Riassunto analitico
Polymeric nanoparticles (NPs) are some of the most advanced and promising systems in nanomedicine for drug delivery and targeting and in the diagnosis and treatment of a wide number of diseases. One of the main challenges in nanomedicine is the successful encapsulation and stabilization of therapeutic proteins and especially enzymes due to their instability under stress conditions. In fact, several critical steps in the manufacturing of these delivery systems may negatively affect enzyme stability and catalytic activity: contact with organic solvents, heat, mechanical stresses, freeze-drying, etc. are all critical factors which could pose a limit to the viability of such a kind of approach. A possible solution to reduce enzymatic instability to technological strains could be the use of stabilizing compounds during the formulative process, such as specific polypeptides and surfactants. The present thesis work focused on a group of non-ionic surfactants, known as polysorbates or Tween®s, to evaluate their potential stabilizing effect on model enzyme β-Glucosidase during its encapsulation into polymeric NPs made of poly-lactide-co-glycolide by double emulsion solvent evaporation method. In particular, Tween® 20, 60 and 80 were studied not only in relation to their interactions with the enzyme and impact on its catalytic activity, but also with regards to their effect on NPs technological properties (size, zeta potential, weight yield, encapsulation efficiency, loading capacity, etc.). Once the most promising stabilizer was selected, different concentrations were compared to determine the optimal Tween®/β-Glucosidase molar ratio. The study then focused on enzyme release profiles and residual activity in biologically significant conditions (pH 7.4 and 4.5). These results were compared to those obtained when another stabilizer (Bovine serum albumin, BSA) was used and the combination of both Tween® 20 and BSA was also explored. Results show that these polysorbates are effective in stabilizing β-Glucosidase, preserving its activity and increasing its encapsulation without compromising nanoparticle properties. Tween® 20 at a 20% v/v concentration proved to be the most promising stabilizer and its combination with BSA 10% w/v lead to even better outcomes as to enzyme stability and activity. Therefore it can be concluded that, as much as compounds such as polysorbates or albumins are effective in stabilizing enzymes during formulation, combinations of such stabilizers with different properties could pave the way for new technological strategies that should be further explored.
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Abstract
Polymeric nanoparticles (NPs) are some of the most advanced and promising systems in nanomedicine for drug delivery and targeting and in the diagnosis and treatment of a wide number of diseases. One of the main challenges in nanomedicine is the successful encapsulation and stabilization of therapeutic proteins and especially enzymes due to their instability under stress conditions. In fact, several critical steps in the manufacturing of these delivery systems may negatively affect enzyme stability and catalytic activity: contact with organic solvents, heat, mechanical stresses, freeze-drying, etc. are all critical factors which could pose a limit to the viability of such a kind of approach. A possible solution to reduce enzymatic instability to technological strains could be the use of stabilizing compounds during the formulative process, such as specific polypeptides and surfactants. The present thesis work focused on a group of non-ionic surfactants, known as polysorbates or Tween®s, to evaluate their potential stabilizing effect on model enzyme β-Glucosidase during its encapsulation into polymeric NPs made of poly-lactide-co-glycolide by double emulsion solvent evaporation method. In particular, Tween® 20, 60 and 80 were studied not only in relation to their interactions with the enzyme and impact on its catalytic activity, but also with regards to their effect on NPs technological properties (size, zeta potential, weight yield, encapsulation efficiency, loading capacity, etc.). Once the most promising stabilizer was selected, different concentrations were compared to determine the optimal Tween®/β-Glucosidase molar ratio. The study then focused on enzyme release profiles and residual activity in biologically significant conditions (pH 7.4 and 4.5). These results were compared to those obtained when another stabilizer (Bovine serum albumin, BSA) was used and the combination of both Tween® 20 and BSA was also explored. Results show that these polysorbates are effective in stabilizing β-Glucosidase, preserving its activity and increasing its encapsulation without compromising nanoparticle properties. Tween® 20 at a 20% v/v concentration proved to be the most promising stabilizer and its combination with BSA 10% w/v lead to even better outcomes as to enzyme stability and activity. Therefore it can be concluded that, as much as compounds such as polysorbates or albumins are effective in stabilizing enzymes during formulation, combinations of such stabilizers with different properties could pave the way for new technological strategies that should be further explored.
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