In order to obtain more selective and tunable Drug Delivery Systems (DDS), “Smart” DDS that are able to 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 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 Ph.D. 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 an ROS-responsive mPEG (methoxy polyethylene glycol) polymer (mPEG-TK-COOH) and, by exploiting Cy5 fluorescent dye, ROS-responsive (mPEG-TK-Cy5) and non-ROS-responsive (mPEG-Cy5) polymer conjugates. Full chemico-physical and technological characterization were performed to confirm the success in polymer conjugation and to describe chemico-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 and with Prof. Boury, University of Angers, during a period of international mobility. Results clearly indicated that mPEG-TK-Cy5 could be released selectively 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 synthesized 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, namely PLGA-TK-COOH and PLGA-TK-PLGA. Aiming 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 PLGA-TK-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 Ph.D. project, could be considered as a promising starting point for their future application in GBM as well as in relevant neurodegenerative diseases as Alzheimer’s disease.

Al fine di ottenere sistemi di somministrazione di farmaci (DDS) più selettivi e regolabili, sono attualmente sotto studio DDS "intelligenti" in grado di rilasciare i farmaci in risposta ad uno stimolo specifico (pH, GSH, ROS, etc). Ad esempio lo stress ossidativo caratterizza malattie infiammatorie, neurodegenerative e il cancro; la progettazione di DDS “ROS-responsive” potrebbe dimostrarsi quindi un approccio interessante per il rilascio selettivo di farmaci per il trattamento di queste malattie. Pertanto, lo scopo di questa tesi di dottorato è stato quello di sviluppare coniugati e profarmaci polimerici “ROS-responsive” collegati a Tiochetale (TK) diacido, come linker sensibile ai ROS, che potrebbe essere sfruttato per il trattamento delle malattie cerebrali. Con l'obiettivo di convalidare l'uso di polimeri e profarmaci ROS-responsive contenenti TK, abbiamo eseguito studi preliminari sintetizzando un polimero mPEG (metossi polietilenglicole) sensibile ai ROS (mPEG-TK-COOH). Sfruttando un marker fluorescente come farmaco modello (Cy5), abbiamo realizzato coniugati polimerici sensibili (mPEG-TK-Cy5) e non sensibili (mPEG-Cy5) ai ROS. La completa caratterizzazione chimico-fisica e tecnologica è stata eseguita per confermare il successo della coniugazione polimerica e per descrivere le proprietà chimico-fisiche dei coniugati ottenuti. La capacità di questi coniugati di rispondere ai ROS è stata validata in condizioni simulate dai ROS e valutata in vitro su linee cellulari di Glioblastoma multiforme (GBM). Questi test sono stati condotti in collaborazione con il Prof. Grabrucker dell’Università di Limerick e con il Prof. Boury dell’ Università di Angers, durante un periodo di mobilità internazionale. I risultati hanno indicato chiaramente che mPEG-TK-Cy5 potrebbe rilasciare selettivamente il farmaco modello in condizioni "patologiche" (cellule C6 GBM) rispetto a condizioni "sane" (cellule di astrociti DI TNC1). In secondo luogo, per il rilascio “ROS-responsive” di Melphalan (MPH), un farmaco antitumorale scarsamente solubile e non selettivo, mirato al trattamento del GBM, è stato sintetizzato un profarmaco (mPEG-TK-MPH) sensibile ai ROS ed il suo controllo non sensibile ai ROS (mPEG-MPH). Entrambi i profarmaci sono stati caratterizzati e si sono sintetizzate strutture nanometriche sferiche autoassemblanti. I saggi di citotossicità in vitro eseguiti su cellule GBM hanno dimostrato come mPEG-TK-MPH sia significativamente più citotossico su cellule GBM con elevati livelli di ROS (cellule C6 e U251MG) rispetto al controllo (mPEG-MPH). Sorprendentemente, nessuno dei profarmaci ha mostrato di essere citotossico su cellule di astrociti con bassi livelli di ROS (DI TNC1), confermando la loro safety. Infine, con l’obiettivo di ottenere un rilascio selettivo (nelle malattie con stress ossidativo) e sfruttando la potenzialità di NPs polimeriche (a base di acido polilattico-co-glicolico, PLGA) abbiamo prodotto e caratterizzato altri coniugati polimerici “ROS-responsive” con PLGA (PLGA-TK-COOH e PLGA-TK-PLGA) e formulati in NPs funzionalizzate. Possiamo concludere quindi che a presciendere dal successo di coniugazione chimica e formulazione tecnologica, per i risultati in vitro ottenuti, l’approccio “ROS-responsive prodrug” si è dimostrato promettente per un possibile trattamento del GBM, caratterizzato da elevati livelli di ROS; approccio che può essere ulteriormente ampliato sfruttando anche altri polimeri (come il PLGA) e applicato non solo al GBM ma anche ad altre patologie caratterizzte da alti livelli di ROS, come le patologie neurodegenerative.

Coniugati e profarmaci polimerici “ROS- responsive” come DDS innovativo per il trattamento delle malattie cerebrali / Natalia Irene Oddone , 2020 Mar 19. 32. ciclo, Anno Accademico 2018/2019.

Coniugati e profarmaci polimerici “ROS- responsive” come DDS innovativo per il trattamento delle malattie cerebrali.

ODDONE, NATALIA IRENE
2020

Abstract

In order to obtain more selective and tunable Drug Delivery Systems (DDS), “Smart” DDS that are able to 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 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 Ph.D. 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 an ROS-responsive mPEG (methoxy polyethylene glycol) polymer (mPEG-TK-COOH) and, by exploiting Cy5 fluorescent dye, ROS-responsive (mPEG-TK-Cy5) and non-ROS-responsive (mPEG-Cy5) polymer conjugates. Full chemico-physical and technological characterization were performed to confirm the success in polymer conjugation and to describe chemico-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 and with Prof. Boury, University of Angers, during a period of international mobility. Results clearly indicated that mPEG-TK-Cy5 could be released selectively 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 synthesized 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, namely PLGA-TK-COOH and PLGA-TK-PLGA. Aiming 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 PLGA-TK-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 Ph.D. project, could be considered as a promising starting point for their future application in GBM as well as in relevant neurodegenerative diseases as Alzheimer’s disease.
ROS-responsive polymer conjugates and prodrugs as innovative DDS aiming for the treatment of brain diseases
19-mar-2020
TOSI, Giovanni
RUOZI, Barbara
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/1201012
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