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Item Photodynamic therapy of cationic and anionic BSA-curcumin nanoparticles on amastigotes of Leishmania braziliensis and Leishmania major and Leishmania amazonensis(Photodiagnosis and Photodynamic Therapy, Elsevier) Marcolino, Luciana Maria Cortez; Ambrósio, Jéssica Aparecida Ribeiro; Pinto, Juliana Guerra; Ferreira, Isabelle; Simioni, Andreza Ribeiro; Ferreira-Strixino, JulianaCutaneous leishmaniasis is a neglected disease prevalent in tropical countries, and conventional treatment can cause several serious side effects. Photodynamic therapy (PDT) can be considered a promising treatment alternative, as it is non-invasive therapy that has no side effects and uses accessible and low-cost substances, such as curcumin. This study evaluated the PDT response with cationic and anionic BSA nanoparticles encapsulated with curcumin in macrophages infected with L. braziliensis, L. major, and L. amazonensis. The nanoparticle system was characterized using a steady-state technique, scanning electron microscopy (SEM) study, and its biological activity was evaluated using macrophage cell lines infected with different Leishmania species. All spectroscopy measurements demonstrated that BSA curcumin (BSACur) has good photophysical properties, and confocal microscopy shows that macrophages and protozoa internalized the nanoparticles. The viability test demonstrated that at low concentrations, such as 0.1, 0.7, and 1.0 μmol. L 1, there was a decrease in cell viability after PDT application. Furthermore, a decrease in the number of parasites recovered was observed in the PDT groups. The results allowed us to conclude that curcumin loaded into BSA nanoparticles may have potential application in drug delivery systems for PDT protocols, demonstrating reduced cell viability at lower concentrations than free curcumin.Item Molecular effects of photodynamic therapy with curcumin on Leishmania major promastigotes(Parasitology Research, Springer-Verlag GmbH Germany) Marcolino, Luciana Maria Cortez; Pinto, Juliana Guerra; Ferreira, Isabelle; Godoi, Bruno Henrique; Canevari, Renata de Azevedo; Ferreira-Strixino, JulianaLeishmaniasis is a neglected disease mainly affecting low-income populations. Conventional treatment involves several side effects, is expensive, and, in addition, protozoa can develop resistance. Photodynamic therapy (PDT) is a promising alternative in treating the disease. PDT involves applying light at a specific wavelength to activate a photosensitive compound (photosensitizer, PS), to produce reactive oxygen species (ROS). Curcumin and its photochemical characteristics make it a good candidate for photodynamic therapy. Studies evaluating gene expression can help to understand the molecular events involved in the cell death caused by PDT. In the present study, RNA was extracted from promastigotes from the control and treated groups after applying PDT. RT-qPCR was performed to verify the expression of the putative ATPase beta subunit (ATPS), ATP synthase subunit A (F0F1), argininosuccinate synthase 1 (ASS), ATP-binding cassette subfamily G member 2 (ABCG2), glycoprotein 63 (GP63), superoxide dismutase (FeSODA), and glucose-6-phosphate dehydrogenase (G6PDH) genes (QR). The results suggest that PDT altered the expression of genes that participate in oxidative stress and cell death pathways, such as ATPS, FeSODA, and G6PD. The ATP-F0F1, ASS, and GP63 genes did not have their expression altered. However, it is essential to highlight that other genes may be involved in the molecular mechanisms of oxidative stress and, consequently, in the death of parasites.Item Vaterite microparticle-loaded methylene blue for photodynamic activity in macrophages infected with Leishmania braziliensis(Springer Nature) Marmo, Vitor Luca Moura; Ambrósio, Jéssica Aparecida Ribeiro; Gonçalves, Érika Peterson; Raniero, Leandro José; Beltrame Junior, Milton; Pinto, Juliana Guerra; Ferreira-Strixino, Juliana; Simioni, Andreza RibeiroCalcium carbonate (CaCO3) exhibits a variety of crystalline phases, including the anhydrous crystalline polymorphs calcite, aragonite, and vaterite. Developing porous calcium carbonate microparticles in the vaterite phase for the encapsulation of methylene blue (MB) as a photosensitizer (PS) for use in photodynamic therapy (PDT) was the goal of this investigation. Using an adsorption approach, the PS was integrated into the CaCO3 microparticles. The vaterite microparticles were characterized by scanning electron microscopy (SEM) and steady-state techniques. The trypan blue exclusion method was used to measure the biological activity of macrophages infected with Leishmania braziliensis in vitro. The vaterite microparticles produced are highly porous, non-aggregated, and uniform in size. After encapsulation, the MB-loaded microparticles kept their photophysical characteristics. The carriers that were captured allowed for dye localization inside the cells. The results obtained in this study indicated that the MB-loaded vaterite microparticles show promising photodynamic activity in macrophages infected with Leishmania braziliensis.Item Cellular and metabolic changes after photodynamic therapy in leishmania promastigotes(Elsevier) Marcolino, Luciana Maria Cortez; Pereira, André Henrique Correia; Pinto, Juliana Guerra; Mamone, Leandro Ariel; Ferreira-Strixino, JulianaLeishmaniasis is a zoonotic disease, regarded by WHO as a public health problem that has presented a significant increase in the recent years. Conventional treatment is toxic and leads to serious side effects. Photodynamic therapy has been studied as a treatment to cutaneous leishmaniasis. This study aimed to evaluate the cell viability, morphological changes, type of cell death, production of reactive oxygen species, and changes in the mitochondrial membrane and DNA fragmentation in Leishmania braziliensis and Leishmania major promastigotes. Confocal microscopy was used to quantify the fluorescence emitted by JC-1, Annexin V, and propidium iodide reagents. The trypan blue exclusion test was used to evaluate the viability of the cells, the mitochondrial activity was verified with MTT, and the morphological changes were analyzed for SEM and DNA damage using the comet assay. PDT using curcumin at 500, 125, and 31,25 μg/mL decreased the viability of the parasites and induced changes in the mitochondrial membrane potential. The production of reactive oxygen species was dose- dependent and was observed only in the groups submitted to PDT. DNA damage was also observed in the parasite cells. The morphology of the cells was affected mainly at the highest curcumin concentration, resulting in rounded cells with a shortened flagellum. When the type of cell death was analyzed, the prevalence of apoptosis was noted. The results support the use of curcumin as photosensitizer in PDT against Leishmania promastigotes in the treatment for cutaneous leishmaniasis.