Navegando por Palavras-chave "Membrana"

Agora exibindo 1 - 5 de 5
  • Imagem de Miniatura
    Item
    Acesso aberto (Open Access)
    Desenvolvimento de uma membrana de quitosana com propriedades de barreira celular e capacidade de liberação de droga
    (Universidade Federal de São Paulo, 2014-03-06) Gondim, Luciano de Rezende [UNIFESP]; Bizeto, Marcos Augusto [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Chitosan is a water-soluble semisynthetic biopolymer with recognized potential application in the medical field because of properties such as bactericidal activity, biocompatibility, drug releasing, among others. Chitosan Membranes were prepared by the casting method. In order to improve mechanical and drug delivery properties of these membranes, different amounts (1 to 90 wt.%) of mesoporous silica MCM-41 were added to the chitosan solution during the preparation process. The MCM-41 presents an extensive network of pores with regular sizes and structural ordering that provide high surface area and drug loading capacity for later release. Membranes as well as their intermediate components were chemically and structurally characterized. The membranes containing less than 20 wt% of MCM-41 have adequate flexibility to manipulation, do not show pore with cell dimensions and surface segregation of the polymer and inorganic phases. A membrane containing 20 wt.% of MCM- 41 and another one composed of pure chitosan were subjected to the in vitro and in vivo assays, comparatively. The drug release property of membranes is influenced by the MCM-41 presence. The membrane where ibuprofen was previously immobilized in MCM-41 showed a release profile with prolonged release over time when compared to pure chitosan membrane. The biocompatibility was evaluated in vivo by surgical insertion between the abdominal wall muscles of Wistar-SEM (n = 8) and subsequent histological analysis of tissue after 7 and 28 days of implantation. The potential inhibition of cell transmigration was confirmed by histology tissue where the membrane has been deployed. The inflammatory response membranes with or without silica was similar to the one observed for the collagen membranes and commercial polypropylene used as controls. After 28 days the chitosan membrane showed integration with the surrounding tissue and no chronic inflammation, thus demonstrating adequate biocompatibility. The hybrid membranes of chitosan prepared with MCM-41 showed cell barrier property, biocompatibility and release capacity of ibuprofen, showing a great potential for medical applications. Microbiological testing of the membranes showed the preservation of antibacterial property of chitosan..
  • Imagem de Miniatura
    Item
    Acesso aberto (Open Access)
    O efeito da composição lipídica da membrana no estudo do mecanismo de ação do peptídeo antimicrobiano esculentina 1b (1-18)
    (Universidade Federal de São Paulo (UNIFESP), 2019-09-26) Silva, Isabela Moreira [UNIFESP]; Perez, Katia Regina [UNIFESP]; http://lattes.cnpq.br/2250091739407083; http://lattes.cnpq.br/7641887893666670; Universidade Federal de São Paulo (UNIFESP)
    The antimicrobial peptides are part of innate immune system of several organisms. They are characterized by the presence of cationic and hydrophobic amino acids that assists in the interaction with plasma membranes. Esculentin 1b (1-18) is an antimicrobial peptide containing 46 amino acids, whose region composed of the first 18 amino acids residues presents bactericidal activity as the whole peptide, without hemolytic activity. The mode of action of antimicrobial peptide Esc 1b (1-18) was studied using mimetic membranes composed of zwitterionic and anionic phospholipids in different physical states. Measurements of carboxyfluorescein leakage, isothermal titration calorimetry and interaction measures using Lagmuir monolayers were made to verify the effect of the peptide on model membranes. To observe the influence of Esc 1b (1-18) in the bilayer, measurements of differential scanning calorimetry and electron paramagnetic resonance were made, and the effect of aggregation caused by the peptide in the membranes was verified using measures of static and dynamic light scattering and Zeta potential. Measurements of circular dichroism and polarization modulation infrared reflection-absorption spectroscopy were made to determine the secondary structure of Esc 1b (1-18) in the presence of vesicles and monolayers, respectively. In general, the results of carboxyfluorescein leakage, isothermal titration calorimetry and maximum insertion pressure show that the peptide interacts mainly with negatively charged and fluid state membranes. Furthermore, it was observed that Esc 1b (1-18) causes a larger influence in the phase transition on DPPG membranes and the peptide inserts deeply on negatively charged membranes. Lastly, it was observed that the peptide presents α-helical secondary structure in the presence of charged membranes. Therefore, the antimicrobial peptide Esculentin 1b (1-18) interacts selectively with negatively charged membranes.
