Nanomedicine is a field of pivotal importance to medicine and healthcare with the aim to create nano-sized carriers able to deliver drugs to the action site selectively. Traditional cancer treatments (e.g. surgical resection, radiotherapy and chemotherapy, either alone or in combination) have demonstrated to dramatically worse the quality life of the patients whereas nanomedicine based therapeutic approaches have benefited millions of patients by relieving suffering and prolonging life. This study aims to evaluate in vitro the non-toxicity and selectivity cellular uptake of polymeric nanoparticles, based on hyaluronic acid (HA) and chitosan (CS) carrying Everolimus (EVE), on cells that overexpress the CD44 receptor. Indeed, CD44 is a receptor widely overexpressed in tumor lines such as breast cancer, pancreatic cancer, prostate and colon-rectal cancer. HA and CS are natural, biocompatible and biodegradable polymers, widely used in the pharmaceutical-medical field; in particular, HA is the natural ligand of CD44 receptor. Everolimus was shown to be an effective inhibitor of mTOR pathway that allows to reduce cell growth, proliferation, differentiation, chemotaxis, angiogenesis and survival of tumor cells. In this study, the nanocarriers were synthesized using a microfluidic technique, a preparation method of recent application in the field of pharmaceutical nanotechnologies, innovative and easy to scale-up. This technique was previously optimized in the laboratory where this thesis work was carried out and proposed for the first time in order to obtain, with a single pass, nanoparticles based on CS coated with HA. All nanoparticles batches prepared were characterized in the size (≤200 nm), size polydispersity (PDI 0.306 ± 0.05) and ζ potential (-20.8 mV). Morphological examination was performed by TEM. Polymeric matrix characterization was carried out by FT-IR analysis. Everolimus was successfully loaded into nanoparticles (88.12 ± 20.76 μg/1 mg nanoparticles). Nanoparticles (NPs) in vitro behavior were investigated on mesenchymal cells isolated from human bone marrow, choice as a model of cells with CD44 overexpressed. Biocompatibility of placebo HA/CS NPs was confirmed by MTT assay. Cell proliferation inhibition of EVE loaded HA/CS NPs was evaluated by Bromodeossiuridine assay. HA/CS NPs endocytosis mechanism were qualitatively and quantitatively studied by using Rhodamine-labelled NPs incubated at different concentration and time. The outcomes of this thesis confirmed the fast, efficient and reproducible NPs synthesis by microfluidic technology and, moreover, they revealed the promising potential of HA / CS nanoparticles as a targeted drug delivery system to treat CD44 overexpressing tumors.
La nanomedicina è una branca di fondamentale importanza della medicina e dell'assistenza sanitaria che si pone l'obiettivo di creare vettori di dimensioni nanometriche in grado di veicolare farmaci sul sito di azione in modo selettivo. I trattamenti tradizionali per la cura del cancro (ad esempio la resezione chirurgica, la radioterapia e la chemioterapia, utilizzate da sole o in combinazione tra loro) hanno dimostrato di peggiorare drammaticamente la qualità della vita del paziente mentre gli approcci terapeutici basati sulla nanomedicina hanno avvantaggiato milioni di pazienti alleviando la sofferenza e prolungando le aspettative di vita. Questo studio si propone l'obiettivo di valutare in vitro la non tossicità e la selettività di uptake cellulare di nanoparticelle polimeriche, a base di acido ialuronico (HA) e chitosano (CS) veicolanti Everolimus (EVE), su cellule che iperesprimono il recettore CD44. CD44 è infatti un recettore ampiamente sovraespresso in linee tumorali quali seno, pancreas, prostata e colon-retto. HA e CS sono polimeri naturali, biocompatibili e biodegradabili ampiamente utilizzati in ambito medico-farmaceutico; in particolare HA è il ligando naturale di CD44. EVE ha dimostrato essere un efficace inibitore del pathway molecolare di mTOR; ciò consente di ridurre la crescita cellulare, la proliferazione, la differenziazione, la chemiotassi, l'angiogenesi e la sopravvivenza delle cellule tumorali. In questo studio, i nanocarriers sono stati sintetizzati mediante una tecnica microfluidica, metodo di preparazione di recente applicazione nell’ambito delle nanotecnologie farmaceutiche, innovativo e di facile scale-up. Tale tecnica è stata precedentemente ottimizzata presso il laboratorio in cui è stato svolto questo lavoro di tesi e proposta per la prima