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Item Zinc pthalocyanine loaded poly (lactic acid) nanoparticles by double emulsion methodology for photodynamic therapy against 9 L/LacZ gliosarcoma cells(Taylor & Francis Group) Oliveira Junior, Benedito Marcio de; Teodoro, Jéssica Beatriz Miranda; Ambrósio, Jéssica Aparecida Ribeiro; Gonçalves, Érika Peterson; Beltrame Junior, Milton; Marcolino, Luciana Maria Cortez; Pinto, Juliana Guerra; Ferreira-Strixino, Juliana; Simioni, Andreza RibeiroDevelopment delivery systems, such as nanoparticles, represent a growing area in biomedical research. Nanoparticles (NP) were prepared using a double-emulsion method to load zinc(II) phthalocyanine (ZnPc). NP were obtained using poly (lactic acid) (PLA). ZnPc is a second generation of photosensitizer used in photodynamic therapy (PDT). ZnPc loaded PLA nanoparticles (NPLAZnPc) were prepared by double-emulsion method, characterized and available in cellular culture. The mean nanoparticle size presented particle size was 384.7 ± 84.2 nm with polydispersity index (PDI) of 0.150 ± 0.015, and the encapsulation efficiency was of 83%. The nanoparticle formulations presented negative zeta potential values (27.5 ± 1.0 mV), explaining their colloidal stability. ZnPc loaded nanoparticles maintain its photophysical behavior after encapsulation. Photosensitizer release from nanoparticles was sustained over 168 h with a biphasic ZnPc release profile. An in vitro phototoxic effect in range of 80% was observed in 9 L/ LacZ gliosarcoma cells at laser light doses (10 J cm2) with 3.0 mg mL1 of NPLA-ZnPc. All the physical–chemical, photophysical and photobiological measurements performed allow us to conclude that ZnPc loaded PLGA nanoparticles is a promising drug deliverysystem for PDT.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 Evaluation of the Photodynamic Therapy with Curcumin on L. braziliensis and L. major Amastigotes(MDPI) Pereira, André Henrique Correia; Marcolino, Luciana Maria Cortez; Pinto, Juliana Guerra; Strixino, Juliana FerreiraCutaneous leishmaniasis (CL) is a neglected disease prevalent in tropical countries with the ability to cause skin lesions. Photodynamic therapy (PDT) represents a specific and topical option for the treatment of these lesions. This study evaluated the response of macrophages infected with L. braziliensis and L. major to PDT with curcumin. Curcumin concentrations were evaluated in serial dilutions from 500.0 to 7.8 μg/mL using LED (λ = 450 ± 5 nm), with a light dose of 10 J/cm2. The Trypan blue viability test, ultrastructural analysis by scanning electron microscopy (SEM), mitochondrial polarity by Rhodamine 123 (Rho 123), curcumin internalization by confocal microscopy, and counting of the recovered parasites after the PDT treatment were performed. The lowest concentrations of curcumin (15.6 and 7.8 μg/mL) presented photodynamic inactivation. Cell destruction and internalization of curcumin in both macrophages and intracellular parasites were observed in microscopy techniques. In addition, an increase in mitochondrial membrane polarity and a decrease in the number of parasites recovered was observed in the PDT groups. This study indicates that PDT with curcumin has the potential to inactivate infected macrophages and might act as a basis for future in vivo studies using the parameters herein discussed.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 Biochemical changes in Leishmania braziliensis after photodynamic therapy with methylene blue assessed by the Fourier transform infrared spectroscopy(Springer Nature Link) Sakane, Kumiko Koibuchi; Bhattacharjee, Tanmoy; Fagundes, Jaciara; Marcolino, Luciana Maria Cortez; Ferreira, Isabelle; Pinto, Juliana Guerra; Ferreira-Strixino, JulianaPhotodynamic therapy (PDT) with photosensitizer methylene blue was applied to Leishmania braziliensis, and Fourier transform infrared (FTIR) spectroscopy was used to study biochemical changes in the parasite after PDT in comparison to untreated (C), only irradiation (I), and only photosensitizer (PS). Spectral analysis suggests increase in lipids, proteins, and protein secondary structures in PDT compared with C and decrease in nucleic acids and carbohydrates. Interestingly, these trends are different from PDT of Leishmania major species, wherein lipids decrease; there are minimal changes in secondary structures and increase in nucleic acids and carbohydrates. The study thus suggests possibility of different biomolecular players/pathways in PDT-induced death of L. braziliensis and L. major.Item Photodynamic Activity of Photogem® in Leishmania Promastigotes and Infected Macrophage(Taylor & Francis) Pinto, Juliana Guerra; Marcolino, Luciana Maria Cortez; Ferreira-Strixino, JulianaObjectives: This study aimed to evaluate the effect of photodynamic therapy (PDT) with Photogem⃝R in promastigotes of Leishmania braziliensis and Leishmania major, and in infected macrophages. Materials & methods: The following parameters were analyzed: Photogem⃝R internalization, mitochondrial activ- ity, viability, tubulin marking and morphological alterations in promastigotes and viability in infected macrophages. Results: Photogem⃝R accumulated in the cytosol and adhered to the flagellum. Changes were observed in the mitochondrial activity in groups maintained in the dark, with no viability alteration. After PDT, viability decreased up to 80%, and morphology was affected. Conclusion: The results point out that PDT with Photogem⃝R can reduce parasite and macrophage viability. Lay abstract: Cutaneous