The following thesis work focuses on the synthesis of redox-responsive fluorinated cationic polymers for biomedical applications, such as gene delivery and fluorine magnetic resonance imaging. To ensure transfection across biological barriers, the use of vectors, such as non-viral polymeric vectors, is necessary. The goal of our systems is to improve the investigation and detection of tumors, and this is where the use of redox responsive and the idea of exploiting the dual fluorinated element as detection signals that allow the discrimination between healthy and nonhealthy tissue comes in. In the first part of the work, redox responsive fluorinated building blocks were developed, having in common a disulfide bridge, for redox-responsive behavior, and one or more fluorinated groups. Starting from this basic structure, rhodaminated redox-responsive systems, useful for investigating the mechanism of internalization and release in cells, and guanidine redox responsive system, useful for better investigating the interaction with the cell membrane, were also studied. Starting from this building block library, cationic polymers were obtained by functionalizing commercial 1.8K bPEI via Michael type addition reaction, achieving different degrees of functionalization. The obtained fluorinated polymers were characterized by 1H-NMR to assess the degree of functionalization and 19F-NMR to confirm the presence of the added fluorinated groups. An in-depth study of the actual redox -responsive ability of the systems was carried out by considering the fluorinated polymers with the best characteristics. This study was carried out by simulating the tumor environment at temperature, pH and glutathione concentration via dialysis studies release of the fluorinated sulfide part broken due GSH reductive action on disulfide bridge. Once the effective redox capacity of the created fluorinated systems was demonstrated, an in vitro cytotoxicity and release study was carried out on a human primary cell line, Huvec, as they are widely used and studied in the tumor setting for the study of angiogenesis and would correspond to the first barrier that the system should encounter i.e., vascular tissue.
Il seguente lavoro di tesi si concentra sulla sintesi di polimeri cationici fluorurati redox -responsive per applicazioni biomedicali, quali gene delivery e imaging al fluoro tramite 19F-MRI. Per assicurare la trasfezione attraverso le barriere biologiche è cruciale l’utilizzo di vettori, quali ad esempio i vettori polimerici. L'obbiettivo dei nostri sistemi è quello di migliorare l'investigazione e l'individuazione dei tumori e in questo contesto entra in gioco l'utilizzo dei redox responsive e l'idea di sfruttare il doppio elemento fluorurato come segnali di rilevamento che permettono il discrimine tra tessuto sano e no. Nella prima parte del lavoro, sono stati sviluppati building block fluorurati redox responsive, avente in comune un ponte disulfuro e uno o più gruppi fluorurati. Partendo da questa struttura di base sono stati studiate anche varianti rodaminate, utili per investigare il meccanismo di internalizzazione e rilascio nelle cellule, e varianti con gruppo guanidino, utili per investigare meglio l'interazione con la membrana cellulare. Partendo da questa libreria di building block si sono ottenuti polimeri cationici funzionalizzando il polimero commerciale bPEI 1.8K con diversi gradi di funzionalizzazione. I polimeri fluorurati ottenuti sono stati caratterizzati mediante 1H NMR per valutarne il grado di funzionalizzazione e 19F-NMR per confermare la presenza dei gruppi fluorurati addizionati. Uno studio approfondito sull'effettiva capacità redox-responsive dei sistemi è stato svolto considerando i polimeri fluorurati con le migliori caratteristiche. Questo studio è stato svolto simulando l'ambiente tumorale tramite temperatura, pH e concentrazione di glutatione. Una volta dimostrato l'effettiva capacità redox dei sistemi fluorurati creati si è proceduto con uno studio in vitro di citotossicità e rilascio, su una linea cellulare primaria umana, Huvec, in quanto sono ampiamente utilizzate e studiate nell'ambito tumorale per lo studio dell'angiogenesi e corrisponderebbe alla prima barriera che il sistema dovrebbe incontrare ovvero il tessuto vascolare.
Synthesis and characterisation of Redox-Responsive fluorinated building blocks to functionalize cationic polymers for cancer therapy
CARUSO, MARTA
2021/2022
Abstract
The following thesis work focuses on the synthesis of redox-responsive fluorinated cationic polymers for biomedical applications, such as gene delivery and fluorine magnetic resonance imaging. To ensure transfection across biological barriers, the use of vectors, such as non-viral polymeric vectors, is necessary. The goal of our systems is to improve the investigation and detection of tumors, and this is where the use of redox responsive and the idea of exploiting the dual fluorinated element as detection signals that allow the discrimination between healthy and nonhealthy tissue comes in. In the first part of the work, redox responsive fluorinated building blocks were developed, having in common a disulfide bridge, for redox-responsive behavior, and one or more fluorinated groups. Starting from this basic structure, rhodaminated redox-responsive systems, useful for investigating the mechanism of internalization and release in cells, and guanidine redox responsive system, useful for better investigating the interaction with the cell membrane, were also studied. Starting from this building block library, cationic polymers were obtained by functionalizing commercial 1.8K bPEI via Michael type addition reaction, achieving different degrees of functionalization. The obtained fluorinated polymers were characterized by 1H-NMR to assess the degree of functionalization and 19F-NMR to confirm the presence of the added fluorinated groups. An in-depth study of the actual redox -responsive ability of the systems was carried out by considering the fluorinated polymers with the best characteristics. This study was carried out by simulating the tumor environment at temperature, pH and glutathione concentration via dialysis studies release of the fluorinated sulfide part broken due GSH reductive action on disulfide bridge. Once the effective redox capacity of the created fluorinated systems was demonstrated, an in vitro cytotoxicity and release study was carried out on a human primary cell line, Huvec, as they are widely used and studied in the tumor setting for the study of angiogenesis and would correspond to the first barrier that the system should encounter i.e., vascular tissue.| File | Dimensione | Formato | |
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2022_05_Caruso_Tesi_01.pdf
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Descrizione: Tesi
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6.52 MB
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2022_05_Caruso_Executive Summary_02.pdf
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Descrizione: Executive Summary
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1.48 MB
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1.48 MB | Adobe PDF | Visualizza/Apri |
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https://hdl.handle.net/10589/205216