The use of therapeutic proteins in the field of medicine is steadily increasing and the new improvements in field of recombinant DNA technology lead to the development of new therapeutic proteins able to treat autoimmune diseases, infection diseases, metabolic disorders and cancer. However, their efficacy decreases once introduced into the body because of negative effects such as activation of immune response and proteolytic degradation, which lead to fast clearance and short half-life. The conjugation of biocompatible pre-synthesized polymers (such as PEG) to proteins (the “grafting to” approach) represents a successful strategy for overcoming these limitations. However, polymer grafting may be affected by steric hindrance as well as non-selective conjugation, and in several cases it may be difficult to obtain high product yield after purification procedures. Therefore, in this work a new approach called “grafting-from” was investigated, where a well-defined polymer can be grown directly from a specific site of the biomolecule. This method may improve purification procedures and yield significantly, thus reducing costs. The “grafting from” approach was combined with a controlled living polymerization and a site-specific protein modification in order to create a new generation of protein-polymer hybrids, synthesized under biologically friendly conditions. For this purpose, a functionalized aldehyde-ATRP initiator was synthesized. Afterwards, the protein - initiator conjugation was obtained by the reductive amination of the N-terminal group, assuming that its modification should not affect the active site of the protein. Lysozyme was selected as model protein for the tests, and different pH and lysozyme/aldehyde-ATRP initiator ratios were studied in order to maximise the N-terminal functionalization. Afterwards, the work focused on the optimization of Activator ReGenerated Electron Transfer ATRP (ARGET ATRP) for polyethylene glycol monomethacrylte (PEGMA) and glycerol monomethacrylate (GMA) monomers in phosphate buffers, condition well tolerated by proteins. The ARGET ATRP overcomes some limitations related to the standard ATRP allowing to obtain a better control throughout the reaction. Both monomers are used in nanomedicine for their low toxicity, biocompatibility and functionality. Finally, the “grafting from” procedure was carried out in presence of sacrificial, easy removable, initiator-containing polymer microbeads (TENTAGEL), which allowed to perform polymerizations from a relatively low quantity of protein. Two different lysozyme-poly(PEGMA) conjugates were obtained by varying the final chain length and their final protein activity was also tested. Preliminary results showed that protein-polymer hybrids were characterized by a higher activity than their counterparts functionalized only with the initiator. The combination of ARGET ATRP under green condition, the ‘grafting from’ strategy and a selective protein functionalization may pave the way to a new generation of protein-polymer conjugates.
L’uso delle proteine terapeutiche nel campo della medicina è in continua crescita e i nuovi miglioramenti nel campo della tecnologia DNA ricombinante portano allo sviluppo di nuove proteine terapeutiche capaci di trattare malattie autoimmuni, malattie infettive, disordini metabolici e cancro. Tuttavia, la loro efficacia diminuisce una volta introdotti nel corpo a causa di effetti negativi come l’attivazione del sistema immunitario e la degradazione proteolitica, che portano ad una veloce eliminazione e una corta emivita. Il legame di polimeri pre-sintetizzati biocompatibili (come il PEG) con le proteine (approccio “grafting to”) rappresenta una strategia efficace per superare queste limitazioni. Tuttavia, l’attacco polimerico può essere soggetto ad ingombro sterico come anche ad una coniugazione non selettiva, e in molti casi può essere difficile ottenere un elevato rendimento a seguito delle procedure di purificazione. Dunque, in questo lavoro è stato messo alla prova un nuovo approccio chiamato “grafting from”, nel quale un polimero predefinito è in grado di crescere direttamente da un sito specifico della biomolecola. Questo metodo può migliorare in modo significativo le procedure di purificazione e il rendimento, riducendo così i costi. L’approccio “grafting from” è stato combinato ad una polimerizzazione controllata e ad una modifica della proteina in un sito specifico in modo da creare una nuova generazione di ibridi proteina-polimero, sintetizzati sotto condizioni biologiche. A questo scopo, un iniziatore funzionalizzato aldeide-ATRP è stato sintetizzato. Successivamente, il coniugato proteina-iniziatore è stato ottenuto tramite amminazione riduttiva del gruppo N-terminale, supponendo che la sua modifica non influenzi il sito