  • Imagem de Miniatura
    Item
    Embargo
    Influência dos glicosaminoglicanos na citotoxidade de peptídeos catiônicos da família dos mastoparanos
    (Universidade Federal de São Paulo, 2021-08-30) Muniz Seif, Elias Jorge [UNIFESP]; Arcísio Miranda, Manoel; http://lattes.cnpq.br/2680038286691388; http://lattes.cnpq.br/8706280549429736; Universidade Federal de São Paulo - UNIFESP
    OBJETIVO: Mensurar a influência dos glicosaminoglicanos no potencial citotóxico dos peptídeos MPX, HR1 e MP1. METODOLOGIA: As células de CHO-K1 (ATCC CCL-61TM), tipo selvagem, e CHO-745 (ATCC CRL-2242TM) com produção reduzida de glicosaminoglicano foram cultivadas em meio F12 suplementado com 5% de soro fetal bovino e 1% de antibióticos (penicilina e estreptomicina).O brometo de 3-(4,5-dimetil-2- tiazolil)-2, 5-difenil-2H-tetrazólio (MTT) foi utilizado para determinar as concentrações de citotoxicidade média de cada peptídeo para o tratamento durante 24 horas. Na sequência, foi realizada citometria de fluxo para determinação da via de morte celular. A interação dos peptídeos com a membrana plasmática foi monitorada através sonda FPE (N-(Fluorescein-5-Thiocarbamoyl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanol-amine, riethyl-ammonium Salt). RESULTADO: Os experimentos de MTT e citometria de fluxo mostraram que os peptídeos foram potencialmente citotóxicos para as duas linhagens estudadas, sendo mais eficaz contra as células de linhagem CHO-K1. Para o experimento de MTT observou-se diferença de 13,77%, 24,73% e 11,79% para a viabilidade celular após tratamento com MPX, HR1 e MP1, respectivamente. Já a citometria de fluxo mostrou diferença de viabilidade celular de 28,52%, 20,34% e 18,40% após o tratamento com MPX, HR1 e MP1, respectivamente. Além disso, a necrose foi observada como a principal via de morte celular para todos os peptídeos nas duas linhagens celulares utilizadas nesse estudo. Os peptídeos foram capazes de se ligar na membrana de ambas as linhagens. Todavia, essa ligação foi maior na linhagem CHO-K1, sendo 58%, 48% e 11% maior para MPX, HR1 e MP1, respetivamente. CONCLUSÃO: Os peptídeos mastoparanos são potencialmente mais citotóxicos à linhagem CHO-K1 quando comparados a linhagem CHO-745, resultando em uma maior ligação de peptídeos em sua membrana. Essa pesquisa sugere que a ausência de glicosaminoglicanos é um fator protetivo contra atividade membranolítica desses peptídeos.
  • Imagem de Miniatura
    Item
    Acesso aberto (Open Access)
    Interação da citosporona-b com modelos de membrana na interface ar-água
    (Universidade Federal de São Paulo, 2023-08-28) Jaroque, Guilherme Nuñez [UNIFESP]; Caseli, Luciano [UNIFESP]; Sartorelli, Patricia [UNIFESP]; http://lattes.cnpq.br/6836392358779448; http://lattes.cnpq.br/8929162910172931; http://lattes.cnpq.br/3306052344968821
    The study of interactions at the molecular level of compounds that have some bioactive properties with membrane models allows us to understand the bioactive-membrane mechanism of action in detail. Among these models, we can use the Langmuir monolayers, which are monomolecular films built at the air-water interface. Therefore, this work aimed to study the interaction between Cytosporone-B (Csn-B), a secondary metabolite isolated from the endophyte fungus Phomopsis sp., with Langmuir films made from six phospholipids: dipalmitoylphosphatidylcholine (DPPC) and palmitoyl-oleoyl phosphatidylcholine (POPC), as phospholipids found in erythrocyte cell membranes; dipalmitoylphosphatidylethanolamine (DPPE) and dioleoyl-phosphatidylethanolamine (DOPE), found in bacterial cell membranes; and, finally, dipalmitoylphosphatidylserine (DPPS) and palmitoyl oleoyl phosphatidylserine (POPS), found in cancer cells. To analyze the interaction effects between Csn-B and lipid monolayers, surface pressure versus area per molecule isotherms, tensiometric stability curves, surface potential isotherms, and Brewster angle micrographs (BAM) were obtained. When we analyze the surface pressure versus area per molecule isotherms, it is possible to observe that, for some films, the curve shifted to larger areas, indicating the possible incorporation of the compound into the film. However, for some unsaturated lipids, it was possible to observe a shift in the curve to smaller areas, which may indicate a possible decrease in the repulsion between the polar heads of the phospholipid. Observing the tensiometric stability tests, it is possible to conclude that there was an increase in the instability of the films with the addition of Csn-B since the surface pressure decay rate was more significant in the films with the addition of the compound. There was also an increase in the surface potential of the films after the addition of Csn-B, showing a possible ordering of the electric dipoles of the phospholipids that make up the Langmuir film. The BAM images showed the formation of some interfacial clusters for some monolayer compositions, while there were no significant changes in their morphologies for others. In short, it was possible to notice thermodynamic, structural, and morphological changes in the phospholipid films after the addition of Csn-B, in addition to observing that the chemical composition of the films ends up altering the physical-chemical phenomenon observed. We believe that such data can contribute to a possible understanding of the molecular action of substances with possible biological activities in natural lipid interfaces.
