Cancer development is associated to the physiological state of the tumor microenvironment. Despite the existing heterogeneity of tumors, there are common features that can be found in tumor microenvironment development, such as genetic changes, resulting in iperplasia, unregulated cell growth and resistance to apoptosis. Alteration of the extracellular matrix, uncontrolled differentiation of the neighbor stromal cells (e.g., fibroblasts), and immune cells modified response are liable of cancer progression, angiogenesis and, ultimately, metastasis. This step involves an important crosstalk between tumor cells and its microenvironment. Among these pathways, HGF is released from many cell types of the tumor microenvironment and it targets its receptor, MET, within the cells of the primary tumor (CAFs) inducing cell growth and metastasis. MET receptor is a tyrosine-kinase receptor that mediates the interaction between cancerous cells and adjacent stroma and it triggers proliferation and angiogenesis in primary tumor and stimulates motility to form micrometastases. The aim of this thesis is to develop a polymeric nanoplatform (NPs) able to recognize MET receptor on the cancer cellular membrane and exploit it as anchor of attachment to enter the cells. To this aim, a newly synthetized ScFv derived from an anti-MET monoclonal antibody with high selectivity for MET receptor was used. Verteporfin (VP) is selected as active pharmaceutical ingredient to ensure an antiproliferative activity against YAP-induced cell growth. VP is an excellent candidate for regulating CAFs activity, reshaping the tumor microenvironment and controlling the ECM deposition. In the actual work, functionalized polymer (Maleimido-PEG-NHCO-PLGA) was synthetized with a reaction yield of 78% and characterized by gel permeation chromatography to assess the weight average molecular weight that resulted to be 79,969 Da, which is consistent with the effective coupling of PLGA 66KDa and PEG 5KDa. The synthesis and purification procedures were preliminary characterized and optimized by proton magnetic resonance spectroscopy (1H-NMR). Functional polymer (Maleimido-PEG-NHCO-PLGA) was used in combination with PLGA48kDa as structural polymer and PLGA-PEG90kDa as stealth polymer to prepare uniform polymeric NPs of 75 nm (PDI = 0.12) characterized by negative charge, with 28 nmol of maleimide groups on the surface (quantified by Ellman’s test). Next, NPs were successfully conjugated with ScFv leading to MET-targeting NPs of around 190 nm (PDI = 0.3) with a neutral surface charge. The enlargement of the NPs size and the neutralization of the surface charge prove the ScFv conjugation. Moreover, the conjugation efficiency, evaluated with BCA assay was of 56 ± 12 %, meaning that 3.4 ± 0.7 nmol of ScFv reacted with the maleimide groups of polymers. Considering that nearly 28 nmol of Maleimido-PEG-NHCO-PLGA were present, it can be concluded that nearly 12% of maleimide groups were bioconjugated with the ScFv. NPs morphology was confirmed by TEM. Furthermore, Verteporfin was loaded into these NPs with a satisfactory encapsulation efficiency (38%, corresponding to 12.2 ± 0.7 µg/mg of NPs). Verteporfin was released from polymeric nanocarriers through a triphasic pattern within 96h of incubation. Finally, NPs internalization was qualitatively assessed on epithelial cells of lung carcinoma (A549) and gastric adenocarcinoma cells (MKN45) by confocal microscopy analysis figuring out MET on the cell membrane by immunocytochemistry assay. Results displayed engineered NPs massively enter the cells after 1.5h of incubation and they were observed on the membrane colocalized with green fluorescence of MET and also inside the nucleus. In conclusion, in this thesis a versatile engineered polymeric platform was developed ensuring the functionalization with specific ScFv and efficiently deliver drug to MET-expressing cells.
