In the last years, the development of new polymeric non-viral vectors for gene delivery able to overcome both the cytotoxicity issues and the transfection efficiency limitation which are often linked to the non-viral vectors exploited so far has gained great attention. The principal goal of this thesis is to develop multifunctional fluorinated systems for gene/drug delivery that could possibly overcome this drawback being, at the same time, highly efficient, low cytotoxic, and with a possible implementation in 19F-MRI imaging. Two main projects characterized the thesis, each one composed of sub-projects, dealing with 1) functionalization of PAMAM dendrimers with ad hoc designed fluorinated Michael acceptors, and 2) synthesis of fluorinated block-copolymers. Polyamidoamine (PAMAM) dendrimers are among the most studied cationic polymers as non-viral gene delivery vectors. An “ideal” PAMAM-based gene delivery vector is still missing because of the paradox efficiency/cytotoxicity associated to their generation (the higher the generation, the higher the efficiency and cytotoxicity of the polymer) along with high manufacturing costs. In this contest, the first project is focused on the synthesis of multifunctional PAMAM conjugates 1) via functionalization of the outer primary amines of low generation and medium generation PAMAM (G2 and G4) with building blocks bearing fluorinated moieties along with a guanidino functional group and 2) by crosslinking the same dendrimers with a fluorinated stimuli responsive linker containing a disulfide bond. For the functionalization, we have designed different fluorinated Michael acceptors able to be straightforwardly “clicked” to PAMAM dendrimers without the need of coupling reagents and/or catalysts. The employed “click” functionalization resulted to be a smooth and tunable tool, guarantying higher control of the reaction outcome by varying the equivalent of fluorinated Michael acceptors with respect to the outer amines of dendrimer. Efficient plasmid DNA complexation and negligible cytotoxicity is observed for the obtained conjugates, showing improved gene transfection efficiency as compared to undecorated PAMAM dendrimers, to the corresponding unfluorinated PAMAM conjugates, and, remarkably, to gold standard bPEI 25 kDa. In a second project, the functionalization of terminal primary amines of PAMAM G2 and G4 with a new fluorinated ibuprofen (IBU)-Arg Michael acceptor is described. The obtained fluorinated IBU PAMAM-Arg conjugates were tested as fluorinated pro-drug systems designed to overcome conventional drug formulation. When comparing the IBU release profiles of these systems with the release of the encapsulated drug using fluorinated Arg-PAMAM conjugates, which were previously synthesized by our team, we observe a superior efficiency in controlled and pH-dependent drug release behavior with the fluorinated prodrugs. This superiority is evident in both weak alkaline and physiological buffer solutions compared to the drug encapsulation strategy. These results emphasize the significance of trifluoromethyl groups and suggest the potential for synergistic biomedical applications, such as drug/gene delivery. Consequently, we embarked on an investigation into the catalytic activity of fluorinated macromolecular species. Specifically, we focused on G2 and G4 PAMAM dendritic structures, linear polyethylenimine (lPEI), and branched polyethylenimine (bPEI), aiming to develop a fully multifunctional medical tool. This research line aligns with antisense therapy, which relies on the selectively binding of antisense oligonucleotide to mRNA sequence to prevent or silence the the transcription of disease-related proteins. The investigation presented in the thesis shows that PAMAM G2 and overall PAMAM G4 dendrimers partially functionalized with the designed linkers incorporating trifluoromethyl and guanidino groups are able to efficiently catalyzes the cleavage of a phosphodiester model compounds demonstrating their possible application in antisense oligonucleotide. The second main project of PhD project relies on the synthesis of biodegradable cationic polymers for biomedical applications, namely gene delivery and 19F-MRI, and is splitted in two sub-projects. In the first study, ionizable amino-polyesters were synthesized via ring opening polymerization and free radical polymerization of lipophilic component, producing positively charged polyesters. Sixteen stable nanoparticles were obtained exploiting fluorinated zwitterionic-based polymers by varying the number of the fluorinated units. Depth study over the size of the different NPs obtained were done, followed by a preliminary transfection test in vitro using the polyplexes that have shown the most suitable characteristics. A degradation study via DLS investigation demonstrated the degradability of NPs. Furthermore, a deeply investigation on the influence of the number of the fluorinated units inside zwitterionic backbone outlined a remarkable independent control linked to linear correlation between fluorine intensity, NPs size, and fluorinated units, enforcing RAFT and ROP coupled strategies in NPs development by simply modulating the stoichiometry of polymerization reaction. The second sub-project reported the synthesis of fluorinated PEGylated polymers as novel nano-contrast agents detectable via 19F-MRI. Two reactive PEGylated fluorinated macro-RAFT agents were prepared through acylation and living polymerization reactions and used for subsequent polymerization induced self-assembly (PISA) reactions to synthesize fluorinated and rhodamine labeled NPs. A preliminary in vitro intracellular investigation of the nanotracers revealed an increased cellular internalization with the number of fluorinated units inside the polymeric systems, guarantying an extremely low cytotoxicity.
