immune-deficient line of pigs as a model for human cancers (Chapter-5)

Cette étude propose l'idée des aimants dits de ferrite tétragonale en rendant la symétrie cristalline des ferrites de spinelle cubique afin d'améliorer l'anisotropie magnétique (et donc, d'améliorer la coercivité). Pour concrétiser cette idée, nous avons synthétisé des particules (Cu, Co) -ferrite à distorsion tétragonale et caractérisé systématiquement les propriétés magnétiques en conséquence avec leurs distorsions de réseau. Les facteurs intrinsèques et extrinsèques contribuant à la coercivité ont été étudiés. Pour élucider l'anisotropie magnétique, nous avons démontré un modèle de couplage physique de l'effet Jahn-Teller (JT) et de l'effet magnétoélastique (ME) au sein de la théorie phénoménologique. Ensuite, nous avons effectué une analyse de coercivité dans deux modèles généraux de coercivité afin de clarifier les paramètres de la microstructure contribuant au mécanisme d'inversion de la magnétisation. À partir de l'analyse du modèle magnétoélastique, nous avons démontré l'expression linéaire de l'anisotropie magnétique en utilisant le paramètre tétragonal obtenu à partir de la distorsion JT. Les valeurs du coefficient magnétoélastique pour Cu (B1Cu = 2 MJ / m3) et Co (B1Co = 40 MJ / m3) déduites de la courbe expérimentale étaient acceptables avec la valeur calculée pour le ferrite de cuivre en vrac (B1Cu en vrac = 4 MJ / m3) et le cobalt. ferrite (masse B1Co = 55 MJ / m3). Les résultats suggèrent que l’anisotropie magnétique peut être attribuée au couplage de la distorsion JT avec l’effet magnétoélastique de Co. Au lieu d’une augmentation indéfinie avec x, l’anisotropie magnétique Ku tend à atteindre une valeur de saturation en raison de la concurrence entre les effet magnétoélastique de Co et le JT de Cu. Entre le x tétragonal x = 0,1 et le x cubique = 0,2, les valeurs de Ku constantes d'anisotropie magnétique intrinsèque ne varient pas de manière aussi significative que la différence entre les champs de coercivité et d'anisotropie. La réduction des champs d'anisotropie supérieurs à x = 0,1 peut alors être attribuée à l'augmentation de l'aimantation spontanée. L'analyse de la coercivité au sein du modèle micromagnétique a révélé une contribution importante à la coercivité de la microstructure et de l'effet démagnétisant local. Le paramètre de microstructure αMM = 0,25 obtenu était une valeur classique de l'analyse micromagnétique, suggérant le départ du champ d'anisotropie avec ce facteur de réduction. Les facteurs démagnétisants locaux effectifs NeffMM d’environ 1,4 obtenus étaient plutôt importants, ce qui suggère un effet démagnétisant significatif. Dans l'analyse du modèle global (GM), les valeurs de NeffGM obtenues étaient were 0,38 pour l'échantillon x = 0,1. La valeur négative suggère la présence d'une interaction d'échange agissant efficacement en opposition à l'interaction dipolaire. En deçà de 100 K, une différence dans le modèle suggère l’idée d’un réchauffement local consécutif à l’activation thermique due au changement d’énergie Zeeman et à une dissipation de chaleur inefficace. Cet événement peut avoir conduit à la réduction du champ coercitif à une température suffisamment basse dans l'échantillon x = 0.1 en supposant que les grains sont fortement couplés en échange. / This study proposes the idea of the so-called tetragonal ferrite magnets by rendering the crystal symmetry of the cubic spinel ferrites to enhance the magnetic anisotropy (and hence, enhance the coercivity). To realize this idea, we synthesized tetragonally distorted (Cu,Co)-ferrite particles and systematically characterized the magnetic properties accordingly with their lattice distortions. The intrinsic and extrinsic factors contributing to coercivity were investigated. To elucidate the magnetic anisotropy, we demonstrated a physical coupling model of the Jahn-Teller (JT) effect and the magnetoelastic (ME) effect within the phenomenological theory. Then, we performed coercivity analysis within two general models of coercivity to clarify the microstructure parameters contributing to the magnetization reversal mechanism. From the magnetoelastic model analysis, we demonstrated the linear expression of the magnetic anisotropy using the tetragonal parameter obtained from the JT distortion. The magnetoelastic coefficient values for Cu (B1Cu = 2 MJ/m3) and Co (B1Co = 40 MJ/m3) deduced from the experimental curve were agreeable with the value calculated for bulk copper ferrite (B1Cu bulk= 4 MJ/m3) and cobalt ferrite (B1Co bulk= 55 MJ/m3). The results suggests that the source of magnetic anisotropy can be attributed to the coupling of the JT distortion with the magnetoelastic effect of Co. Instead of an indefinite increase with x, the magnetic anisotropy Ku tends to reach a saturation value due to the competition between the magnetoelastic effect of Co and the JT effect of Cu. Between the tetragonal x = 0.1 and the cubic x = 0.2 samples, the intrinsic magnetic anisotropy constant Ku values do not vary as significantly compared to the difference in the coercivity and the anisotropy fields. The reduction of anisotropy fields above x = 0.1 then can be attributed to the increase in the spontaneous magnetization.The coercivity analysis within the micromagnetic model revealed significant contribution to the coercivity by the microstructure and the local demagnetizing effect. The microstructure parameter αMM = 0.25 obtained was a classical value in the micromagnetic analysis, suggesting the departure of anisotropy field with this reduction factor. The effective local demagnetizing factor NeffMM of about 1.4 obtained were rather large suggesting a significant demagnetizing effect. Within the global model (GM) analysis, the values of NeffGM obtained were －0.38 for the x = 0.1 sample. The negative value suggests the presence of an exchange interaction acting effectively in opposition to the dipolar interaction. Below 100 K, discrepancy in the GM suggests the idea of a local heating event following the thermal activation due to the change in Zeeman energy and ineffective heat dissipation. This event may have led to the reduction of coercive field at sufficiently low temperature in the x = 0.1 sample assuming the grains are strongly exchange-coupled.