Lo sviluppo tumorale è associato allo stato fisiologico del microambiente circostante. Nonostante esista eterogeneità tra diversi tumori, caratteristiche comuni vengono riscontrate nello sviluppo del microambiente tumorale, come variazioni genetiche, risultanti in iperplasia e crescita cellulare non regolata. Alterazioni della ECM, differenziazione incontrollata delle vicine cellule stromali (come i fibroblasti), e modifica della funzionalità delle cellule del sistema immunitario sono responsabili di crescita tumorale, neoangiogenesi e metastasi. Questo passaggio coinvolge un’importante cooperazione tra cellule tumorali e microambiente. Ad esempio, HGF è secreto da molte cellule presenti nel microambiente e ha come recettore MET, presente sulle cellule del tumore primario. HGF, legandosi a MET, induce crescita cellulare e sviluppo di metastasi. MET appartiene alla famiglia dei recettori tirosin-chinasici e media l'interazione tra le cellule cancerose e lo stroma adiacente, innesca proliferazione e angiogenesi nel tumore primario e stimola la motilità cellulare, formando micrometastasi. Lo scopo della tesi è sviluppare nanoparticelle polimeriche (NP) in grado di riconoscere MET sulla membrana tumorale e sfruttarlo come ancora di attacco per entrare nelle cellule. A tale scopo è stato utilizzato un ScFv recentemente sintetizzato con elevata selettività per MET. Verteporfin è stato selezionato per garantire attività antiproliferativa rivolta contro la crescita cellulare indotta da YAP. VP è anche un ottimo candidato per regolare la deposizione di ECM e rimodellare il microambiente. Nel concreto, è stato sintetizzato un polimero funzionalizzato (Maleimido-PEG-NHCO-PLGA) con una resa di reazione del 78%. Il polimero è stato caratterizzato mediante Gel Permeation Chromatography per valutare il peso molecolare medio ponderale che è risultato essere 79,969 Da, coerente con l'effettivo legame tra PLGA 66KDa e PEG 5KDa. Le procedure di sintesi e purificazione sono state caratterizzate in via preliminare mediante spettroscopia di risonanza magnetica protonica (1H-NMR). Il polimero funzionalizzato è stato utilizzato in combinazione con PLGA48kDa come polimero strutturale e PLGA-PEG90kDa come polimero di “stealth” per preparare NP di 75 nm (PDI = 0,12), con carica superficiale negativa e 28 nmol di gruppi maleimmidici sulla superficie (quantificati tramite test di Ellman). Successivamente, le NP sono state coniugate con ScFv, portando alla formazione di NP di 190 nm (PDI = 0, 3) con carica superficiale neutra. L'aumento delle dimensioni e la neutralizzazione della carica superficiale dimostrano la coniugazione con ScFv. L'efficienza di coniugazione, valutata con il saggio BCA era del 56 ± 12 %, il che significa che 3,4 ± 0,7 nmol di ScFv hanno reagito con i gruppi maleimmidici presenti sulla superficie delle NP. Considerando che erano presenti 28 nmol di Maleimido-PEG-NHCO-PLGA, si può concludere che il 12% dei gruppi di maleimmidici è stato coniugato con ScFv. La morfologia delle NP è stata confermata dall’analisi al TEM. Inoltre, VP è stato incapsulato con un'efficienza soddisfacente (38%, corrispondente a 12.2 ± 0.7 µg/mg di NP) ed è stato rilasciato seguendo un pattern trifasico entro 96 ore di incubazione. L'internalizzazione delle NP è stata valutata qualitativamente su cellule di carcinoma polmonare e su cellule di adenocarcinoma gastrico mediante microscopia confocale, visualizzando MET sulla membrana cellulare mediante test di immunocitochimica. Le NP ingegnerizzate entrano massicciamente nelle cellule dopo 1.5 ore di incubazione e sono state osservate sulla membrana cellulare colocalizzate con la fluorescenza verde di MET e anche all'interno del nucleo. In conclusione, in questa tesi è stata sviluppata una piattaforma polimerica ingegnerizzata versatile che garantisce la funzionalizzazione con ScFv specifico e veicola in modo efficiente il farmaco alle cellule che esprimono MET.