volta al fine di ottenere con un unico passaggio nanoparticelle a base di CS rivestite con HA. Tutti i lotti di nanoparticelle (NPs) preparati sono stati caratterizzati nelle dimensioni (≤200 nm), relativa polidispersità della distribuzione dimensionale (PDI 0,306 ± 0,05) e nel potenziale ζ (-20,8 mV). L'esame morfologico è stato eseguito mediante analisi TEM. La caratterizzazione della matrice polimerica è stata effettuata mediante analisi FT-IR. Everolimus è stato caricato con successo all’interno delle nanoparticelle (88,12 ± 20,76 μg / 1 mg di nanoparticelle). Il comportamento in vitro delle NPs è stato studiato su cellule mesenchimali isolate dal midollo osseo umano, scelte come modello di cellule con CD44 sovraespresso. La biocompatibilità delle nanoparticelle placebo a base di HA e CS è stata confermata dal saggio MTT. L'inibizione della proliferazione cellulare dovuta all’azione delle stesse nanoparticelle caricate con EVE è stata valutata mediante il saggio della Bromodeossiuridina. Il meccanismo di endocitosi delle nanoparticelle a base CS e rivestite con HA è stato valutato qualitativamente e quantitativamente utilizzando nanoparticelle marcate con rodamina, incubate a tempi e concentrazioni differenti. I risultati di questa tesi hanno confermato la rapidità, efficienza e riproducibilità della sintesi di NPs mediante la tecnologia microfluidica e, inoltre, hanno rivelato la promettente potenzialità delle nanoparticelle a base di HA e CS come un sistema di somministrazione di farmaci mirato per il trattamento di forme tumorali che sovraesprimono CD44.
CD44 Active Targeting for Cancer Therapy Implemented through Microfluidic Technique: in vitro evaluation
GRECO, ANTONIETTA
2017/2018
Abstract
Nanomedicine is a field of pivotal importance to medicine and healthcare with the aim to create nano-sized carriers able to deliver drugs to the action site selectively. Traditional cancer treatments (e.g. surgical resection, radiotherapy and chemotherapy, either alone or in combination) have demonstrated to dramatically worse the quality life of the patients whereas nanomedicine based therapeutic approaches have benefited millions of patients by relieving suffering and prolonging life. This study aims to evaluate in vitro the non-toxicity and selectivity cellular uptake of polymeric nanoparticles, based on hyaluronic acid (HA) and chitosan (CS) carrying Everolimus (EVE), on cells that overexpress the CD44 receptor. Indeed, CD44 is a receptor widely overexpressed in tumor lines such as breast cancer, pancreatic cancer, prostate and colon-rectal cancer. HA and CS are natural, biocompatible and biodegradable polymers, widely used in the pharmaceutical-medical field; in particular, HA is the natural ligand of CD44 receptor. Everolimus was shown to be an effective inhibitor of mTOR pathway that allows to reduce cell growth, proliferation, differentiation, chemotaxis, angiogenesis and survival of tumor cells. In this study, the nanocarriers were synthesized using a microfluidic technique, a preparation method of recent application in the field of pharmaceutical nanotechnologies, innovative and easy to scale-up. This technique was previously optimized in the laboratory where this thesis work was carried out and proposed for the first time in order to obtain, with a single pass, nanoparticles based on CS coated with HA. All nanoparticles batches prepared were characterized in the size (≤200 nm), size polydispersity (PDI 0.306 ± 0.05) and ζ potential (-20.8 mV). Morphological examination was performed by TEM. Polymeric matrix characterization was carried out by FT-IR analysis. Everolimus was successfully loaded into nanoparticles (88.12 ± 20.76 μg/1 mg nanoparticles). Nanoparticles (NPs) in vitro behavior were investigated on mesenchymal cells isolated from human bone marrow, choice as a model of cells with CD44 overexpressed. Biocompatibility of placebo HA/CS NPs was confirmed by MTT assay. Cell proliferation inhibition of EVE loaded HA/CS NPs was evaluated by Bromodeossiuridine assay. HA/CS NPs endocytosis mechanism were qualitatively and quantitatively studied by using Rhodamine-labelled NPs incubated at different concentration and time. The outcomes of this thesis confirmed the fast, efficient and reproducible NPs synthesis by microfluidic technology and, moreover, they revealed the promising potential of HA / CS nanoparticles as a targeted drug delivery system to treat CD44 overexpressing tumors.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/20.500.14239/22406