Negli ultimi anni, lo sviluppo di vettori non-virali polimerici per gene delivery attira sempre più l’interesse della comunità scientifica, per la possibile capacità di superare problemi di citotossicità e biodegradabilità che caratterizzano i sistemi considerati come gold standard attualmente presenti in letteratura. Tuttavia, l’efficienza di trasfezione rimane ancora l’ostacolo principale da superare. L’obiettivo principale di questa tesi è lo sviluppo di sistemi fluorurati multifunzionali per gene/drug delivery, the potrebbero superare i problemi sopracitati e, allo stesso tempo, essere molto efficienti, con una bassa citotossicità e una possibile implementazione nell’imaging al fluoro. La tesi presenta due progetti principali, ognuno costituito da sotto progetti, caratterizzati da 1) funzionalizzazione di PAMAM mediante accettori di Michael fluorurati appositamente sviluppati e 2) la sintesi di polimeri a blocchi. Poliammidoammine (PAMAM) risultano essere tra i sistemi policationici più studiati e investigati come possibili vettori non-virali. Tuttavia, lo sviluppo di un PAMAM “ideale” nell’ambito del gene delivery continua a mancare a causa degli elevati costi di produzione e dalla marcata citotossicità dovuta all’uso di PAMAM ad elevata generazione. Di contro, i PAMAM a bassa generazione mostrano una ridotta citotossicità, ma bassa efficienza di trasfezione. Per superare questa disparità, nel primo progetto di questo dottorato proponiamo la funzionalizzazione e la reticolazione delle ammine primarie poste sulla superficie dei PAMAM a bassa generazione G2 e media G4, mediante l’uso di sistemi modulari fluorurati multifunzionali agenti come accettori di Michael, in grado di legarsi direttamente al PAMAM senza l’ausilio di reagenti di coupling e/o catalizzatori. La strategia di sintesi sviluppata si mostra estremamente efficace, garantendo una funzionalizzazione controllata e organizzata ad hoc dall’operatore variando a priori gli equivalenti di accettore di Michael fluorurato adoperato. I derivati ottenuti mostrano un’efficiente abilità di complessazione del materiale genetico, con una trascurabile citotossicità ed incrementata efficienza di trasfezione rispetto ai sistemi non funzionalizzati e al golden standard 2bPEI5kDa. In un secondo progetto, per superare i problemi legati alla formulazione e alla somministrazione di farmaci, proponiamo la funzionalizzazione dei PAMAM mediante accettori di Michael fluorurati IBU-derivati come sistemi prodrug. Il rilascio di ibuprofene (IBU) è stato analizzato considerando l’incapsulamento del farmaco mediante i coniugati precedentemente sintetizzati, comparandolo al comportamento pH-responsive dei sistemi PAMAM-IBU prodrug. I sistemi fluorurati prodrug ottenuti garantiscono un rilascio controllato del farmaco sia in ambiente fisiologico che in condizioni alcaline. L’importanza della presenza dell’unità trifluorometilica e la possibilità di sviluppare sistemi sinergici per applicazioni biomedicali, ha portato all’investigazione dell’attività catalitica di sistemi macromolecolari fluorurati, quali PAMAM e PEI derivati, agenti come fosfodiesterasi allo scopo di ottenere sistemi medicali multifunzionali completi. Questa linea di ricerca è legata alle terapie antisenso, che si basano sul legame selettivo di oligonucleotidi antisenso alla sequenza di mRNA per prevenire ed inibire la trascrizione di proteine patologiche. Questo studio sottolinea la marcata efficienza dei materiali da noi sintetizzati, mostrando una capacità di catalisi nella rottura dei legami fosfodiesterici del ribonucleotide, incrementando di conseguenza l’efficienza degli oligonucleotidi antisenso. Il secondo progetto si basa sullo sviluppo di sistemi fluorurati biodegradabili cationici per il gene delivery e di innovativi sistemi di contrasto per l’imaging al fluoro, ed è sviluppato in due sotto progetti. Nella prima parte del