Browsing by Author "Rodrigues, Susana"
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- Bacillus subtilis 168 Carboxylesterase A: characterization and selection of improved variants towards IPG-C4Publication . Rodrigues, Susana; Quax, Wim. J.; Godinho, Luís; Melo, EduardoEnzymes had become an attractive tool for the kinetic resolution of racemic mixtures for the enantiopure production of chiral pharmaceuticals. Sometimes the existing enzymes are not suitable for the desired process which led to the development of directed evolution. In directed evolution experiments, success strongly depends on the availability of genetic screening or selection methods. However enantioselectivity is a difficult parameter to select for. In 2008 Boersma, et al. had developed a new selection system based on bacterial growth that not only selects for catalytic activity but also for enantioselectivity. Despite of having been used with Bacillus subtilis 168 Lipase A (LipA) the author suggests that other enzymes could be applied to the method. Growth selection designed for LipA were applied to Bacillus subtilis 168 Carboxilesterase A (CesA), despite of the low activity of CesA to the substrate that led to a longer incubation time (4 days instead of 2), and false positives of about 25 % the method seems to work and some enantioselective mutants were obtained. The preliminary studies in the gas chromatography (GC) analysis of the periplasm fraction of some mutants revealed that the enantioselectivity had increased to the desired enantiomer, R-1,2-O-isopropilideno-sn-glicerol-butyrate. Despite of this work was a preliminary study the results showed that the developed method of selection system based on bacterial growth, designed for LipA, can be performed with other enzymes. In the present work it was obtained high values for the first round of screening, enantiomeric excess values (ee) around 60%.
- Biocompatibility and stability of polysaccharide polyelectrolyte complexes aimed at respiratory deliveryPublication . Rodrigues, Susana; Cardoso, Lurdes; Costa, Ana M. Rosa da; Grenha, AnaChitosan (CS) and chondroitin sulfate (CHS) are natural polymers with demonstrated applicability in drug delivery, while nanoparticles are one of the most explored carriers for transmucosal delivery of biopharmaceuticals. In this work we have prepared CS/CHS nanoparticles and associated for the first time the therapeutic protein insulin. Fluorescein isothiocyanate bovine serum albumin (FITC-BSA) was also used to enable comparison of behaviors regarding differences in molecular weight (5.7 kDa versus 67 kDa). Nanoparticles of approximately 200 nm and positive zeta potential around +20 mV were obtained. These parameters remained stable for up to 1 month at 4 C. Proteins were associated with efficiencies of more than 50%. The release of FITC-BSA in PBS pH 7.4 was more sustained (50% in 24 h) than that of insulin (85% in 24 h). The biocompatibility of nanoparticles was tested in Calu-3 and A549 cells by means of three different assays. The metabolic assay MTT, the determination of lactate dehydrogenase release, and the quantification of the inflammatory response generated by cell exposure to nanoparticles have indicated an absence of overt toxicity. Overall, the results suggest good indications on the application of CS/CHS nanoparticles in respiratory transmucosal protein delivery, but the set of assays should be widened to clarify obtained results.
- Biocompatibility of chitosan carriers with application in drug deliveryPublication . Rodrigues, Susana; Dionísio, Marita; Remuñán-López, Carmen; Grenha, AnaChitosan is one of the most used polysaccharides in the design of drug delivery strategies for administration of either biomacromolecules or low molecular weight drugs. For these purposes, it is frequently used as matrix forming material in both nano and micron-sized particles. In addition to its interesting physicochemical and biopharmaceutical properties, which include high mucoadhesion and a great capacity to produce drug delivery systems, ensuring the biocompatibility of the drug delivery vehicles is a highly relevant issue. Nevertheless, this subject is not addressed as frequently as desired and even though the application of chitosan carriers has been widely explored, the demonstration of systems biocompatibility is still in its infancy. In this review, addressing the biocompatibility of chitosan carriers with application in drug delivery is discussed and the methods used in vitro and in vivo, exploring the effect of different variables, are described. We further provide a discussion on the pros and cons of used methodologies, as well as on the difficulties arising from the absence of standardization of procedures.
