Programa de Pós-graduação em Física

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Navegando Programa de Pós-graduação em Física por Por Orientador "Alonso, Antonio"

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    Estudo das interações da miltefosina com membranas de L. (Leishmania) amazonensis e macrófagos peritoneais
    (Universidade Federal de Goiás, 2016-02-15) Fernandes, Kelly de Souza; Dorta, Miriam Cristina Leandro; ttp://lattes.cnpq.br/3933395097851681; Alonso, Antonio; http://lattes.cnpq.br/5013069863616789; Alonso, Antonio; Dorta, Miriam Cristina Leandro; Izumi, Erika; Souza, Paulo Eduardo Narcizo de; Oliveira, Valéria de
    Miltefosine (MT) is a alkylphospholipid originally developed for treatment of breast cancer and other solid tumors. It is currently used in the treatment of leishmaniasis, an infectious parasitic disease caused by protozoa of the genus Leishmania, being the first oral drug adopted for this purpose. However, its mechanism of action remains unclear. Electron paramagnetic resonance (EPR) spectroscopy of a spin-labeled lipid (5-DOXIL stearate) and a thiol-specific spin label (4-maleimido-TEMPO) in the membrane of axenic amastigotes of L.(Leishmania) amazonensis and peritoneal macrophages from Balb/c mice showed that MT causes significant increase in membrane dynamics at similar concentrations that inhibit parasite growth or are cytotoxic to macrophage. Although these alterations can be detected using a spin-labeled lipid, our experimental results indicated that MT interacts predominantly with the protein component of the membrane. Using a method for the rapid incorporation of MT into the membrane, these effects were measured immediately after treatment. Cytotoxicity, estimated via microscopic counting of living and dead cells, indicated ~80% parasites and macrophages death at the concentration of MT at which EPR spectroscopy detected a significant change in membrane dynamics. Cell viability, analyzed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide tetrazolium) reduction assay, showed that 50% inhibitory concentration (IC50) of MT depends on the cell concentration used in the assay. This dependence was analyzed using a theoretical equation involving biophysical parameters such as the partition coefficient of watermembrane and MT concentrations on the membrane and in the aqueous medium. The data showed that cells more sensitive to MT are respectively: erythrocytes, Leishmania promastigotes and Leishmania amastigotes and macrophage. The IC50 value of MT for 4 x 107 parasites/mL was 24,35 M. For the same cell concentration, a significant alteration was detected in the membrane lipid fluidity of parasites to 15 M of MT. The EPR spectra of spinlabeled membrane-bound proteins were consistent with more expanded and solvent exposed protein conformations, suggesting a detergent-like action, with a possible formation of micelle-like structures around polypeptide chains.
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    Rigidez de membrana em leishmania amazonensis causada por lipossomas contendo miltefosina e/ou anfotericina B
    (Universidade Federal de Goiás, 2024-09-06) Silva, Jean Carlo de Sousa e; Alonso, Antonio; http://lattes.cnpq.br/5013069863616789; Alonso, Antonio; Gomes, Rodrigo Saar; Lima, Eliana Martins; Silva, Kleber Santiago Freitas e; Mendanha Neto, Sebastião Antônio
    Leishmaniasis is a tropical disease, still neglected, caused by protozoa of the genus Leishmania, representing a serious public health problem in many regions of the world. In this study, liposome formulations containing the leishmanicidal drugs miltefosine (MTF) and amphotericin B (AmB) were prepared. The liposomes are intended to function as carriers for the drugs, eliminating the need for potentially toxic organic solvents and aiding in their delivery to target cells. To characterize the liposomes, Dynamic Light Scattering (DLS) analysis was performed to assess vesicle size, and Zeta potential was measured to evaluate liposome stability. Drug quantification was carried out to estimate losses during the preparation process, using HPLC for MTF-containing liposomes and absorbance for AmB-containing liposomes. Electron Paramagnetic Resonance (EPR) was employed to uncover the crucial interactions of the drugs with the liposome membrane, the stratum corneum (SC), parasites, and macrophages, paving the way for significant advancements in the understanding of their mechanism of action. The formulations were applied to the SC to evaluate the outcome of their interaction with the liposomes. EPR data revealed that formulations containing soybean phosphatidylcholine (PC) caused an increase in stratum corneum fluidity, while those containing distearoylphosphatidylglycerol (a lipid found in commercial liposomal AmB) did not cause significant changes. The increase in fluidity provided by PC is expected to facilitate the targeted delivery of the compounds to the parasites, thereby enhancing therapeutic efficacy in the topical treatment of cutaneous leishmaniasis. The drugs, when encapsulated in liposomes, also caused increased rigidity in Leishmania membranes after 24 hours of exposure, suggesting that both drugs generate oxidative stress in the parasite. However, the formulations did not cause changes in the membrane of the uninfected macrophage. On the other hand, they caused membrane rigidity in the system of Leishmania-infected macrophages at concentrations in the range of their IC50 values in promastigotes. The EPR data also indicated that the membranes of the macrophageamastigote system can also undergo oxidative processes even without treatment. This work further showed that both MTF and AmB are active drugs at the plasma membrane of the Leishmania parasite and suggest that their antileishmanial mechanisms of activity are associated with their primary effects on the cell membrane. The increase in fluidity caused by MTF or the pore formation produced by AmB are membrane alterations that can likely result in ionic leakage, leading to plasma membrane depolarization, which in turn should hyperpolarize the mitochondrial membrane and thus increase the formation of reactive oxygen species (ROS), triggering other events such as membrane rigidity, which result in the death of the parasite. These findings have the potential to significantly contribute to the development of more effective and safer therapies for the treatment of leishmaniasis. However, additional studies are needed to validate these results in clinical trials and to further improve the liposomal formulations.