According to the World Health Organization, cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020. It’s officially considered the major burden disease worldwide and, in many countries, it’s the second most common cause of death after cardiovascular diseases. In cancer therapy, beside to the primary mass growth, it is now well described as special attention must be paid also to the tumor metastasis appearance and spreading. Tumor metastasis (or the mechanism by which cancer cells spread from their original location to other parts of the body) is caused not only by intrinsic alteration of primary tumor cells but also by the altered cancer microenvironment where, under a histological point of view, macrophages represent more than 50% of cells. The tumor-associated macrophages (TAMs) are considered immunosuppressive, facilitating the metastatic cascade of cancer cells producing cytokines and growth factors, and triggering the inhibitory immune checkpoint proteins released in T cells. Therefore, in the last years, the drug-mediated promotion of the selective impairment of the TAMs proliferation (by targeting their mannose receptor) became an interesting strategy for the development of novel anticancer treatments. In the last decades, owing to their intrinsic characteristics as synthetic flexibility and enhanced site-selectivity, liposomes have encountered great application in different biomedical fields and especially as site-selective drug-nanocarriers in advanced cancer therapy. In this work, we synthesized and characterized a tailor-made azide-mannose as potentially selective targeting agent of mannose receptor of TAMs. This synthetic carbohydrate has been used to decorate the surface of liposomes showing a dibenzocyclooctyne group (DBCO) on their surface with the aim to impart them the desired selectivity toward TAMs. As the first part of the work, the synthesis and characterization of a specific DBCO-liposome able to promote the subsequent ‘click’ coupling with the azide-modified mannose was carried out. In a second stage, the characterization of the cellular uptake has been carried out by comparing in vitro with confocal laser microscopy imaging techniques the incubation of cultured TAMs with mannosylated or bare liposomes. The achieved results successfully demonstrate the potential of the hypothesis of this thesis since an highly increased cellular uptake of mannose-based liposomes within cultured macrophages has been observed.
Secondo l'Organizzazione Mondiale della Sanità, il cancro è una delle principali cause di morte nel mondo, con quasi 10 milioni di decessi nel 2020. È ufficialmente considerato la principale malattia da carico in tutto il mondo e, in molti paesi, è la seconda causa di morte più comune dopo le malattie cardiovascolari . Nella terapia oncologica, oltre all'accrescimento di massa primaria, è ormai ben descritto poiché occorre prestare particolare attenzione anche alla comparsa e alla diffusione delle metastasi tumorali. La metastasi tumorale (ovvero il meccanismo attraverso il quale le cellule tumorali si diffondono dalla loro sede originaria ad altre parti del corpo) è causata non solo dall'alterazione intrinseca delle cellule tumorali primarie ma anche dall'alterato microambiente tumorale dove, dal punto di vista istologico, i macrofagi rappresentano più del 50% delle cellule. I macrofagi associati al tumore (TAM) sono considerati immunosoppressori, facilitano la cascata metastatica delle cellule tumorali che producono citochine e fattori di crescita e attivano le proteine del checkpoint immunitario inibitorie rilasciate nelle cellule T. Pertanto, negli ultimi anni, la promozione farmaco-mediata della compromissione selettiva della proliferazione dei TAM (mirando al loro recettore del mannosio) è diventata una strategia interessante per lo sviluppo di nuovi trattamenti antitumorali. Negli ultimi decenni, a causa delle loro caratteristiche intrinseche di flessibilità sintetica e maggiore selettività del sito, i liposomi hanno trovato grande applicazione in diversi campi biomedici e soprattutto come nanocarrier selettivi per il sito nella terapia avanzata del cancro. In questo lavoro, abbiamo sintetizzato e caratterizzato un azide-mannosio su misura come agente di targeting potenzialmente selettivo del recettore del mannosio dei TAM. Questo carboidrato sintetico è stato utilizzato per decorare la superficie dei liposomi che mostrano un gruppo dibenzocicloottino (DBCO) sulla loro superficie con l'obiettivo di conferire loro la selettività desiderata verso i TAM. Come prima parte del lavoro è stata effettuata la sintesi e la caratterizzazione di uno specifico DBCO-liposoma in grado di promuovere il successivo accoppiamento “click” con il mannosio modificato con azide. In una seconda fase, la caratterizzazione dell'assorbimento cellulare è stata effettuata confrontando in vitro con tecniche di imaging di microscopia laser confocale l'incubazione di TAM in coltura con liposomi mannosilati o nudi. I risultati ottenuti dimostrano con successo il potenziale dell'ipotesi di questa tesi poiché è stato osservato un assorbimento cellulare altamente aumentato di liposomi a base di mannosio all'interno di macrofagi in coltura.
Sintesi di liposomi innestati con mannosio come nanocarrier selettivi potenziati nella terapia del cancro mirata ai macrofagi
AYASH, FATIMA
2021/2022
Abstract
According to the World Health Organization, cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020. It’s officially considered the major burden disease worldwide and, in many countries, it’s the second most common cause of death after cardiovascular diseases. In cancer therapy, beside to the primary mass growth, it is now well described as special attention must be paid also to the tumor metastasis appearance and spreading. Tumor metastasis (or the mechanism by which cancer cells spread from their original location to other parts of the body) is caused not only by intrinsic alteration of primary tumor cells but also by the altered cancer microenvironment where, under a histological point of view, macrophages represent more than 50% of cells. The tumor-associated macrophages (TAMs) are considered immunosuppressive, facilitating the metastatic cascade of cancer cells producing cytokines and growth factors, and triggering the inhibitory immune checkpoint proteins released in T cells. Therefore, in the last years, the drug-mediated promotion of the selective impairment of the TAMs proliferation (by targeting their mannose receptor) became an interesting strategy for the development of novel anticancer treatments. In the last decades, owing to their intrinsic characteristics as synthetic flexibility and enhanced site-selectivity, liposomes have encountered great application in different biomedical fields and especially as site-selective drug-nanocarriers in advanced cancer therapy. In this work, we synthesized and characterized a tailor-made azide-mannose as potentially selective targeting agent of mannose receptor of TAMs. This synthetic carbohydrate has been used to decorate the surface of liposomes showing a dibenzocyclooctyne group (DBCO) on their surface with the aim to impart them the desired selectivity toward TAMs. As the first part of the work, the synthesis and characterization of a specific DBCO-liposome able to promote the subsequent ‘click’ coupling with the azide-modified mannose was carried out. In a second stage, the characterization of the cellular uptake has been carried out by comparing in vitro with confocal laser microscopy imaging techniques the incubation of cultured TAMs with mannosylated or bare liposomes. The achieved results successfully demonstrate the potential of the hypothesis of this thesis since an highly increased cellular uptake of mannose-based liposomes within cultured macrophages has been observed.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.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/15191