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Dissertação IPEN-doc 27698 Modelo tridimensional in vitro de adenocarcinoma prostático humano produzido por levitação magnética2020 - PASSOS, PRISCILA de Q.S.Culturas celulares tradicionais em monocamada nem sempre são suficientes quando aplicadas a ensaios toxicológicos pré-clínicos in vitro, pois podem frequentemente perder as características e funções quando não integradas em um tecido ou órgão. A utilização de PIONs funcionalizadas para a construção de esferoides por levitação magnética é uma ferramenta de baixo custo, de fácil manipulação e biocompatível, permitindo sua associação a linhagens celulares. Neste trabalho, as propriedades biofuncionais de esferoides celulares construídos a partir de células tumorais e nanopartículas magnéticas de óxido de ferro (PIONs) biofuncionalizadas foram estudadas, cujas nanopartículas de óxido de ferro foram sintetizadas por co-precipitação, funcionalizadas com os aminoácidos glicina e poli-L-lisina e então submetidas a caracterização físico-química por MET, DRX, DLS, potencial zeta e FTIR. A partir dessas análises foi possível identificar nanopartículas de magnetita com tamanho ideal e carga eletrostática positiva estável para adsorção às células. Essas nanopartículas também foram submetidas a uma avaliação biológica quanto a adsorção à membrana de células tumorais de próstata (LNCaP), citotoxicidade e formação de esferoides. Foi observada boa adsorção dos PIONs, sem desprendimento das células durante o cultivo celular, não foi observada citotoxicidade das nanopartículas e houve resposta magnética suficiente a fim de permitir a formação dos esferoides quando na presença de um campo magnético. Os esferoides tumorais obtidos a partir da adsorção das nanopartículas foram avaliados estruturalmente por microscopia de fluorescência e analise histológica com resultados satisfatórios para uma estrutura tumoral. Foram também utilizados como sistema-teste na avaliação do potencial tóxico de substâncias farmacológicas e não-farmacológicas de ação conhecida com resultados reprodutíveis. Portanto, o produto final deste trabalho pode ser utilizado em testes posteriores de eficácia de fármacos que visem tratar os tumores prostáticos humanos.Artigo IPEN-doc 26645 Microwave-mediated synthesis of iron-oxide nanoparticles for use in magnetic levitation cell cultures2019 - BONFIM, LETICIA; PASSOS, PRISCILA de Q.S.; GONÇALVES, KARINA de O.; COURROL, LILIA C.; SILVA, FLAVIA R. de O.; VIEIRA, DANIEL P.The use of three-dimensional cell cultures has been widely used for efficacy and/or toxicity testing of compounds. One of the most promising systems, based on magnetic levitation, is dependent on proper cell magnetization, achieved through adsorption of iron-oxide nanoparticles on cell membranes. These particles must bare not only significant responses to magnetic fields, but also a stable mechanism to attachment to cells. This work proposes a simple, one-pot synthesis method to produce magnetite nanoparticles, using a Fe2+ precursor associated with amino acids under microwave heating, and successive steps to confer positive charges to particles. X-ray diffraction could confirm Fe3O4 composition, and TEM analysis showed cubic-like crystallites with less than 50 nm. Zeta-potential experiments showed that particles remained positively charged (20.98 ± 0.28 mV) in physiological pH, suggesting ability to attach to (negatively charged) cell membranes, observed through optical microscopy. Iron colloid was found to be non-cytotoxic in concentrations up to 8% in cell culture media. Finally, human prostate cancer cells were cultured in 96-well plates using magnetic levitation and could be kept 8 days in culture. The results showed a feasible way to produce spheroids relying on magnetic levitation, using a newly described method of magnetic and cell adherent nanoparticle production.Artigo IPEN-doc 26273 Synthesis of paramagnetic iron oxide nanoparticles for application in in vitro three-dimensional biological models through electron beam irradiation and microwave reduction of iron ions2019 - PASSOS, PRISCILA de Q.S.; CORAZZA, FULVIO G.; LIMA, MAYELLE M.P.; TOMINAGA, FLAVIO K.; SAKATA, SOLANGE K.; GONÇALVES, KARINA O.; COURROL, LILIA C.; VIEIRA, DANIEL P.Three-dimensional (3D) cell culture is increasingly being used in assays to assess the safety and efficacy of new drug candidates. Tumor cell spheroids can mimic with high precision the biological complexity of cellular interactions with their tumor microenvironment. Currently, several techniques can be used to construct 3D spheroids. Among them, magnetic levitation is one of the most used in biomedical research. This technique consists in the magnetization of cells through the adsorption of magnetic nanoparticles of iron oxide (Fe3O4) that are produced by the reaction of Fe2+ and Fe3+ ions in alkaline medium. In this work, nanoparticles of paramagnetic iron oxide (PIONS) were synthesized by coprecipitation through electron beam irradiation at 15 and 30 kGy doses. After functionalization with polar amino acids, nanoparticle suspensions were characterized by physical-chemical assays that showed the successful attachment of the carboxylate groups to the iron, explaining the ability of the particles to adsorb the membranes. Cytotoxicity assay showed that the nanoparticles synthesized by microwave (MW) and electron beam had no toxicity. Others biological assays have also shown efficient adsorption of the particles by human prostate tumor cells, allowing the in vitro application of a biomimetic 3D biological model with potential utilization regarding the development and evaluation of antitumor drugs and radiopharmaceuticals for the treatment of prostate cancer.Artigo IPEN-doc 26272 Synthesis of paramagnetic iron oxide nanoparticles for application in in vitro three-dimensional biological models through gamma radiation and microwave reduction of iron ions2019 - CORAZZA, FULVIO G.; PASSOS, PRISCILA de Q.S.; LIMA, MAYELLE M.P.; TOMINAGA, FLAVIO K.; SAKATA, SOLANGE K.; GONÇALVES, KARINA O.; COURROL, LILIA C.; VIEIRA, DANIEL P.Two-dimensional (2D) cell models are extensively used in biomedical research to evaluate the efficacy and safety of new drugs. However, these conventional approaches do not precisely mimic the complexity of the organ microenvironment. To overcome this obstacle, three-dimensional (3D) spheroid cell structures usually referred to as spheroids are being developed to better represent the morphological and functional similarity to the tissues. Among several techniques currently employed to produce three-dimensional cell cultures, one of the most promising is the magnetic levitation, which consists of the magnetization of the cells through adsorption of magnetic nanoparticles of iron oxide (Fe3O4), which are produced by the reaction of Fe2+ and Fe3+ ions in alkaline medium. This work produced paramagnetic iron oxide nanoparticles (PIONs) by coprecipitation from an Fe2+ source. The reduction to Fe3+ was obtained by the ionization caused by gamma radiation (60Co) at 15 or 30 kGy radiation absorbed doses. After functionalization with poly-lysine, the nanoparticle suspensions were characterized by XRD, FTIR, zeta potential analysis, DLS and TEM which showed the successful attachment of the carboxylate groups to iron, explaining the ability of the particles to be adsorbed by the membranes. Biological assays showed that these PIONs were biocompatible and efficiently could be applied to develop prostate 3D tumor spheroids model for drug screening.