Nanomateriais apresentam propriedades ajustáveis pelo seu tamanho e forma, como o fenômeno de superparamagnetismo em nanopartículas magnéticas ou o confinamento quântico dos portadores de carga em pontos quânticos (quantum dots). Assim, a síntese de nanopartículas esféricas monodispersas torna-se um fator extremamente importante, haja visto que tais propriedades podem ser ajustáveis para diferentes aplicações na área de tecnologia e biomedicina. Nanopartículas magnéticas e quantum dots podem ser apontados como promissores materiais para diagnóstico e terapia de neoplasias (câncer), e o desenvolvimento desses sistemas busca, atualmente, intensificar a magnetização e a eficiência de emissão, respectivamente, relativo às propriedades magnéticas e ópticas, além de outros requisitos. Neste trabalho, nanopartículas esféricas de cobalto metálico foram sintetizadas com diâmetro médio de 5,3 nm e desvio padrão de 0,4 nm, distribuição de tamanhos lognormal. A equação de Langevin modificada pelo modelo de partículas interagentes foi utilizada no ajuste da curva de magnetização M(H) para obtenção do diâmetro magnético médio e desvio padrão, 4,7 nm e 1,0 nm, respectivamente. Comparando os dois diâmetros, encontra-se uma camada morta de magnetização de aproximadamente 3,0 Å a qual, praticamente, não contribui para a magnetização da amostra, sendo a magnetização de saturação de 125 emu g-1. Nanoestruturas core/shell de Co/Au apresentaram a propriedade de ressonância plamon de superfície, uma propriedade adicional também desejada para aplicações biomédicas, sendo este sistema denominado magnetoplasmônico. Quantum dots de CdSe foram sintetizados como elevado controle de tamanho e forma. Utilizando rotas de síntese diferentes dos clássicos procedimentos denominados TOP-TOPO, e dióxido de selênio como precursor, estudos mostraram que na presença de um agente redutor no meio de reação e do solvente 1-octadeceno, as amostras apresentaram melhores propriedades óticas. A estrutura cristalina das amostras de CdSe corresponde à formação da fase blenda de zinco, diferentemente das sínteses TOP-TOPO que levam à formação da fase hexagonal wurtzita. A cinética de crescimento dos quantum dots de CdSe também foram avaliadas através de alíquotas retiras com o tempo de reação mostrando um crescimento exponencial do diâmetro das partículas, como previsto pelas teorias de nucleação e crescimento. Estudos por microscopia de fluorescência mostraram que os quantum dots apresentaram o comportamento de intermitência de fluorescência relatado na literatura como um dos fatores que levam a uma diminuição do rendimento quântico de fluorescência. Nanoestruturas core/shell de CdSe/ZnS foram obtidas com elevado controle da espessura da camada de recobrimento e a intensificação das propriedades de fotoluminescência foram mostradas. Os objetivos do trabalho foram alcançados com sucesso, onde foi possível observar a estabilização e a intensificação da magnetização da fase de cobalto metálico, pouco relatado na literatura. Ainda, foi possível conferir maior estabilidade química, versatilidade de funcionalização da superfície e uma segunda propriedade de ressonância plasmônica com o recobrimento com ouro, sem grande prejuízo da propriedade magnética. Em relação aos sistemas ópticos, os semicondutores de CdSe foram obtidos por nova rota de síntese com expressivo controle de tamanho e forma, recobertos com ZnS intensificando as propriedades ópticas do sistema.  / Nanomaterials properties are size- and shape-controlled, such as the superparamagnetism phenomenon of magnetic nanoparticles or the quantum confinement of charge carriers of quantum dots. Therefore, synthesis of monodisperse spherical nanoparticles became extremely important over the past few deacades, since nanoparticles can be used for plenty of applications in technology and biomedicine. Magnetic nanoparticles and quantum dots are promising materials for diagnosis and therapy of cancer. Spherical nanoparticles of metallic cobalt were synthesized with mean diameter of 5,3 nm and standard deviation of 0,4 nm, lognormal distribution. A modified Langevin equation using the interacting superparamagnetic model was used to fit magnetization curves obtaining the mean magnetic diameter and standard deviation, 4,7 nm and 1,0 nm, respectively. The difference between these two diameters was assigned to the magnetic dead layer (∼3.0 Å), which does not contribute to the sample magnetization, being the saturation magnetization of cobalt nanoparticles around 125 emu g-1. Co/Au core/shell nanostructures were synthesized and the surface plasmon ressonance property was observed, an additional property also desired for biomedical applications, being the Co/Au core/shell system called magnetoplasmonic. CdSe quantum dots were synthesized with high size- and shape-controlled. Using different synthetic routes from the classic TOP-TOPO synthesis, and selenium dioxide as a precursor, the results show that and reducing agent is necessary and 1-octadecene solvent leads to better optical properties. CdSe samples showed a zinc blend (cubic phase) crystal structure, different from TOP-TOPO syntheses that leads to wurtzite structure (hexagonal phase). The growth kinetics of CdSe particles were also evaluated through aliquots from reaction showing exponential growth of particles diameter, as predicted on the theory of nucleation and growth. Fluorescence microscopy studies showed that quantum dots exhibited fluorescence intermittence behavior already reported in the literature as one fo the reasons for the quantum yield decrease. CdSe/ZnS core/shell nanostructures were obtained with high control of the coating layer thickness and the increase of the photoluminescence properties were shown.

