Glioblastoma (GBM) is the most common primary malignant brain tumor characterized by a very poor response to standard surgery and chemotherapeutic treatments. The integrity of the BBB at the peripheral regions of the tumor limits the amount of chemotherapeutic drug that can reach GBM cells, and an inevitable recidivism is generally observed even after surgery. Furthermore, drugs used for chemotherapeutic treatment are toxic and can cause side effects and kill normal cells. In this thesis, new drug-loaded engineered polymeric nanocarriers, which are specifically targeted to glioblastoma cells, were developed as an alternative approach to classical treatments of GBM. Firstly, polyester-PEG based nanoparticles (NPs) were successfully obtained through Free Radical Polymerization (FRP) and a nanoprecipitation method. Surface NPs were functionalised with a cysteine containing cyclic RGD peptide, thus demonstrating the versatility of Michael type addition for an efficient surface modification of this type of biocompatible nanoparticles. In vitro biological tests confirmed the capability of these peptide-conjugated nanocarriers to bind endothelial cells and the possibility to achieve an active cell targeting by using this peptide conjugation approach. Secondly, we investigated how different polymeric structures, molecular weight, and number of Poly(ethylene glycol) (PEG) and polyester blocks could influence nanoparticle self-assembly in aqueous environment, as well as the ability to encapsulate and release hydrophobic therapeutic drugs. Well-defined amphiphilic Poly(ε-caprolactone) (PCL) - PEG copolymers with complex molecular architectures (i.e., linear, 4-arm comb-like, and brush block comb-like), were synthesized through a combination of Ring Opening Polymerization (ROP) and Atom Transfer Radical Polymerization (ATRP) and used to produce drug-loaded NPs. In particular, dexamethasone was chosen as a model drug for these studies, due to its relatively low cost, hydrophobicity, and easy quantification. A 4-arm comb-like copolymer made of a PCL core and a brush-like PEG shell proved to be an optimal compromise between small particle size and stability together with a relatively high drug loading. Therefore, polymeric NPs based on a star-shaped PCL-PEG copolymer were produced and tested as drug delivery system for targeting glioblastoma tumors. In particular, these macromolecules were functionalized through Michael-type addition with an Activable Low Molecular Weight Protamine (ALMWP) and an ApoE-derived peptide (mApoE). ALMWP was identified because of its ability to be activated and internalized by glioblastoma tumor selectively, while mApoE for its capability to cross BBB as well as binding lipoprotein receptors in glioblastoma cells. In this study, doxorubicin (i.e. an effective and traceable chemotherapic drug) was selected as payload. NPs were characterized in vitro on glioblastoma-derived cell lines and on an endothelial cell line to assess their cytotoxicity, cellular uptake and therapeutic effects. Finally, more conventional ALMWP-conjugated polymeric NPs obtained from linear polyester-PEG copolymer were synthesized. Two different drug encapsulation strategies were investigated: doxorubicin was either physically entrapped in the NPs, or chemically conjugated to the terminal group of the polyester block. A pH-sensitive (hydrazone) group was inserted to allow the release of the active compound in acidic conditions, i.e. in a tumor environment or in the endosomal compartment after cellular uptake, aiming to minimize unspecific drug-release. Preliminary in vitro tests were also carried out with these nanocarriers to evaluate their cytotoxicity and uptake by glioblastoma cell lines.
