Glioblastoma (GBM) is the most common malignant tumor of the brain, for which there are no curative therapies. The tumor aggressiveness, the insufficient drug accumulation in the brain, and resistance to treatments, are major obstacles limiting the effectiveness of conventional therapies. Nanomedicine (NMed) delivery systems are a promising strategy to improve current therapies for GBM, by encapsulating traditional drugs to ensure their protection, site-specific delivery via targeting, enhanced accumulation in the brain, as well as controlled release at the target site. In the context of GBM, two main NMed-based drug delivery approaches can be exploited: systemic or locoregional ones. The aim of this project was to design, characterize and test novel NMeds for both locoregional and systemic delivery of drugs to GBM. In the systemic approach, NMed can be targeted with specific ligands on their surface to deliver the cargo across the blood brain barrier (BBB) and/or target GBM cells. To that aim, in the first part of this work, an injectable polymeric NMed was decorated onto the surface with novel ligands selected for their GBM-targeting potential. Different targeted NMeds were therefore produced, characterized, and tested in vitro to evaluate their GBM cell specificity and ability to deliver an anticancer drug (paclitaxel) to GBM cells. By optimizing surface modifications, this study demonstrated the potential of this ligands for BBB and/or GBM targeting. In addition, a monoclonal antibody (M08) that can specifically enter GBM cells over healthy cells and cause synergistic effects when delivering paclitaxel was identified. The improved GBM specificity sets the basis to create NMeds for systemic administration with higher therapeutic potential and lower off-target toxicity to healthy cells. In the second part of this work, novel NMeds were designed to deliver cutting-edge molecules with a therapeutic potential for GBM. In particular, small interfering RNA (siRNA) was selected as a promising drug candidate. Two novel polymer-lipid hybrid NMed platforms were developed, intended to complex a model siRNA onto their surface and deliver it in the form of NMed/siRNA complexes. NMed and their complexes were characterized for physicochemical properties, siRNA binding ability, and siRNA protection from enzymatic degradation. Cell uptake in GBM cells, efficacy and biocompatibility were assessed in vitro. Both complexes showed high siRNA binding, enhanced siRNA protection, and high cell uptake in a GBM cell model in vitro. One of them also displayed superior gene silencing activity and good cytocompatibility in vitro. Given the promising properties, we decided to explore the potential of the developed siRNA/NMed formulation for a locoregional drug delivery strategy for GBM. For such approach, the use of biocompatible scaffolds for the local delivery of siRNA into the tumor resection cavity is a promising means to bypass the blood–brain barrier, increase siRNA concentration at the tumor site, and prolong its local release. Therefore, the most promising siRNA/NMed formulation, in terms of cytocompatibility and efficacy in vitro, was incorporated into an injectable and thermo-sensitive hydrogel, intended to be injected into the resection cavity and act as a depot for the NMeds to slow down siRNA release in situ. The NMed-hydrogel nanocomposites were characterized for physicochemical and rheological properties, and siRNA release kinetics. The optimized system gelified at the body temperature and possessed high cytocompatibility in vitro. In vitro studies are ongoing to assess whether the nanocomposite can ensure prolonged siRNA gene silencing activity in vitro on GBM model cell lines.
Il glioblastoma (GBM) è la forma più comune di tumore cerebrale maligno. Ad oggi, non esistono terapie curative. L’aggressività del tumore, l’insufficiente accumulo di farmaci al cervello e la resistenza ai trattamenti sono ostacoli maggiori che limitano l’efficacia delle terapie convenzionali. Le nanomedicine (NMeds) rappresentano una strategia promettente per migliorare le attuali terapie per il GBM, incapsulando i farmaci tradizionali per garantirne la protezione, il delivery sito-specifico tramite targeting e il rilascio controllato nel sito target. Nell'ambito del GBM, possono essere sfruttati due principali approcci di delivery di farmaci basati su NMeds: sistemico e locoregionale. Questo lavoro di tesi mira a progettare, caratterizzare e testare nuove NMeds per il delivery sia locoregionale che sistemico di farmaci al GBM. Nell'approccio sistemico, le NMeds possono essere funzionalizzate in superficie con ligandi specifici per promuovere l’attraversamento della barriera emato-encefalica (BEE) e/o per il targeting alle cellule tumorali. A questo scopo, nella prima parte di questo lavoro, una nanoparticella polimerica è stata decorata in superficie con nuovi ligandi con potenziale abilità di targeting alle cellule di GBM. Diverse NMeds “mirate” sono state formulate, caratterizzate e testate in vitro per valutare la loro specificità verso le cellule di GBM e la capacità di veicolare un agente chemioterapico (paclitaxel). Lo studio ha dimostrato il potenziale di tali ligandi per il targeting al GBM. Tra questi, è stato identificato un anticorpo monoclonale (M08) in grado sia di essere internalizzato selettivamente nelle cellule di GBM rispetto alle cellule sane, sia di causare effetti sinergici con il paclitaxel veicolato. La maggiore specificità verso il GBM costituisce la base per la creazione di NMeds per il delivery sistemico con un maggiore potenziale terapeutico e una minore tossicità sulle cellule sane. Nella seconda parte di questo lavoro, sono state progettate nuove NMed per il delivery di molecole innovative