Il tumore e il suo microambiente come bersaglio: nanoparticelle polimeriche direzionate verso il recettore MET per la veicolazione di Verteporfin
GIGLIO, ALESSIA
2021/2022
Abstract
Cancer development is associated to the physiological state of the tumor microenvironment. Despite the existing heterogeneity of tumors, there are common features that can be found in tumor microenvironment development, such as genetic changes, resulting in iperplasia, unregulated cell growth and resistance to apoptosis. Alteration of the extracellular matrix, uncontrolled differentiation of the neighbor stromal cells (e.g., fibroblasts), and immune cells modified response are liable of cancer progression, angiogenesis and, ultimately, metastasis. This step involves an important crosstalk between tumor cells and its microenvironment. Among these pathways, HGF is released from many cell types of the tumor microenvironment and it targets its receptor, MET, within the cells of the primary tumor (CAFs) inducing cell growth and metastasis. MET receptor is a tyrosine-kinase receptor that mediates the interaction between cancerous cells and adjacent stroma and it triggers proliferation and angiogenesis in primary tumor and stimulates motility to form micrometastases. The aim of this thesis is to develop a polymeric nanoplatform (NPs) able to recognize MET receptor on the cancer cellular membrane and exploit it as anchor of attachment to enter the cells. To this aim, a newly synthetized ScFv derived from an anti-MET monoclonal antibody with high selectivity for MET receptor was used. Verteporfin (VP) is selected as active pharmaceutical ingredient to ensure an antiproliferative activity against YAP-induced cell growth. VP is an excellent candidate for regulating CAFs activity, reshaping the tumor microenvironment and controlling the ECM deposition. In the actual work, functionalized polymer (Maleimido-PEG-NHCO-PLGA) was synthetized with a reaction yield of 78% and characterized by gel permeation chromatography to assess the weight average molecular weight that resulted to be 79,969 Da, which is consistent with the effective coupling of PLGA 66KDa and PEG 5KDa. The synthesis and purification procedures were preliminary characterized and optimized by proton magnetic resonance spectroscopy (1H-NMR). Functional polymer (Maleimido-PEG-NHCO-PLGA) was used in combination with PLGA48kDa as structural polymer and PLGA-PEG90kDa as stealth polymer to prepare uniform polymeric NPs of 75 nm (PDI = 0.12) characterized by negative charge, with 28 nmol of maleimide groups on the surface (quantified by Ellman’s test). Next, NPs were successfully conjugated with ScFv leading to MET-targeting NPs of around 190 nm (PDI = 0.3) with a neutral surface charge. The enlargement of the NPs size and the neutralization of the surface charge prove the ScFv conjugation. Moreover, the conjugation efficiency, evaluated with BCA assay was of 56 ± 12 %, meaning that 3.4 ± 0.7 nmol of ScFv reacted with the maleimide groups of polymers. Considering that nearly 28 nmol of Maleimido-PEG-NHCO-PLGA were present, it can be concluded that nearly 12% of maleimide groups were bioconjugated with the ScFv. NPs morphology was confirmed by TEM. Furthermore, Verteporfin was loaded into these NPs with a satisfactory encapsulation efficiency (38%, corresponding to 12.2 ± 0.7 µg/mg of NPs). Verteporfin was released from polymeric nanocarriers through a triphasic pattern within 96h of incubation. Finally, NPs internalization was qualitatively assessed on epithelial cells of lung carcinoma (A549) and gastric adenocarcinoma cells (MKN45) by confocal microscopy analysis figuring out MET on the cell membrane by immunocytochemistry assay. Results displayed engineered NPs massively enter the cells after 1.5h of incubation and they were observed on the membrane colocalized with green fluorescence of MET and also inside the nucleus. In conclusion, in this thesis a versatile engineered polymeric platform was developed ensuring the functionalization with specific ScFv and efficiently deliver drug to MET-expressing cells.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/14544