Il glioblastoma (GBM) è il tumore cerebrale primario e maligno più comune caratterizzato da una risposta molto scarsa ai trattamenti standard di chirurgia e chemioterapia. L'integrità della barriera emato-encefalica (blood-brain barrier, BBB) nelle regioni periferiche del tumore limita la quantità di farmaco chemioterapico che riesce a raggiungere le cellule di glioblastoma e generalmente si osserva un'inevitabile recidiva anche dopo l'intervento chirurgico. Inoltre, i farmaci utilizzati per il trattamento chemioterapico sono tossici e possono causare effetti collaterali e uccidere le cellule normali. In questa tesi, sono stati sviluppati nuovi nanocarriers polimerici ingegnerizzati caricati con farmaci, come approccio alternativo ai trattamenti classici per la cura del GBM, potenzialmente in grado di raggiungere le cellule tumorali di glioblastoma e rilasciare selettivamente un agente chemioterapico, al fine di trattare efficacemente il tumore. In primo luogo, sono state ottenute con successo nanoparticelle (NPs) a base di poliestere-PEG mediante la polimerizzazione a radicali liberi (Free Radical Polymerization, FRP) e il metodo di nanoprecipitazione. La superficie esterna delle NP è stata funzionalizzata con un peptide ciclico RGD contenente cisteina, dimostrando così la versatilità della reazione di addizione di Michael per un'efficiente modifica della superficie di questo tipo di NPs biocompatibili. I test biologici in vitro hanno confermato la capacità di questi nanocarriers coniugati con peptidi di legare le cellule endoteliali e la possibilità di ottenere un targeting cellulare attivo utilizzando questo approccio di coniugazione dei peptidi. In secondo luogo, abbiamo studiato come diverse strutture polimeriche, peso molecolare e numero di poli(etilene glicole) (PEG) e blocchi di poliestere potrebbero influenzare l'autoassemblaggio delle nanoparticelle in ambiente acquoso, nonché la capacità di incapsulare e rilasciare farmaci terapeutici idrofobici. Copolimeri anfifilici a base di poli(ε-caprolattone) (PCL) e PEG con architetture molecolari complesse (ad esempio lineare, a pettine a 4 braccia e a pettine a blocco), sono stati sintetizzati attraverso una combinazione di polimerizzazione ad apertura di anello (Ring Opening Polymerization, ROP) e polimerizzazione radicalica per trasferimento atomico (Atom Transfer Radical Polymerization, ARTP), e utilizzati per produrre NPs caricate con farmaci. In particolare, il desametasone è stato scelto come farmaco modello per questi studi, per il suo costo relativamente basso, la sua idrofobicità e la facile quantificazione. Un copolimero a pettine avente 4 braccia costituito da un nucleo di PCL e un guscio di PEG è risultato essere un ottimo compromesso per ottenere un accurato controllo delle dimensioni delle particelle e dell'incapsulamento di farmaci idrofobici a basso peso molecolare. Pertanto, sono state prodotte NPs polimeriche con un copolimero di PCL-PEG a forma di stella e successivamente testate come sistema di somministrazione di farmaci per un targeting attivo. In particolare, queste macromolecole sono state funzionalizzate attraverso la reazione di addizione di Michael con una protamina attivabile a basso peso molecolare (ALMWP) e un peptide derivato da ApoE (mApoE). Il peptide ALMWP è stato scelto per la sua capacità di essere attivato e interiorizzato selettivamente dalle cellule di glioblastoma, mentre il peptide mApoE per la sua capacità di attraversare la BBB e legare i recettori delle lipoproteine nelle cellule di glioblastoma. In questo studio, la doxorubicina (un farmaco chemioterapico efficace e tracciabile) è stata selezionata come molecola attiva da incapsulare. Le NPs sono state testate in vitro su linee cellulari derivate da glioblastoma e su una linea cellulare endoteliale per valutarne la citotossicità, l'assorbimento cellulare e gli effetti terapeutici. Infine, sono state sintetizzate NPs polimeriche coniugate con ALMWP più convenzionali ottenute da copolimeri lineari di poliestere-PEG. Sono state studiate due diverse strategie di incapsulamento del farmaco: la doxorubicina è stata intrappolata nelle NP fisicamente oppure coniugata chimicamente al gruppo terminale della catena di poliestere. Nel secondo caso, è stato inserito un gruppo sensibile al pH (idrazone) per consentire il rilascio del composto attivo in condizioni acide, cioè in un ambiente tumorale o nel compartimento endosomiale dopo l'assorbimento cellulare, con l'obiettivo di ridurre al minimo il rilascio aspecifico del farmaco. Sono stati inoltre effettuati dei test preliminari in vitro con questi nanocarriers per valutarne la citotossicità e l'assorbimento da parte delle linee cellulari di glioblastoma.