Leishmaniasis is a disease that can cause deforming lesions, the treatment of which is highly toxic. It is considered a neglected disease and little progress has been made in the treatment of this disease. Photodynamic therapy can be an alternative treatment which is less costly, and involves local treatment of the lesion, thereby reducing side effects for the patient. The present study aims to test the photodynamic therapy with Photogem, a photo sensitive drug, and to verify if the parasites can be affected by this therapy, aiming to apply this therapy in lesions in patients in the near future.Item Methylene blue functionalized ZnO nanoparticles: a promising approach for photodynamic therapy in the treatment of leishmaniasis(Sage) Gouvea, Thainara Alves; Ambrósio, Jéssica Aparecida Ribeiro; Carvalho, Janicy Arantes; Marmo, Vitor Luca Moura; Marcolino, Luciana Maria Cortez; Pinto, Juliana Guerra; Ferreira-Strixino, Juliana; Simioni, Andreza Ribeiro; Gonçalves, Erika PetersonZinc oxide (ZnO) has wide application in engineering, but its use in medical sciences has aroused growing interest. In this context, ZnO nanoparticles were investigated as vehicles for the delivery of methylene blue (MB), a photosensitizer (PS) used in photodynamic therapy (PDT) against Leishmania braziliensis. ZnO-NPs were produced by a coprecipitation method and characterized by several techniques, including scanning electron microscopy (SEM), UV-VIS spectroscopy, Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The results showed that the ZnO-NPs presented uniform spheroidal morphology with open porosity, allowing an efficient methylene blue (MB) encapsulation without significant structural changes, ensuring stability and the absence of aggregation. The PS was adsorbed on the porous surface of the ZnO nanoparticles, characterized by scanning electron microscopy (SEM) and steady-state analysis techniques. Spectroscopic analysis confirmed the maintenance of the photosensitizing properties of MB. The biological activity was evaluated in vitro using the trypan blue exclusion method in macrophages infected with Leishmania braziliensis. After loading with the photosensitizer, they maintained their photophysical properties, ensuring the proper location of the dye within the cells. In vitro assays demonstrated the internalization of ZnO/MB-NPs by infected macrophages and a significant reduction in parasite viability after light activation. Thus, the results showed that the developed system exhibits a promising photodynamic activity with relevant therapeutic potential in treating macrophages infected by Leishmania braziliensis.Item N-Doped Carbon Dot-Based Nanoconjugates with Simultaneous Generation of Nitric Oxide and Singlet Oxygen for Phototherapeutic Applications(ACS Publications) Laneri, Francesca; Parisi, Cristina; Andrigo, Vittoria; Pinto, Juliana Guerra; Marcolino, Luciana Maria Cortez; Ferreira-Strixino, Juliana; Natile, Marta Maria; Sortino, SalvatoreNitric oxide (NO) and singlet oxygen (O2) represent two of the most intriguing agents for unconventional phototherapeutic applications in cancer. In this contribution, N-doped carbon dots (NCDs) with strong absorption in the biocompatible green region have been synthesized and covalently decorated with an otherwise blue-light-activatable NO photodonor (NOPD), leading to a nanoconjugate ca. 3.5 nm in diameter. The NCD core of the nanoconstruct acts as a green light antenna, permitting the release of NO from the NOPD by an intramolecular photoinduced electron transfer, with an improvement of more than 100 nm in the excitation wavelength. Simultaneously, green light excitation generates O2 by collisional energy transfer with molecular oxygen. Due to its emissive properties, the nanoconjugate can be visualized in 9L/LacZ brain cancer cells, where it localizes mainly in the cytoplasm. Amplified mortality of cancer cells is observed upon green light irradiation due to the mutual photodynamic action of NO and O2.Item Photodynamic treatment in glioma: Metabolic and structural evaluation after therapy(Wiley) Teixeira, Marina Gabriela; Marcolino, Luciana Maria Cortez; Pinto, Juliana Guerra; Almeida, Rainara Moreno Sanches de; Ferreira, Isabelle; Ferreira-Strixino, JulianaGliomas are malignant tumors of the central nervous system, and one severe variant is called gliosarcoma. Photodynamic therapy (PDT) is a technique that stands out in the oncology area for minimizing side effects for the patient, triggering cell death at the site of irradiation, and can be used concomitantly with conventional treatments. This study aimed to evaluate the interaction of chlorine e6 with the cytoskeleton and mitochondria, as well as morphological changes and the death mechanism trig- gered after PDT. Chlorin e6 was used at concentrations of 200, 12.5, and 6.25 μg/mL, and cytoskeletal changes were analyzed by alpha-tubulin staining and mitochondrial membrane potential (MMP) analysis by JC-1 and Rhodamine 123 in flow cytom- etry. Surface features were examined using scanning electron microscopy, and the type of cell death mechanism was determined by flow cytometry with annexin and propidium iodide. Changes in the cytoskeleton were observed after PDT. Cytometry showed that cell death occurred predominantly via the apoptosis pathway, followed by the necrosis pathway. Chlorin e6 associated with PDT causes damage to gliosar- coma cells, regardless of concentration, showing cytoskeletal disruption, a decrease in MMP, and the percentage of cell death varies according to the concentration of PS.