- Carrageenan from red algae: an application in the development of inhalable tuberculosis therapy targeting the macrophagesPublication . Rodrigues, Susana; Cunha, Ludmylla Costa; Martins Rico, João; Rosa Da Costa, Ana; Almeida, Antonio J.; Faleiro, ML; Buttini, Francesca; Grenha, AnaMacrophages have unique surface receptors that might recognize preferentially several moieties present on the surface of infecting organisms, including in the bacterial cell wall. Benefiting from a similar composition regarding the referred moieties, polysaccharides might be good candidates to compose the matrix of drug carriers aimed at macrophage targeting, as they can use the same recognition pathways of the infecting organisms. Carrageenan (CRG), a polysaccharide extracted from red edible seaweed, is an interesting possibility for the approach of directly targeting alveolar macrophages, as its composition is reported to be recognized by several macrophage lectin receptors. Inhalable starch/CRG microparticles were successfully produced, effectively associating isoniazid (96%) and rifabutin (74%) simultaneously. Furthermore, the produced microparticles presented adequate aerodynamic properties for pulmonary delivery with potential to reach the respiratory zone, with a mass median aerodynamic diameter (MMAD) between 3.3 and 3.9 mu m. It was further demonstrated that the antitubercular activity of the drugs remained unchanged after encapsulation. The formulation evidenced no cytotoxic effects on lung epithelial cells (A549), although mild toxicity was observed on macrophage-differentiated THP-1 cells for the drug-loaded formulation. Starch/CRG microparticles also exhibited a propensity to be captured by macrophages in a dose-dependent manner, as well as an ability to activate the target cells. This work provides indications on the potential of the starch/CRG carriers to interact with macrophages, thus providing a platform for drug delivery in the context of macrophage intracellular diseases. Additionally, if tuberculosis is focused, these microparticles can be used as inhalable drug carriers.
- Chitosan/carrageenan nanoparticles: effect of cross-linking with tripolyphosphatePublication . Rodrigues, Susana; Costa, Ana M. Rosa da; Grenha, AnaChitosan/carrageenan/tripolyphosphate nanoparticles were prepared by polyelectrolyte complexation/ionic gelation, the latter compound acting as cross-linker. The incorporation of the three components in the nanoparticle matrix was assessed by analytical techniques (FTIR, XPS and TOF-SIMS). Using chitosan/carrageenan nanoparticles as control, the effect of the cross-linker in the particles properties was studied. A decrease in size (from 450-500 nm to 150-300 nm) and in zeta potential (from +75 - +85 mV to +50 - +60 mV), and an increase in production yield (from 15-20% to 25-35%), and in stability (from one week to up to 9 months) were observed. Also, a correlation between positive to negative charge ratios in the formulations and the above characteristics was established. The small size and high positive surface charge make the developed chitosan/carrageenan/tripolyphosphate nanoparticles potential tools for an application in mucosal delivery of macromolecules.
- Chondroitin sulphate microparticles for tuberculosis treatment: a way to target macrophagesPublication . Rodrigues, Susana; Rosa Da Costa, Ana; Grenha, AnaTuberculosis remains a leading cause of death; therapeutic failure being mainly due to non-compliance with prolonged treatments, often associated with severe side-effects. New therapeutic strategies are demanded and, considering that the lung is the primary site of infection, direct lung delivery of antibiotics is an interesting and, possibly, effective approach. Therapeutic success in this context depends on suitable carriers that reach the alveoli where Mycobacterium hosts (macrophages) reside, as well as on their ability to promote macrophage capture and intracellular accumulation of drugs. In this work, we propose inhalable chondroitin sulphate microparticles produced by spray-drying and tailored to suitable aerodynamic properties to reach the alveoli. Macrophage targeting will be driven by microparticle size, which is favoured for carriers of 1-2 μm, and composition based on chondroitin sulphate, a glycosaminoglycan comprised of alternating units of sulphated N-acetylgalactosamine and glucuronic acid residues, the former recognized by macrophage receptors. Spray-drying of chondroitin sulphate in combination with two first-line antitubercular drugs (isoniazid and rifabutin) was successful with a satisfactory production yield (> 70%). Microparticles have Feret’s diameter of 1.6 μm, potentiating macrophage uptake. Chondroitin sulphate as solution or microparticulate form and drug-loaded microparticles appear to not have a cytotoxic effect on alveolar epithelial cells. A more extended biocompatibility/safety assessment of this formulation needs to be performed. Taking into account the general trend of the results obtained so far, good indications are given that encourage the continuation of the studies in order to establish the potential of these microparticles as inhalable carriers in tuberculosis treatment.
- Cytocompatibility and cellular interactions of chondroitin sulfate microparticles designed for inhaled tuberculosis treatmentPublication . Rodrigues, Susana; Cunha, Ludmylla Costa; Kollan, Julia; Neumann, Paul Robert; Costa, Ana Rosa da; Dailey, Lea Ann; Grenha, AnaTuberculosis remains a leading cause of death, therapeutic failure being mainly due to non-compliance with prolonged treatments, often associated with severe side-effects. New therapeutic strategies are demanded and, considering that the lung is the primary site of infection, direct lung delivery of antibiotics is possibly an effective approach. Therapeutic success in this context depends on suitable carriers that reach the alveoli where Mycobacterium hosts (macrophages) reside, as well as on their ability to promote macrophage capture and intracellular accumulation of drugs. In this work, we propose inhalable polymeric microparticles produced from chondroitin sulfate, a polymer composed by moieties recognized by macrophage receptors. Spray-drying of chondroitin sulfate in combination with two first-line antitubercular drugs (isoniazid and rifabutin) yielded respirable microparticles that evidenced no cytotoxic effects on lung epithelial cells (A549) and macrophages (dTHP1 and J744A.1). The microparticles exhibited tendency for macrophage capture in a dose-dependent manner, which was validated through imaging. High content image analysis revealed that rifabutin induced a dose-dependent increase in phospholipid content of macrophages, which could be prevented by formulation in chondroitin sulfate microparticles. This work provides indications on the potential of chondroitin sulfate carriers to interact with macrophages, thus providing a platform for drug delivery in the context of macrophage intracellular diseases, namely tuberculosis.
