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Item Síntese e Caracterização de Au@SPIONs Funcionalizadas com EGF para Diagnóstico do Câncer de Mama e Tratamento por Terapias Fotodinâmica e Fototérmica(2023-03-06) Raniero, Leandro José; Soares, Cristina Pacheco; Oliveira, Ivone Regina de; Tada, Dayane Batista; Campos, Andrea Porto Carreiro; Cândido, Marcela Aparecida; São José dos CamposO câncer de mama é caracterizado pela presença de neoplasias malignas, com alto grau de proliferação e, sendo a principal causa de morte em mulheres nos últimos anos. Os tratamentos disponíveis são geralmente agressivos e com baixa eficácia, dependendo do subtipo molecular. Sendo assim, se faz necessário desenvolver novos métodos de detecção, quando a nanotecnologia pode proporcionar diagnósticos precoces e tratamentos efetivos. As nanopartículas superparamagnéticas de óxido de ferro (Superparamagnetic Iron Oxide Nanoparticles, SPIONs) podem ser revestidas com fina camada de ouro e funcionalizadas, o que potencializa sua aplicação biomédica para formação de um biossensor sensível, rápido e preciso como agentes de terapia do câncer. As terapias fotodinâmica (TFD) e fototérmica (TFT) têm sido utilizadas no tratamento do câncer, sendo o seu mecanismo de ação baseado na interação de moléculas fotossensíveis com radiação de comprimento de onda adequado, na presença de oxigênio molecular. No entanto, cada passo deve ser controlado com precisão para obter um tratamento eficiente. Nesse contexto, a presente tese propõe a síntese de SPIONs pelo método de coprecipitação, o revestimento com ouro para obter Au@SPIONs, a funcionalização com a proteína EGF e o fotossensibilizador Clorina e6 para produção de Nanossondas Teranósticas (NTs) e sua aplicação na TFD e TFT. As dimensões das nanoestruturas foram caracterizadas por tamanhos físico e hidrodinâmico, bem como a morfologia, estabilidade coloidal, estrutura cristalina, composição química e propriedades magnéticas. Posteriormente foram realizados estudos in vitro para avaliação da atividade antitumoral e toxicológica da TFT e TFD nas linhas celulares MCF 10A e MDA-MB-468. As NTs apresentaram internalização no citoplasma e no núcleo de algumas células da linhagem celular MDA-MB-468. Após os tratamentos com as NTs obteve-se percentuais significativos de apoptose na linhagem celular MDA-MB-468 e níveis crescentes de ERO?s após TFD. No estudo com a cultura 3D da linhagem celular MDA-MB-468, as NTs apresentaram resultados que corroboraram com o estudo inicial 2D, sendo observado a capacidade de penetração das NTs nos esferoides com a variação do tempo de incubação.Item Specific nanomarkers fluorescence: in vitro analysis for EGFR overexpressed cells in triple-negative breast cancer and malignant glioblastoma(Elsevier) Vieira, Paula Fonseca Antunes; Jesus, Viviane Paula dos Santos; Cândido, Marcela Aparecida; Pacheco-Soares, Cristina; Castilho, Maiara Lima; Raniero, Leandro JoséBackground: Epidermal Growth Factor Receptor (EGFR receptor) is encoded by the EGFR gene. EGFR receptor signaling pathways are activated by EGF protein, regulating cell actions. Overexpression of EGFR receptor may be linked to malignancies with a poor prognosis. As a result, EGFR receptor is being studied for a variety of tumor diagnostics, spurring the development of innovative approaches to increase quality and efficiency. Nanomaterials can recognize cancer cells by specifically targeting of molecular pathways, underscoring the importance of nanomedicine. In this study, we synthesized EGFR-specific nanomarkers by functionalizing EGF protein and Chlorin e6 in gold nanoparticles. These nanoparticles use active targeting to deliver EGF protein to EGFR receptor, and Chlorin e6 serves as a fluorescent marker molecule Methods: Nanomarkers were examined in vitro in MDA-MB-468 and M059J cell lines. Confocal microscopy and flow cytometry were used to examine the distribution, uptake, internalization, and fluorescence intensity of nanomarkers in vitro Results: The results show that both lines examined accumulate nanomarkers. However, MDA-MB-468 had the highest intensity due to its EGFR receptor overexpression properties Conclusion: The findings point to ideal properties for detecting EGFR receptor overexpressed cells.Item Synthesis of iron oxide nanoparticles stabilized with sodium citrate and TMAOH(CDRR Editors) Cândido, Marcela Aparecida; Rost, Nathanne Cristina Vilela; Ferreira, Virginia Rezende; Raniero, Leandro JoséIron oxide nanoparticles (IONPs) represents a class of magnetic and biocompatible nanomaterials that have been widely used in research and medical applications, such as hyperthermia studies, as contrast agents for magnetic resonance imaging, biosensors, among others. However, their application depends on factors as surface properties, size, and morphology being appropriately balanced. IONPs can be obtained by different synthesis methods, however, chemical coprecipitation represents a simpler, easier and faster route, in which aqueous solutions of precursors containing iron (Fe3+) and ferrous (Fe2+) ions are alkalized under control of temperature and pH. This study proposes to synthesize iron oxide nanoparticles by the chemical coprecipitation methodand to stabilize them with sodium citrate (IONPs-CIT) and tetramethylammonium hydroxide (IONPs-TMAOH). Furthermore, to characterize the hydrodynamic diameter and the Zeta Potential