“In order to obtain more selective and tunable Drug Delivery Systems (DDS), “Smart” DDS that can release their drugs in response to a specific stimulus (e.g. pH, GSH and ROS), are currently under investigation. Inflammatory diseases, neurodegenerative diseases and cancer, including Glioblastoma (GBM) are all sharing a relevant oxidative stress; therefore the design of ROS- responsive DDS for the treatment of these conditions could be a smart and very encouraging approach to access to a selective and specific delivery mediated by a pathological stimulus. Thus, the aim of this PhD thesis was to develop ROS-responsive polymeric conjugates and prodrugs linked to a ROS cleavable group, namely Thioketal (TK) diacid linker that could be used for the treatment of brain diseases. Aiming to validate the use of TK- containing ROS- responsive polymers and prodrugs, we firstly performed proof-of-concept studies by synthesizing a ROSresponsive methoxy polyethylene glycol (mPEG) polymer (mPEG-TK-COOH) and, by exploiting Cy5 fluorescent dye, ROS-responsive (mPEG-TK-Cy5) and non-ROSresponsive (mPEG-Cy5) polymer conjugates. Full chemical-physical and technological characterization was performed to confirm the success in polymer conjugation and to describe chemical-physical properties of the obtained conjugates; then the ability of these conjugates to respond to ROS was validated in ROS-simulated conditions as well as assessed in vitro on Glioblastoma multiforme (GBM) cell lines. These tests were performed in close collaboration with Prof. Grabrucker, University of Limerick, Ireland, and with Prof. Boury, University of Angers, France, during a period of international mobility. Results clearly indicated that mPEG-TK-Cy5 could be selectively released in “pathological” conditions (C6 GBM cells) over “healthy” conditions (DI TNC1 astrocyte cells). Secondly, a prodrug (mPEG-TK-MPH) for the ROS- responsive release of Melphalan (MPH), which is a poorly soluble and non-selective anticancer drug, was synthetized aiming to GBM treatment. A non-ROS responsive prodrug (mPEG-MPH) was also prepared through a similar synthetic procedure. Both prodrugs were characterized and demonstrated to undergo spontaneous auto- assembling into spherical nanometric structures. In vitro cytotoxicity assays performed on GBM cells, showed that mPEG-TK-MPH was significantly more cytotoxic than mPEG-MPH on High- ROS GBM cells (C6 and U251MG cells). Remarkably, none of the prodrugs showed to be cytotoxic on Low- ROS astrocyte cells (DI TNC1), demonstrating their safety. Finally, since PLGA (polylactic-co-glycolic acid) NPs demonstrated to be promising DDS for their application in several diseases, we produced and characterized other ROS-responsive polymeric conjugates with PLGA: PLGA-TK-COOH and PLGATK-PLGA, for the selective release of surface attached and encapsulated drugs into oxidative stress featuring diseases. We were able (starting from the PLGA conjugates produced) to formulate ROS-responsive TK-surface functionalized PLGA and PLGATK-PLGA NPs, respectively. We can conclude that due to its selective cytotoxicity in High-ROS GBM cells without being toxic to “healthy” cells, our developed ROS-responsive prodrugs show encouraging results for GBM treatment. On the other hand, the ROS-responsive PLGA NPs developed during this PhD project, could be considered as promising starting point for their future application in GBM as well as in relevant neurodegenerative diseases as Alzheimer’s disease.”
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