- Dificuldades de aprendizagem da leitura e da escrita: a perceção dos professores de 1.º ciclo da região do AlgarvePublication . Baptista, Ana Catarina; Rodrigues, Susana; Hilário, Ana; Silva, Andreia; Leal, VerónicaA leitura e a escrita são consideradas ferramentas básicas e cruciais para a aprendizagem escolar. Os professores devem ter conhecimento das componentes da leitura, da estrutura da linguagem, das dificuldades de aprendizagem e de práticas efetivas de avaliação e de intervenção pedagógicas de crianças em risco de insucesso escolar. Com este estudo pretende-se conhecer a perceção dos professores do 1º Ciclo do Ensino Básico (CEB) da região do Algarve sobre as crianças com dificuldades de leitura e escrita. Foi construído um questionário difundido via correio eletrónico aos professores do 1º CEB da região do Algarve, tendo-se obtido 46 respostas. Apenas 13% dos professores que responderam a este estudo, refere que tem formação específica na área das dificuldades de leitura e escrita. É também referido o 2º ano de escolaridade como aquele que se verifica mais insucesso escolar, o qual os professores associam às dificuldades de aprendizagem de leitura e escrita.
- Dual antibiotherapy of tuberculosis mediated by inhalable locust bean gum microparticlesPublication . Rodrigues, Susana; Alves, Ana D.; Cavaco, Joana S.; Pontes, Jorge Filipe; Guerreiro, Filipa; Rosa Da Costa, Ana; Buttini, Francesca; Grenha, AnaDespite the existence of effective oral therapy, tuberculosis remains a deadly pathology, namely because of bacterial resistance and incompliance with treatments. Establishing alternative therapeutic approaches is urgently needed and inhalable therapy has a great potential in this regard. As pathogenic bacteria are hosted by alveolar macrophages, the co-localisation of antitubercular drugs and pathogens is thus potentiated by this strategy. This work proposes inhalable therapy of pulmonary tuberculosis mediated by a single locust bean gum (LBG) formulation of microparticles associating both isoniazid and rifabutin, complying with requisites of the World Health Organisation of combined therapy. Microparticles were produced by spray-drying, at LBG/INH/RFB mass ratio of 10/1/0.5. The aerodynamic characterisation of microparticles revealed emitted doses of more than 90% and fine particle fraction of 38%, thus indicating the adequacy of the system to reach the respiratory lung area, thus partially the alveolar region. Cytotoxicity results indicate moderate toxicity (cell viability around 60%), with a concentration-dependent effect. Additionally, rat alveolar macrophages evidenced preferential capture of LBG microparticles, possibly due to chemical composition comprising mannose and galactose units that are specifically recognised by macrophage surface receptors. (C) 2017 Elsevier B.V. All rights reserved.
- Effect of Erica australis extract on Caco-2 cells, fibroblasts and selected pathogenic bacteria responsible for wound infectionPublication . Nunes, Ricardo; Rodrigues, Susana; Pasko, Pawel; Tyszka-Czochara, Malgorzata; Grenha, Ana; Carvalho, Isabel Saraiva dePlants from the genus Erica are used in many countries to treat several ailments. In this work we intend to evaluate the potential in vivo benefits of Erica australis L. by testing in vitro the effect induced by the plant extract when in contact with BJ fibroblasts (3 and 9 hours) and Caco-2 cells (3, 6 and 24 hours). Effects on five pathogenic microorganisms(Enterococcus faecalis, Bacillus cereus, Escherichia coli, Staphylococcus aureus and Listeria monocytogenes) were also determined. It was found that the extracts enhanced fibroblast proliferation (maximum of 484% of control at 6 hour exposure) while Caco-2 cells viability was reduced in a concentration and time dependent manner (minimum of 22.3% of control at 24 hour exposure). Antimicrobial effects were also detected, with differences registered among the plant parts and solvent used, with the lowest minimum concentration for diffusion inhibition (MCDI) of 1 mg/mL. Results obtained with the fibroblasts and bacteria strongly show that this plant has potential to be used in wound healing as a stimulant of fibroblast growth and disinfection, as well as an antibiotic. Results obtained with Caco-2 cells indicate this plant also has some potential for and application as anticancer agent.