con potenziale terapeutico per il GBM: i siRNA (small interfering RNA). Sono state sviluppate due nuove NMeds ibride polimero-lipide, destinate a complessare un siRNA modello in superficie e a veicolarlo sotto forma di complessi NMed/siRNA. Le NMed e i complessi sono stati caratterizzati per proprietà fisico-chimiche, capacità di complessare il siRNA e protezione del siRNA dalla degradazione enzimatica. L’uptake, l'efficacia e la biocompatibilità sono state valutate in vitro su linee cellulari di GBM. Entrambi i complessi hanno mostrato un'elevata capacità di legare a proteggere il siRNA. Uno di essi ha inoltre mostrato un'elevata attività di silenziamento genico e una buona citocompatibilità in vitro. Date le proprietà promettenti, i complessi NMed/siRNA sono stati sfruttati per una strategia di delivery locoregionale al GBM. In particolare, l'uso di scaffold biopolimerici per la somministrazione locale di siRNA nella cavità di resezione tumorale rappresenta una strategia utile per bypassare la BEE, aumentare la concentrazione di siRNA nel sito tumorale e prolungarne il rilascio. Pertanto, la formulazione di NMed/siRNA più promettente, in termini di efficacia e citocompatibilità in vitro, è stata incorporata in un idrogel iniettabile e termosensibile, destinato ad essere iniettato nella cavità di resezione e agire come sistema di depot per rallentare il rilascio di siRNA in-situ. I nanocompositi NMed-idrogel sono stati caratterizzati per proprietà fisico-chimiche e reologiche e cinetica di rilascio del siRNA. Il sistema sviluppato, che gelifica alla temperatura corporea, ha dimostrato inoltre un'elevata citocompatibilità in vitro. Studi di efficacia su modelli di GBM in vitro sono in corso.
Approcci nanotecnologici innovativi per il targeting del glioblastoma
RINALDI, ARIANNA
2024
Abstract
Glioblastoma (GBM) is the most common malignant tumor of the brain, for which there are no curative therapies. The tumor aggressiveness, the insufficient drug accumulation in the brain, and resistance to treatments, are major obstacles limiting the effectiveness of conventional therapies. Nanomedicine (NMed) delivery systems are a promising strategy to improve current therapies for GBM, by encapsulating traditional drugs to ensure their protection, site-specific delivery via targeting, enhanced accumulation in the brain, as well as controlled release at the target site. In the context of GBM, two main NMed-based drug delivery approaches can be exploited: systemic or locoregional ones. The aim of this project was to design, characterize and test novel NMeds for both locoregional and systemic delivery of drugs to GBM. In the systemic approach, NMed can be targeted with specific ligands on their surface to deliver the cargo across the blood brain barrier (BBB) and/or target GBM cells. To that aim, in the first part of this work, an injectable polymeric NMed was decorated onto the surface with novel ligands selected for their GBM-targeting potential. Different targeted NMeds were therefore produced, characterized, and tested in vitro to evaluate their GBM cell specificity and ability to deliver an anticancer drug (paclitaxel) to GBM cells. By optimizing surface modifications, this study demonstrated the potential of this ligands for BBB and/or GBM targeting. In addition, a monoclonal antibody (M08) that can specifically enter GBM cells over healthy cells and cause synergistic effects when delivering paclitaxel was identified. The improved GBM specificity sets the basis to create NMeds for systemic administration with higher therapeutic potential and lower off-target toxicity to healthy cells. In the second part of this work, novel NMeds were designed to deliver cutting-edge molecules with a therapeutic potential for GBM. In particular, small interfering RNA (siRNA) was selected as a promising drug candidate. Two novel polymer-lipid hybrid NMed platforms were developed, intended to complex a model siRNA onto their surface and deliver it in the form of NMed/siRNA complexes. NMed and their complexes were characterized for physicochemical properties, siRNA binding ability, and siRNA protection from enzymatic degradation. Cell uptake in GBM cells, efficacy and biocompatibility were assessed in vitro. Both complexes showed high siRNA binding, enhanced siRNA protection, and high cell uptake in a GBM cell model in vitro. One of them also displayed superior gene silencing activity and good cytocompatibility in vitro. Given the promising properties, we decided to explore the potential of the developed siRNA/NMed formulation for a locoregional drug delivery strategy for GBM. For such approach, the use of biocompatible scaffolds for the local delivery of siRNA into the tumor resection cavity is a promising means to bypass the blood–brain barrier, increase siRNA concentration at the tumor site, and prolong its local release. Therefore, the most promising siRNA/NMed formulation, in terms of cytocompatibility and efficacy in vitro, was incorporated into an injectable and thermo-sensitive hydrogel, intended to be injected into the resection cavity and act as a depot for the NMeds to slow down siRNA release in situ. The NMed-hydrogel nanocomposites were characterized for physicochemical and rheological properties, and siRNA release kinetics. The optimized system gelified at the body temperature and possessed high cytocompatibility in vitro. In vitro studies are ongoing to assess whether the nanocomposite can ensure prolonged siRNA gene silencing activity in vitro on GBM model cell lines.| File | Dimensione | Formato | |
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Tesi Arianna Rinaldi UIF.pdf
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https://hdl.handle.net/20.500.14242/157231
URN:NBN:IT:UNIMORE-157231