PRISCILA DE QUEIROZ SOUZA PASSOS

PRISCILA DE QUEIROZ SOUZA PASSOS

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In vitro and in vivo response of PSMA-617 radiolabeled with CA and NCA lutetium-177
2022 - 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. de
The 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.
Modelo tridimensional in vitro de adenocarcinoma prostático humano produzido por levitação magnética
2020 - 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.
Mouse mioblast (C2C12) spheroids structured using paramagnetic iron nanoparticles as an in vitro culture system of Toxoplasma gondii tachyzoites
2020 - 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.
In vitro response of 177Lu-PSMA-617 with two different specific activities
2020 - VILLAS BOAS, CRISTIAN A.W.; MENGATTI, JAIR; PASSOS, PRISCILA; VIEIRA, DANIEL; ARAUJO, ELAINE B. de
Introduction: 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.
Microwave-mediated synthesis of iron-oxide nanoparticles for use in magnetic levitation cell cultures
2019 - 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.
Synthesis of paramagnetic iron oxide nanoparticles for application in in vitro three-dimensional biological models through electron beam irradiation and microwave reduction of iron ions
2019 - 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.
Synthesis of paramagnetic iron oxide nanoparticles for application in in vitro three-dimensional biological models through gamma radiation and microwave reduction of iron ions
2019 - 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.