PRISCILA DE QUEIROZ SOUZA PASSOS
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Artigo IPEN-doc 28654 In vitro and in vivo response of PSMA-617 radiolabeled with CA and NCA lutetium-1772022 - BOAS, CRISTIAN A.W.V.; SILVA, JEFFERSON de J.; DIAS, LUIS A.P.; FREIRE, MARIA R.B.; BALIEIRO, LUIZA M.; SANTOS, CAROLINA S.F. dos; VIVALDINI, BIANCA F.; BENEDETTO, RAQUEL; VIEIRA, DANIEL P.; PASSOS, PRISCILA de Q.S.; MARUMO, MARIA H.; TEIXEIRA, LUIS F.S.; ARAUJO, ELAINE B. deThe PSMA-targeted radionuclide therapy has been explored since 2015 with radioisotope lutetium-177, whose β− emission range is adequate for micrometastases treatment. This radioisotope is obtained by two different production routes that directly affect the specific activity of lutetium-177 (non-carrier added and carrier added) and, consequently, the specific activity of radiopharmaceuticals, like 177Lu-PSMA-617. The influence of the specific activity of lutetium-177 on the properties of the radiopharmaceutical PSMA-617 was evaluated through pre-clinical studies. The in vitro study pointed to a lower constant of dissociation with non-carrier added lutetium-177 due to the difference in the specific activity. However, competition and internalization assays resulted in similar results for both lutetium-177. Based on these pre-clinical experiments, the total in vitro tumor cell binding and tumor uptake in vivo were similar, with no influence of the specific activity of the 177Lu-PSMA-617. Regardless the specific activity did not directly affect tumor uptake, the tumor/non-target organs ratios were higher for the radiopharmaceutical labeled with carrier added lutetium-177, which had the lowest specific activity.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.Resumo IPEN-doc 27653 Mouse mioblast (C2C12) spheroids structured using paramagnetic iron nanoparticles as an in vitro culture system of Toxoplasma gondii tachyzoites2020 - NASCIMENTO, A.C.; PASSOS, P.d.; LIMA, M.M.; GALISTEO JUNIOR, A.J.; VIEIRA, D.P.Tridimensional cell culture techniques became essential for understanding physiological processes that are obliterated or fainted in conventional bi-dimensional cultures. These techniques are prone to produce more realistic modeling of the complex environment of living tissues, leading to much better understanding of mammalian tissue organization. This work used magnetic levitation of cell aggregates (spheroids) by adsorbing iron nanoparticles to C2C12 mouse (Mus musculus) mouse line cells (ATCC # CRL-1772), which are suspended with magnetic fields. The cells formed three-dimensional bodies that were cultivated suspended in the air-liquid interface. Magnetite (Fe3O4) nanoparticles with mean diameter of approximately 50 nm were produced by an alkaline coprecipitation methodology under reduction by microwave energy. Composition and size of crystallites were determined by DRX analysis. Adsorption on cell membranes occurred after functionalization with poly-L-lysine. Work concentrations of nanoparticles did no induce cytotoxicity in C2C12 monolayer cultures. Transmission electron microscopy of spheroid sections showed some findings morphologically compatible to the shape of reproductive intracellular vacuoli of T.gondii after cell invasion, demonstrating an interaction of cells with parasites in three-dimensional models.Resumo IPEN-doc 27446 In vitro response of 177Lu-PSMA-617 with two different specific activities2020 - VILLAS BOAS, CRISTIAN A.W.; MENGATTI, JAIR; PASSOS, PRISCILA; VIEIRA, DANIEL; ARAUJO, ELAINE B. deIntroduction: PSMA-617 radiolabeled with lutetium-177 has shown good results in compassionate studies around the world. Being a receptor-specific radiopharmaceutical, the specific activity (SA) of the preparation may represent an important factor for therapeutic efficacy. Lutetium-177 can be produced by two different routes: with ytterbium-176 (Non-carrier-added or NCA) and with lutetium-176 (Carrier-added or CA). The SA (MBq/ug) of the labeled PSMA varies accordingly to each lutetium. For NCA lutetium, the radiolabeling procedure is based on the SA of 74 MBq/ug. When the radiolabeling is performed with CA lutetium, SA is determined by the molar ratio of 2.1:1 (PSMA moles/lutetium moles declared in the certificate), resulting in lower SA than NCA. This work evaluated the influence of specific activity of 177Lu-PSMA-617 on in vitro specific binding assays (saturation, competition and internalization). Materials and Methods: Radiolabeling of PSMA-617 (ABX, Germany) with lutetium-177 was performed in heating block at 90°C for 30 minutes with sodium ascorbate (0.5 M pH 4.7) as buffer. For NCA lutetium (JSC, Russia) the radiopharmaceutical specific activity was 74 MBq/ug. For CA lutetium (IDB, Netherlands), the specific activity was 41 MBq/ug. The radiochemical purity was analyzed with HPLC. For all experiments, 6-well plates were used for adherence cells with 200,000 LNCaP per well. Molar concentration of saturation curves experiments were 0.01; 0.05; 0.6; 1.5; 3.0 and 3.5 for CA lutetium and 0.1; 0.6; 1.5; 2.0; 2.5 and 3.0 for NCA lutetium. After 1 hour of incubation at 8 ºC, supernatant was removed, then washed with PBS (phosphate buffer saline) and finally cells were burst with NaOH 1 M, and activity was measured in gama counter; the experiments were performed in octuplicate. Competition experiments were performed adding in all wells 5 nM of radiolabeled PSMA-617 and in the competition well (non-specific binding) were added an excess of 15 times (76 ug) of non radiolabeled PSMA-617. After 1 hour of incubation at 8 ºC, supernatant was removed, then washed with PBS and finally cells were burst with NaOH 1 M, and activity was measured in gama counter, these experiments were performed in triplicate. The specific binding was obtained by the difference between total binding and non-specific binding. Internalization experiments were performed at Kd of NCA and CA lutetium. After 1 hour of incubation at 37 ºC, supernatant was removed, washed with PBS, then washed again with 0.05 M glycine solution pH 2.8 and finally cells were burst with NaOH 1 M. Activity was measured in gama counter, these experiment were performed in sextuplicate. Results and discussion: The radiochemical purity were 98% and 99% for labeling with NCA and CA lutetium, respectively. Saturation curve assay with NCA lutetium shown a Kd of 0.7 ± 0.15 nM and a Bmax of 857 ± 55.79 pMol/ng, and with CA lutetium resulted in a Kd of 1.71 ± 0.45 nM and a Bmax of 1156 ± 113.8 pMol/ng. The variation between both Kd curves were statistically different (P value = 0.0058). Competition assay demonstrated an effective blocking for both types of lutetium, for NCA unpaired T test shown a P value of 0.0011. For CA lutetium, the unpaired test disclosed a P value of 0.0258. The comparison between both results revealed a P value of 0.01 at the specific binding. Internalization assay shown for both types of lutetium similar results, 27.1 ± 2.45% and 30.6 ± 4.97%, for CA and NCA lutetium, respectively, and was not statistically significant (P value = 0.17). Conclusions: These experiments demonstrated that the type of lutetium (CA or NCA) directly affects in vitro binding of 177Lu-PSMA-617 to receptors expressed in LNCaP cells. It was statistically demonstrated that the higher specific activity of 177Lu-PSMA-617, more radiolabeled peptide can bind to cells at saturation and competition assays.