of the samples by Dynamic Light Scattering. The cytotoxicity of IONPs-CIT in the MDA-MB-468 cell line was evaluated through the analysis of mitochondrial activity.Item Gold-Coated Superparamagnetic Iron Oxide Nanoparticles Functionalized to EGF and Ce6 Complexes for Breast Cancer Diagnoses and Therapy(MDPI) Cândido, Marcela Aparecida; Vieira, Paula Fonseca Antunes; Campos, Andrea; Soares, Cristina Pacheco; Raniero, Leandro JoséSuperparamagnetic iron oxide nanoparticles (SPIONs) have some limitations in the physio- logical environment, however, a modification on their surface, such as a core–shell structure with gold (SPIONs@Au), can enhance their applicability. In this study, SPIONs were synthesized by the chemical coprecipitation method, stabilized by sodium citrate, and followed by the gold-coating process. SPIONs@Au were functionalized with EGF-α-lipoic acid and chlorin e6 (Ce6)-cysteamine complexes, composing a Theranostic Nanoprobe (TP). The outcomes showed that the SPIONs@Au had changed in color to red and had an absorption band centered at 530 nm. The coating was verified in the TEM micrographs in bright and dark fields by EDS mapping, which indicated the presence of Au and Fe. The Ce6-cysteamine complex had a resonant band at 670 nm that enabled the diagnosis of biological samples using fluorescence analysis. In the measure of TNBC cell uptake, the maximum value of TP fluorescence intensity was obtained within 4 h of internalization. At 2 h, the incorporation of the TP in the cytoplasm as well as in the nuclei was observed, suggesting that it could be employed as a diagnostic marker. The PTT results showed significant percentages of apoptosis in the TNBC cell line, which confirms the efficacy of the TP.Item Chemical and cellular characterization of SPIONs stabilized with sodium citrate(Springer Nature Link) Cândido, Marcela Aparecida; Jesus, Viviane Paula dos Santos; Vieira, Paula Fonseca Antunes; Campos, Andrea; Raniero, Leandro JoséIntroduction Iron oxide nanoparticles (IONPs) represent a class of magnetic and biocompatible nanomaterials that have been widely used in research and medical applications, such as hyperthermia studies, as contrast agents for magnetic reso- nance imaging, biosensors, among others. Some studies have already evaluated several formulations containing IONPs in preclinical and clinical trials under the approval of the US Federal Agency. Methods IONPs were obtained by chemical coprecipitation, in which aqueous solutions of precursors containing iron (Fe3+) and ferrous (Fe2+) ions are alkalized under control of temperature and pH. The stabilization process of iron oxide nano- particles was done by sodium citrate and characterize the hydrodynamic diameter and the zeta potential by dynamic light scattering (DLS), the morphology and the diameter by transmission electron microscopy (TEM), crystal structure by X-ray diffraction (XRD), chemical composition by Fourier transform infrared spectroscopy (FT-IR), and magnetic properties by vibrating sample magnetometer (VSM). Results The DLS, MET, and FT-IR analyses confirmed that the superparamagnetic IONPs (SPIONs) syntheses had diameters smaller than 100 nm and were stabilized since the vibrational bands were present both in the sodium citrate and SPIONs spectra. IONPs exhibit superparamagnetic behavior due to the magnetic properties of the crystalline phase. Finally, pho- tothermal therapy study was carried out to determine the cell viability of MDA-MB-468 cell line after the application of SPIONs at concentrations of 0.5, 10, and 75 μg mL−1 by flow cytometry. Conclusion IONPs were synthesized in the nanometric range, and photothermal therapy shows promising results for triple- negative breast cancer treatment.Item Nano-Enabled Colorimetric Assay for the Detection of Paracoccidioides lutzii: Advancing Diagnostics with Nanotechnology(MDPI) Comparato Filho, Olavo de Osti; Cândido, Marcela Aparecida; Ventura, Aveline; Morais, Flavia Villaça; Raniero, Leandro JoséDeforestation is a common occurrence driven by agricultural expansion, urbanization, and infrastructure development. These activities often lead to increased human interaction with ecosys- tems, potentially exposing individuals to Paracoccidioides spores (P. brasiliensis and P. lutzii) found in the soil, resulting in Paracoccidioidomycosis (PCM). This fungal infection is endemic to specific regions in Latin America, such as Brazil, Colombia, Venezuela, and Argentina. Diagnosis typically involves a combination of clinical assessment, imaging techniques, and laboratory examinations. P. lutzii lacks the glycoprotein Gp43, a key antigenic protein utilized in serological tests for PCM diagnosis. In this study, a colorimetric test employing gold nanoparticles (AuNPs) and label-free methodology was employed for P. lutzii detection. The effectiveness of the label-free colorimetric test was assessed using a total of 100 samples. This detection was achieved through the amplification of the gp43 gene and the use of a specific probe (5′CAGGGGTGCG3′) in conjunction with AuNPs. The receiver operating characteristic curve was employed to assess the test, revealing that the method can accurately detect P. lutzii with a sensitivity of 100% and a specificity of 100%. The findings indicate a substantial impact on remote endemic regions attributable to the implementation of cost-effective diagnostic methodologies.