lavoro, sono stati sviluppati poli-aminoesteri ionizzabili mediante polimerizzazione ad apertura di anello e polimerizzazione radicalica libera. La sintesi di una libreria di polimeri fluorurati zwitterionici, ottenuta variando le unità fluorurate nel backbone polimerico, ha permesso di sfruttare tali sistemi per sintetizzare nanoparticelle stabili in ambiente acquoso. Un test preliminare di trasfezione in vitro è stato performato considerando le nanoparticelle con le migliori caratteristiche dimensionali. Per dimostrare il comportamento degradabile delle NP, è stata studiata la cinetica di degradazione in condizioni di elevata basicità. In più, approfondendo lo studio sull’incremento delle unità fluorurate nel polimero zwitterionico, è emersa una correlazione lineare tra l’incremento delle unità fluorurate, l’intensità del segnale del fluoro e la dimensione delle nanoparticelle. Infatti, mediante semplice modulazione della stechiometria della reazione di polimerizzazione è possibile sviluppare le NPs in modo controllato. Nella seconda parte del lavoro, sono stati sintetizzati agenti di contrasto PEG-derivati rilevabili mediante risonanza magnetica a 19F. Due macro-agenti RAFT PEGylati fluorurati sono stati ottenuti ed utilizzati nelle successive sintesi di due nanotracer rodaminati e fluorurati mediante polimerizzazione indotta da “self-assembly” (PISA). I composti ottenuti sono stati caratterizzati mediante spettroscopia nucleare e sistemi di analisi della dimensione alla nano-scala. Successivamente, test in vitro sull’internalizzazione cellulare hanno sottolineato una ridotta citotossicità e una buona internalizzazione cellulare che aumenta all’incrementare del numero di unità fluorurate.
Synthesis of fluorinated polymers for drug/gene delivery and 19F-MRI imaging
Romani, Carola
2023/2024
Abstract
In the last years, the development of new polymeric non-viral vectors for gene delivery able to overcome both the cytotoxicity issues and the transfection efficiency limitation which are often linked to the non-viral vectors exploited so far has gained great attention. The principal goal of this thesis is to develop multifunctional fluorinated systems for gene/drug delivery that could possibly overcome this drawback being, at the same time, highly efficient, low cytotoxic, and with a possible implementation in 19F-MRI imaging. Two main projects characterized the thesis, each one composed of sub-projects, dealing with 1) functionalization of PAMAM dendrimers with ad hoc designed fluorinated Michael acceptors, and 2) synthesis of fluorinated block-copolymers. Polyamidoamine (PAMAM) dendrimers are among the most studied cationic polymers as non-viral gene delivery vectors. An “ideal” PAMAM-based gene delivery vector is still missing because of the paradox efficiency/cytotoxicity associated to their generation (the higher the generation, the higher the efficiency and cytotoxicity of the polymer) along with high manufacturing costs. In this contest, the first project is focused on the synthesis of multifunctional PAMAM conjugates 1) via functionalization of the outer primary amines of low generation and medium generation PAMAM (G2 and G4) with building blocks bearing fluorinated moieties along with a guanidino functional group and 2) by crosslinking the same dendrimers with a fluorinated stimuli responsive linker containing a disulfide bond. For the functionalization, we have designed different fluorinated Michael acceptors able to be straightforwardly “clicked” to PAMAM dendrimers without the need of coupling reagents and/or catalysts. The employed “click” functionalization resulted to be a smooth and tunable tool, guarantying higher control of the reaction outcome by varying the equivalent of fluorinated Michael acceptors with respect to the outer amines of dendrimer. Efficient plasmid DNA complexation and negligible cytotoxicity is observed