Atualmente com o advento da nanociência e nanotecnologia, as nanopartículas magnéticas têm encontrado inúmeras aplicações nos campos da biomedicina, diagnóstico, biologia molecular, bioquímica, catálise, etc. As nanopartículas magnéticas funcionalizadas são constituídas de um núcleo magnético, envolvido por uma camada polimérica com sítios ativos, que podem ancorar metais ou compostos orgânicos seletivos. Estas nanopartículas são consideradas materiais híbridos orgânico-inorgânicos de grande interesse em aplicações comerciais devido à particularidade das propriedades obtidas. Entre as aplicações importantes podemos citar: tratamento por magnetohipertermia, carregadores de fármacos para áreas específicas do corpo, seleção de moléculas específicas, biossensores, melhoria da qualidade de imagens por RMN, etc. O trabalho foi desenvolvido em duas partes: 1) a síntese do núcleo constituído de nanopartículas superparamagnéticas de ferrita de cobalto e, 2) o recobrimento do núcleo por um polímero bifuncional o 3-aminopropiltrietoxissilano. Os parâmetros estudados na primeira parte da pesquisa foram: pH, concentração molar da base, tipo de base, ordem de adição dos reagentes, modo de adição dos reagentes, velocidade de agitação, concentração inicial dos metais, fração molar de cobalto e tratamento térmico. Na segunda parte estudou-se: o pH, a temperatura, o catalisador, a concentração do catalisador, o tempo de reação, a relação H2O/silano, o tipo de meio, o agente umectante e a eficiência do recobrimento em relação ao pH. Os produtos obtidos foram caracterizados pelas técnicas de difratometria de raios-X (DRX), microscopia eletrônica de transmissão (MET), microscopia eletrônica de varredura (MEV), espectroscopia de energia dispersiva (EDS), espectroscopia de emissão atômica (ICP-AES), espectroscopia por infravermelho (FTIR), análises termogravimétricas (TGA/DTGA), calorimetria exploratória diferencial (DSC) e curvas de magnetização (MAV) / Nowadays with the appear of nanoscience and nanotechnology, magnetic nanoparticles have been finding a variety of applications in the fields of biomedicine, diagnosis, molecular biology, biochemistry, catalysis, etc. The magnetic functionalized nanoparticles are constituted of a magnetic nucleus, involved by a polymeric layer with active sites, which ones could anchor metals or selective organic compounds. These nanoparticles are considered organic-inorganic hybrid materials and have great interest as materials for commercial applications due to the specific properties. Among the important applications it can be mentioned: magnetohyperthermia treatment, drugs delivery in specific local of the body, molecular recognition, biossensors, enhancement of nuclear magnetic ressonance images quality, etc. This work was developed in two parts: 1) the synthesis of the nucleus composed by superparamagnetic nanoparticles of cobalt ferrite and, 2) the recovering of nucleus by a polymeric bifunctional 3-aminepropyltriethoxysilane. The parameters studied in the first part of the research were: pH, hydroxide molar concentration, hydroxide type, reagent order of addition, reagent way of addition, speed of shake, metals initial concentrations, molar fraction of cobalt and thermal treatment. In the second part it was studied: pH, temperature, catalyst type, catalyst concentration, time of reaction, relation ratios of H2O/silane, type of medium and the efficiency of the recovering regarding to pH. The products obtained were characterized using the following techniques X-ray powder diffraction (DRX), transmission electronic microscopy (MET), scanning electronic microscopy (MEV), spectroscopy of scatterbrained energy spectroscopy (DES), atomic emission spectroscopy (ICP-AES), themogravimetric analysis (TGA/DTGA), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and magnetization curves (VSM)