attivo della proteina. Il lisozima è stato selezionato come proteina modello per gli esperimenti, e diversi pH e rapporti lisozima/iniziatore aldeide-ATRP sono stati studiati in modo da massimizzare la funzionalizzazione all’N-terminale. In seguito, il lavoro si è focalizzato sull’ottimizzazione della Activator ReGenerated Electron Transfer ATRP (ARGET ATRP) per i monomeri polietilene glicole mono-metacrilato (PEGMA) e glicerolo mono-metacrilato (GMA) in tampone fosfato salino (PBS), condizione ben tollerata dalle proteine. L’ARGET ATRP supera alcune limitazioni della standard ATRP permettendo di avere un migliore controllo sulla reazione di polimerizzazione. Entrambi i monomeri sono usati in nanomedicina per la loro bassa tossicità, biocompatibilità e funzionalità. Infine, la procedura “grafting from” è stata effettuata in presenza di iniziatori sacrificali, facilmente removibili, contenenti microsfere polimeriche (TENTAGEL), che hanno permesso di eseguire polimerizzazioni da una quantità relativamente bassa di proteina. Due diversi coniugati polimero-proteina sono stati ottenuti cambiando la lunghezza finale di poly(PEGMA) e la loro attività è stata inoltre testata. I risultati preliminari hanno mostrato che gli ibridi proteina-polimero erano caratterizzati da un'attività superiore rispetto alle loro controparti funzionalizzate solo con l'iniziatore. La combinazione di ARGET ATRP in condizioni che non denaturano le proteine, la strategia di "grafting from" e una funzionalizzazione selettiva delle proteine aprirà la strada a una nuova generazione di coniugati proteina-polimero.
Protein-polymer conjugates synthesized via a grafting from approach
LACROCE, ELISA
2019/2020
Abstract
The use of therapeutic proteins in the field of medicine is steadily increasing and the new improvements in field of recombinant DNA technology lead to the development of new therapeutic proteins able to treat autoimmune diseases, infection diseases, metabolic disorders and cancer. However, their efficacy decreases once introduced into the body because of negative effects such as activation of immune response and proteolytic degradation, which lead to fast clearance and short half-life. The conjugation of biocompatible pre-synthesized polymers (such as PEG) to proteins (the “grafting to” approach) represents a successful strategy for overcoming these limitations. However, polymer grafting may be affected by steric hindrance as well as non-selective conjugation, and in several cases it may be difficult to obtain high product yield after purification procedures. Therefore, in this work a new approach called “grafting-from” was investigated, where a well-defined polymer can be grown directly from a specific site of the biomolecule. This method may improve purification procedures and yield significantly, thus reducing costs. The “grafting from” approach was combined with a controlled living polymerization and a site-specific protein modification in order to create a new generation of protein-polymer hybrids, synthesized under biologically friendly conditions. For this purpose, a functionalized aldehyde-ATRP initiator was synthesized. Afterwards, the protein - initiator conjugation was obtained by the reductive amination of the N-terminal group, assuming that its modification should not affect the active site of the protein. Lysozyme was selected as model protein for the tests, and different pH and lysozyme/aldehyde-ATRP initiator ratios were studied in order to maximise the N-terminal functionalization. Afterwards, the work focused on the optimization of Activator ReGenerated Electron Transfer ATRP (ARGET ATRP) for polyethylene glycol monomethacrylte (PEGMA) and glycerol monomethacrylate (GMA) monomers in phosphate buffers, condition well tolerated by proteins. The ARGET ATRP overcomes some limitations related to the standard ATRP allowing to obtain a better control throughout the reaction. Both monomers are used in nanomedicine for their low toxicity, biocompatibility and functionality. Finally, the “grafting from” procedure was carried out in presence of sacrificial, easy removable, initiator-containing polymer microbeads (TENTAGEL), which allowed to perform polymerizations from a relatively low quantity of protein. Two different lysozyme-poly(PEGMA) conjugates were obtained by varying the final chain length and their final protein activity was also tested. Preliminary results showed that protein-polymer hybrids were characterized by a higher activity than their counterparts functionalized only with the initiator. The combination of ARGET ATRP under green condition, the ‘grafting from’ strategy and a selective protein functionalization may pave the way to a new generation of protein-polymer conjugates.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/166603