RESUMEN
Electrospinning is an attractive method to generate drug releasing systems. In this work, we encapsulated the cell death-inducing drug Diclofenac (DCF) in an electrospun poly-L-lactide (PLA) scaffold. The scaffold offers a system for a sustained and controlled delivery of the cytotoxic DCF over time making it clinically favourable by achieving a prolonged therapeutic effect. We exposed human dermal fibroblasts (HDFs) to the drug-eluting scaffold and employed multiphoton microscopy and fluorescence lifetime imaging microscopy. These methods were suitable for non-invasive and marker-independent assessment of the cytotoxic effects. Released DCF induced changes in cell morphology and glycolytic activity. Furthermore, we showed that drug release can be influenced by adding dimethyl sulfoxide as a co-solvent for electrospinning. Interestingly, without affecting the drug diffusion mechanism, the resulting PLA scaffolds showed altered fibre morphology and enhanced initial DCF burst release. The here described model could represent an interesting way to control the diffusion of encapsulated bio-active molecules and test them using a marker-independent, non-invasive approach.
Asunto(s)
Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Supervivencia Celular/efectos de los fármacos , Diclofenaco/administración & dosificación , Diclofenaco/química , Diclofenaco/farmacología , Fibroblastos/efectos de los fármacos , Humanos , Poliésteres/químicaRESUMEN
Mesenchymal stem cells have attracted great interest in the field of tissue engineering and regenerative medicine because of their multipotentiality and relative ease of isolation from adult tissues. The medical application of this cellular system requires the inclusion in a growth and delivery scaffold that is crucial for the clinical effectiveness of the therapy. In particular, the ideal scaffolding material should have the needed porosity and mechanical strength to allow a good integration with the surrounding tissues, but it should also assure high biocompatibility and full resorbability. For such a purpose, protein-inspired biomaterials and, in particular, elastomeric-derived polypeptides are playing a major role, in which they are expected to fulfil many of the biological and mechanical requirements. A specific chimeric protein, designed starting from elastin, resilin and collagen sequences, was characterized over different length scales. Single-molecule mechanics, aggregation properties and compatibility with human mesenchymal stem cells were tested, showing that the engineered compound is a good candidate as a stem cell scaffold to be used in tissue engineering applications.
Asunto(s)
Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Péptidos/síntesis química , Péptidos/metabolismo , Andamios del Tejido , Proliferación Celular , Supervivencia Celular/fisiología , Células Cultivadas , Proteínas de la Matriz Extracelular/genética , Humanos , Péptidos/genética , Ingeniería de Proteínas/métodosRESUMEN
Calorimetric studies were performed on exon 6 in powdered form and in solution [water and 2,2,2-trifluoroethanol (TFE), a structure-inducing solvent or cosolvent]. Dynamic dielectric spectroscopy (DDS) analyses were realized in water and 20% TFE. The major role of solvent-peptide organization is evidenced with these techniques. Calorimetric measurements reveal the structural water organization around the polypeptide as well as the presence of hydrophobic interactions in TFE solution. Dielectric measurements showed for exon 6/water a decrease of relaxations times of bulk solvent implying a faster dynamics with a slight increase of the activation entropy, suggesting that exon 6 probably creates disorder within the solvent. For TFE/water mixtures, an influence of exon 6 on its environment was seen with a relaxation associated with the exon 6/solvent interactions reinforced by storage of 72 h. Finally, exon 6/solvent interactions were clearly observed with addition of TFE.
Asunto(s)
Exones , Fragmentos de Péptidos/química , Solventes/química , Trifluoroetanol/química , Tropoelastina/química , Secuencia de Aminoácidos , Rastreo Diferencial de Calorimetría , Técnicas Electroquímicas , Humanos , Datos de Secuencia Molecular , Nefelometría y Turbidimetría , Fragmentos de Péptidos/síntesis química , Estructura Terciaria de Proteína , Espectrometría de Masa por Ionización de Electrospray , Temperatura , Tropoelastina/genética , Agua/químicaRESUMEN
The repetitive sequence GGLGY was found in lamprin, the most important matrix protein of lamprey annular cartilage by Keeley and co-workers. Similar sequences appear also in other proteins, i.e. elastin, spidroin, spider minor ampullate silk proteins, in matrix proteins of the chorion or egg shell membrane of insects and others. We synthesized (GGLGY)n, n=1, 2, 6, because the sequence is repeated six times in the aggregated protein. The peptides were studied both in solution and in the solid state. Because the CD spectra were dominated by aromatic contribution, we synthesized GGLGF and GGLGA in order to carefully interpret the CD spectra. The conformational analysis suggests that all synthetic peptides do adopt the same secondary structure. In solution the peptides present a flexible conformation with a significant amount of PPII structure. In the solid state PPII, beta-pleated-sheets and beta-turns possibly co-exist.