  • Nenhuma Miniatura disponível
    Item
    Somente Metadadados
    Obtenção, caracterização e reconstituição da protease PgtE de Salmonella enterica sorovar Typhimurium em lipossomos: um estudo do mecanismo de resistência aos peptídeos antimicrobianos
    (Universidade Federal de São Paulo (UNIFESP), 2020-09-24) Silva, Pamela Jacque De Souza Nascimento Da [UNIFESP]; Perez, Katia Regina [UNIFESP]; Universidade Federal de São Paulo
    Multidrug-resistant gram-negative bacteria represents one of the most prominent public health problems worldwide. Omptin family of the Outer membrane protein, such as PgtE from Salmonella spp. Enterica serovar Typhimurium, are able to cleave a variety of protein substrates, including antimicrobial peptides that have shown promise in the treatment of multi-resistant bacteria. Objective: to obtain and characterize the PAM-resistant proteoliposomes of S. typhi from the incorporation of the recombinant PgtE protein in liposomes. Methods: In this work, we describe how to use recombinant PgtE in its soluble and active form by cloning the gene encoding native PgtE S. typhi in the pET 28a (+) expression vector. The recombinant proteins expression was conducted in the hosted type E. coli BL21 (Dε3) under moderate conditions to avoid protein aggregation in inclusion bodies. It also describes the rational development of the protocols used for the extraction of proteins from the membrane through solubilization with the surfactant SDS. After protein solubilization, establish a convenient purification protocol to obtain a protein with a high degree of homogeneity and proteolytic activity used with SDS and CHAPS surfactant. Determined as kinetic constants (kcat, KM and kcat/KM) against a peptide substrate with intramolecular suppression of fluorescence in order to analyze the specific activity of the recombinant protein used in its solubilized and purified form. To use the proteoliposomes, we perform the incorporation of the recombinant protein in the DPPC or DPPC:DPPG 1:1 liposomes using the co-solubilization method that uses the BioBeads® resin. And it characterizes these proteoliposomes that are conducted by biophysical approaches of calorimetry through DSC and fluorescence to evaluate how changes in the incorporation in the thermodynamic characteristics of the membranes, as well as in the recovery of enzymatic activity when a substance is reconstituted. Results: The use of surfactant was important to obtain recombinant PgtE in its active soluble form after being removed from the lipid bilayer. The optimization of the solubilization and purification protocol using the SDS and CHAPS surfactants allowed obtaining the soluble and active protein with the highest possible yield and a high degree of homogeneity. The incorporation by the method of co-solubilization of lipids, protein and detergent with subsequent selective adsorption of the detergent using the Biobeads® resin was a methodology for obtaining the DPPC and DPPC proteoliposomes: effective DPPG, since we reached values between 87,1 and 90,7% xix protein incorporation yield. The DSC assays showed a more significant change in the profile of the membrane lipids phase transition thermogram, greater changes in the ∆H and in the cooperativity of the lipids of proteoliposomes made up of DPPC: DPPG. Kinetic assays suggest that negative charge on the membrane may influence PgtE activity. Conclusion: The strategies adopted in all stages, from obtaining the protein and purification to the characterization of the proteoliposome system, allowed to study the proteolytic mode of action of PgtE in a less complex and non-pathogenic system and opportunity to allowing the study of candidate peptides to treatment of infections by multi-resistant bacteria as an alternative to treatment using antibiotics.