Targeting glioblastoma cells by drug-loaded polymeric nanocarriers: macromolecular design and nanomaterial development
CELENTANO, WANDA
2020/2021
Abstract
Glioblastoma (GBM) is the most common primary malignant brain tumor characterized by a very poor response to standard surgery and chemotherapeutic treatments. The integrity of the BBB at the peripheral regions of the tumor limits the amount of chemotherapeutic drug that can reach GBM cells, and an inevitable recidivism is generally observed even after surgery. Furthermore, drugs used for chemotherapeutic treatment are toxic and can cause side effects and kill normal cells. In this thesis, new drug-loaded engineered polymeric nanocarriers, which are specifically targeted to glioblastoma cells, were developed as an alternative approach to classical treatments of GBM. Firstly, polyester-PEG based nanoparticles (NPs) were successfully obtained through Free Radical Polymerization (FRP) and a nanoprecipitation method. Surface NPs were functionalised with a cysteine containing cyclic RGD peptide, thus demonstrating the versatility of Michael type addition for an efficient surface modification of this type of biocompatible nanoparticles. In vitro biological tests confirmed the capability of these peptide-conjugated nanocarriers to bind endothelial cells and the possibility to achieve an active cell targeting by using this peptide conjugation approach. Secondly, we investigated how different polymeric structures, molecular weight, and number of Poly(ethylene glycol) (PEG) and polyester blocks could influence nanoparticle self-assembly in aqueous environment, as well as the ability to encapsulate and release hydrophobic therapeutic drugs. Well-defined amphiphilic Poly(ε-caprolactone) (PCL) - PEG copolymers with complex molecular architectures (i.e., linear, 4-arm comb-like, and brush block comb-like), were synthesized through a combination of Ring Opening Polymerization (ROP) and Atom Transfer Radical Polymerization (ATRP) and used to produce drug-loaded NPs. In particular, dexamethasone was chosen as a model drug for these studies, due to its relatively low cost, hydrophobicity, and easy quantification. A 4-arm comb-like copolymer made of a PCL core and a brush-like PEG shell proved to be an optimal compromise between small particle size and stability together with a relatively high drug loading. Therefore, polymeric NPs based on a star-shaped PCL-PEG copolymer were produced and tested as drug delivery system for targeting glioblastoma tumors. In particular, these macromolecules were functionalized through Michael-type addition with an Activable Low Molecular Weight Protamine (ALMWP) and an ApoE-derived peptide (mApoE). ALMWP was identified because of its ability to be activated and internalized by glioblastoma tumor selectively, while mApoE for its capability to cross BBB as well as binding lipoprotein receptors in glioblastoma cells. In this study, doxorubicin (i.e. an effective and traceable chemotherapic drug) was selected as payload. NPs were characterized in vitro on glioblastoma-derived cell lines and on an endothelial cell line to assess their cytotoxicity, cellular uptake and therapeutic effects. Finally, more conventional ALMWP-conjugated polymeric NPs obtained from linear polyester-PEG copolymer were synthesized. Two different drug encapsulation strategies were investigated: doxorubicin was either physically entrapped in the NPs, or chemically conjugated to the terminal group of the polyester block. A pH-sensitive (hydrazone) group was inserted to allow the release of the active compound in acidic conditions, i.e. in a tumor environment or in the endosomal compartment after cellular uptake, aiming to minimize unspecific drug-release. Preliminary in vitro tests were also carried out with these nanocarriers to evaluate their cytotoxicity and uptake by glioblastoma cell lines.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/174239