for the obtained conjugates, showing improved gene transfection efficiency as compared to undecorated PAMAM dendrimers, to the corresponding unfluorinated PAMAM conjugates, and, remarkably, to gold standard bPEI 25 kDa. In a second project, the functionalization of terminal primary amines of PAMAM G2 and G4 with a new fluorinated ibuprofen (IBU)-Arg Michael acceptor is described. The obtained fluorinated IBU PAMAM-Arg conjugates were tested as fluorinated pro-drug systems designed to overcome conventional drug formulation. When comparing the IBU release profiles of these systems with the release of the encapsulated drug using fluorinated Arg-PAMAM conjugates, which were previously synthesized by our team, we observe a superior efficiency in controlled and pH-dependent drug release behavior with the fluorinated prodrugs. This superiority is evident in both weak alkaline and physiological buffer solutions compared to the drug encapsulation strategy. These results emphasize the significance of trifluoromethyl groups and suggest the potential for synergistic biomedical applications, such as drug/gene delivery. Consequently, we embarked on an investigation into the catalytic activity of fluorinated macromolecular species. Specifically, we focused on G2 and G4 PAMAM dendritic structures, linear polyethylenimine (lPEI), and branched polyethylenimine (bPEI), aiming to develop a fully multifunctional medical tool. This research line aligns with antisense therapy, which relies on the selectively binding of antisense oligonucleotide to mRNA sequence to prevent or silence the the transcription of disease-related proteins. The investigation presented in the thesis shows that PAMAM G2 and overall PAMAM G4 dendrimers partially functionalized with the designed linkers incorporating trifluoromethyl and guanidino groups are able to efficiently catalyzes the cleavage of a phosphodiester model compounds demonstrating their possible application in antisense oligonucleotide. The second main project of PhD project relies on the synthesis of biodegradable cationic polymers for biomedical applications, namely gene delivery and 19F-MRI, and is splitted in two sub-projects. In the first study, ionizable amino-polyesters were synthesized via ring opening polymerization and free radical polymerization of lipophilic component, producing positively charged polyesters. Sixteen stable nanoparticles were obtained exploiting fluorinated zwitterionic-based polymers by varying the number of the fluorinated units. Depth study over the size of the different NPs obtained were done, followed by a preliminary transfection test in vitro using the polyplexes that have shown the most suitable characteristics. A degradation study via DLS investigation demonstrated the degradability of NPs. Furthermore, a deeply investigation on the influence of the number of the fluorinated units inside zwitterionic backbone outlined a remarkable independent control linked to linear correlation between fluorine intensity, NPs size, and fluorinated units, enforcing RAFT and ROP coupled strategies in NPs development by simply modulating the stoichiometry of polymerization reaction. The second sub-project reported the synthesis of fluorinated PEGylated polymers as novel nano-contrast agents detectable via 19F-MRI. Two reactive PEGylated fluorinated macro-RAFT agents were prepared through acylation and living polymerization reactions and used for subsequent polymerization induced self-assembly (PISA) reactions to synthesize fluorinated and rhodamine labeled NPs. A preliminary in vitro intracellular investigation of the nanotracers revealed an increased cellular internalization with the number of fluorinated units inside the polymeric systems, guarantying an extremely low cytotoxicity.File | Dimensione | Formato | |
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https://hdl.handle.net/10589/220432