Dissertação para obtenção do Grau de Doutor em
Bioengenharia (MIT)Metalloproteinases are endopeptidases involved in mediating interactions between cells and the extracellular microenvironment, being ascribed to several cellular processes and signaling events. The dysregulation of protease activity has been correlated with several diseased states, such as cancer and arthritis; hence metalloproteinases constitute potential therapeutic targets. However, these therapies have thus far shown little success due to an ill-defined knowledge of the complex regulatory network dictating protease activity. The availability of 3D in vitro tissues in which cells are able to closely recapitulate native physiological behavior, has been essential for studying tissue physiology and for drug development. Therefore, the design of real-time specific protease activity sensors amenable to 3D tissue constructs would be a valuable tool for understanding fundamental aspects of protease biology as well as monitor drug activity towards proteases. Herein, bio-friendly technologies were combined for the preparation of cell-interactive hydrogel particles functionalized with metalloproteinase fluorogenic sensors to monitor local protease activity in 3D cell cultures. Well-defined and cell-sized smart microgels were prepared by synthesis in supercritical carbon dioxide, a green chemistry approach that enables pure and biocompatible materials, without extensive purification steps. Further aqueous complexation of macromolecules or layer-by-layer assembly of polyelectrolytes was used for the preparation of cell-interactive coatings and for fine-tuning the microbeads physical properties of swelling, stimuli-responsive behavior and overall net charge; being a potential strategy to adjust microbead differential permeability to proteases and thus the sensing construct specificity. The impact of the macromolecules deposition on the smart microbeads behavior was assessed by FT-IR spectroscopy analysis. Finally, protease sensing function was conferred to the microbeads by complexation of a modified polymer or by directly tethering probes on the native microbead. The successful activation of the microbeads by a model protease constitutes a significant step towards the validation of this technological basis for preparing local protease probes for 3D engineered tissues
