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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 Effect of serial photodynamic therapy with curcumin on Leishmania braziliensis and Leishmania amazonensis promastigotes(CDRR Editors) Maciel, Lucas Tobias Rodrigues; Marcolino, Luciana Maria Cortez; Maciel, Fernanda Bueno Sant’Anna Pereira; Pinto, Juliana Guerra; Ferreira-Strixino, JulianaPhotodynamic Therapy (PDT) consists of using a light source and a photosensitive drug at an appropriate wavelength and molecular oxygen to trigger cell death through the production of reactive oxygen species. Because it is a localised therapy, PDT is shown to be ideal for skin diseases. American cutaneous Leishmaniasis (ACL) is a highly prevalent protozoan disease worldwide that presents different clinical evolutions and may result in ulcerations and disfiguring lesions on the skin and cartilage. This study was aimed at evaluating the effect in vitro of PDT applied serially using curcumin as a photosensitiser. For this, a concentration of 125 µg.mL-1 of curcumin was used on Leishmania braziliensis and Leishmania amazonensis strains, with a light fluence of 10 J.cm-2 and irradiance of 110 mW.cm-2. The tests done were viability analysis by trypan blue exclusion test, analysis of photosensitizer (PS) internalization by confocal microscopy and morphological alterations by May-Grunwald/Giemsa staining. We observed that there was internalisation of the PS before the first and second application of PDT, with L. braziliensis and L. amazonensis strains mortality of 92% and 82% respectively, after the second application, and induction of alterations in the structural conformation, such as cell size and non-evidence of nucleus and flagellum, demonstrating that PDT was effective. We conclude that serial PDT was effective in inducing the mortality of promastigotes forms of L. braziliensis and L. amazonensis in vitro, thus highlighting its potential for the treatment of leishmaniasis.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.Item Curcumin-loaded bovine serum albumin (BSA) nanoparticles: photoreactivity and photodynamic action against Acinetobacter baumannii and Staphylococcus aureus(Royal Society of Chemistry) Pinto, Juliana Guerra; Laneri, Francesca; Brambilla, Isabelle de Paula Ribeiro; Azevedo, Maria Clara Barbosa Silva de; Ambrósio, Jéssica Aparecida Ribeiro; Simioni, Andreza Ribeiro; Fraix, Aurore; Sortino, Salvatore; Ferreira-Strixino, JulianaCurcumin (CUR) is a naturally occurring pigment, poorly soluble in water and an object of intense interest due to its multifaceted therapeutic and phototherapeutic activity. In this contribution, bovine serum albumin nanoparticles (BSA-NPs) with oppositely charged surfaces have been used as suitable nanocarriers both to overcome the poor water solubility of CUR and to encourage its interaction with Gram-positive and Gram-negative bacteria. The different surface charges of the BSA-NPs do not affect either the spectroscopic or the photochemical behaviour of the encapsulated CUR which in all cases is almost exclusively present in a diketo form rather than the enolic one. This strictly dictates the response of CUR to blue light excitation which, under these conditions, exhibits the usual reactivity of carbonyl compounds toward molecules that behave as hydrogen donors such as the BSA-NP components. Steady-state and time-resolved photochemical experiments show that the encapsulated CUR under- goes photodecomposition with rate basically independent of the presence of oxygen. The photo- decomposition seems to be mediated mainly by an intermolecular H-abstraction from the components of the nanocarrier by the lowest excited triplet state of CUR with the formation of the corresponding ketyl radical occurring in less than 0.5 ms. This radical is oxidized by molecular oxygen, likely leading to peroxyl and hydroperoxyl radical species, probably responsible for the photodynamic action. Accordingly, with the short triplet lifetime, no singlet oxygen photogeneration is observed. Concentrations of 0.2, 0.3 and 0.4 mM were selected for in vitro tests. Internalization of both formulations was observed in strains of A. baumannii and S. aureus, after 15 minutes of interaction. After irradiation, a reduction in the viability of bacteria was observed, at the different concentrations tested, with concentrations of 0.4 and 0.3 mM being the most efficient.Item Analysis of the biomolecular profile by Fourier transform vibrational spectroscopy (FT-IR) in Acinetobacter baumannii after application of photodynamic therapy with curcumin "in vitro "(Elsevier) Marques, Camila Monteiro Santos; Lyra, Lucas Ferreira; Pinto, Juliana Guerra; Sakane, Kumiko Koibuchi; Ferreira-Strixino, JulianaAcinetobacter baumannii stands out for its antimicrobial resistance and high capacity to cause hospital infections, posing a severe threat to global public health. Thus, there is an urgent need for new therapeutic strategies. This work applied photodynamic therapy (PDT) with curcumin to Acinetobacter baumannii, and bacterial cell viability was assessed. Fourier transform infrared spectroscopy (FT-IR) was used to study changes in biomolecules after PDT application, comparing them with control groups that were only irradiated and only with curcumin photosensitizer. According to the results, there was an increase in the total amount of lipids (~0.3 %), total proteins (~0.6 %), and nuclear material (~8.6 %), with a decrease in carbohydrates (~3.0 %) after the appli- cation of PDT, compared to the control group. In the secondary structure of the proteins, a reduction in α-helix (~3.0 %), disordered structure (~7.0 %), and turns (~5.0 %) was observed, with an increase in β-sheets (~8.8 %). The analysis of biomolecular changes by FT-IR after the application of PDT can contribute to understanding the mechanisms of action of cell death, enabling therapeutic strategies to improve diagnosis and clinical treatment.Item Curcumin-coated iron oxide nanoparticles for photodynamic therapy of breast cancer(Springer Nature Link) Ferreira, Virginia Rezende; Ventura, Aveline; Candido, Marcela Aparecida; Ferreira-Strixino, Juliana; Raniero, Leandro JoséBreast cancer is the deadliest cancer among women and its treatment using traditional methods leads the patient to experience adverse effects. However, photodynamic therapy (PDT) is a non-invasive therapy modality that works through a photosensi- tizing agent, which treating activated by a suitable light source, releases reactive oxygen species capable of treating cancer. Furthermore, recent research indicates that combining PDT and nanoparticles can enhance therapeutic effects. In this way, the synthesis of IONPs (iron oxide nanoparticles) was carried out, and their subsequent coating was done with curcumin (IONPs@curcumin) so that they could act as therapeutic agents against breast cancer. Curcumin solubility tests were carried out to achieve the best results, with ethanol as a solvent, in different concentrations of ethanolic curcumin solution, with the optimal outcome observed at a concentration of 1 mM. Subsequently, the stability analysis was conducted by adjusting the pH of the medium, revealing that at pH 10, the IONPs@curcumin exhibited the best stability and dispersion conditions. Then, cytotoxicity tests of IONPs@curcumin were carried out on the MDA-MB-468 triple-negative breast cancer cell line, under experimental conditions without irradiation and subjected to PDT. The results revealed a viability greater than 70%, as it did not exhibit cytotoxicity for cells in the dark. After 1 h of incubation, the PDT associated with IONPs@curcumin showed 32% of cell viability at a concentration of 30 mg/mL.