Reversible electroporation (R-EP) utilizes electric pulses to induce the formation of transient pores in the cell membrane, facilitating the uptake of hydrophilic molecules, peptides, and nucleic acids. This mechanism forms the basis of electrochemotherapy (ECT), a therapeutic approach designed to enhance chemotherapy drug uptake in cancer cells via EP. Currently, ECT following the ESOPE standard operating procedure (SOP) is employed in the treatment of skin and mucosal cancers, improving the uptake of bleomycin or cisplatin. However, drug uptake remains poorly selective, and systemic toxic effects are still present. To address these limitations, the use of nanocarriers in ECT treatment has been proposed to further enhance therapeutic outcomes. In this study, liposomes composed of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) cholesterol and the cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) were prepared using a microfluidic technique and subsequently characterized. Liposomes were selected due to their phospholipid bilayer structure, which closely resembles that of cell membranes, making them responsive to the electric field applied during EP. FaDu cells, a hypopharingeal head and neck squamous cell carcinoma (HNSCC) line, were used as an in vitro 2D model. To evaluate intracellular delivery efficiency, liposomes were loaded with propidium iodide (PI), a fluorescent hydrophilic marker used as a model molecule and tested in combination with EP. Liposomes were characterized in terms of size (nm ± SD), polidispersity index (PDI), encapsulation efficiency (EE%) and cell viability. Additionally, fluorescein-labeled liposomes were utilized to evaluate the uptake by cancer cells following the EP process at different voltages (0V, 160V, 200V, and 250V), using DHPE (N-(Fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt). This study demonstrates the feasibility of employing liposomal nanosystems in combination with electroporation to enhance drug uptake in FaDu cells. Through EP, a higher number of liposomes was detected while maintaining suitable cell viability. However, the need to optimize encapsulation efficiency strategies to improve the efficacy of liposome-based delivery systems in cancer therapy was highlighted and requires further investigation.
L’elettroporazione reversibile (R-EP) utilizza impulsi elettrici per indurre la formazione di pori transitori nella membrana cellulare, facilitando l’ingresso di molecole idrofile, peptidi, acidi nucleici. Questo meccanismo è alla base dell’elettrochemioterapia (ECT), una tecnica terapeutica mirata a migliorare l’assorbimento di farmaci chemioterapici nelle cellule tumorali attraverso l’elettroporazione. Attualmente, l’ECT viene impiegata secondo le Standard Operating Procedure (SOP) ESOPE nel trattamento dei tumori della pelle e delle mucose, aumentando l’assorbimento di bleomicina o cisplatino. Tuttavia, l’internalizzazione del farmaco rimane poco selettiva e sono ancora presenti effetti tossici sistemici. Per superare queste limitazioni è stata proposta l’integrazione di nanocarrier in ECT per potenziare ulteriormente l’efficacia terapeutica. In questo studio sono stati preparati e caratterizzati liposomi composti da DSPC (1,2-distearoil-sn-glicero-3-fosfocolina), colesterolo e il lipide cationico DOTAP (1,2-dioleil-3-trimetilammonio-propano) mediante una tecnica microfluidica. I liposomi sono stati scelti per la loro struttura a doppio strato fosfolipidico, simile a quella delle membrane cellulari, che li rende particolarmente reattivi al campo elettrico applicato durante l’elettroporazione. Come modello in vitro 2D sono state utilizzate cellule FaDu, una linea cellulare di carcinoma a cellule squamose dell’ipofaringe, appartenente alla categoria di tumori testa-collo (HNSCC). Per valutare l’efficienza di internalizzazione nelle cellule tumorali, i liposomi sono stati caricati con ioduro di propidio (PI), un marcatore fluorescente idrofilo utilizzato come modello di molecola, e testati in combinazione con l’elettroporazione. I liposomi sono stati caratterizzati per dimensione (nm ± SD), indice di polidispersità (PDI), efficienza di incapsulamento (EE%) e citotossicità. Inoltre, sono stati preparati liposomi marcati con fluoresceina per valutare l’uptake cellulare dopo il processo di elettroporazione a diversi voltaggi (0V, 160V, 200V, 250V), utilizzando DHPE (N-(Fluoresceina-5-tiocarbamoil)-1,2-diesadecanoil-sn-glicero-3-fosfoetanolamina, sale di trietilammonio). Questo studio dimostra la fattibilità di impiego dei sistemi liposomiali in combinazione con l’elettroporazione, per migliorare l’assorbimento nelle cellule FaDu. Attraverso l’EP, è stata rilevata una maggiore quantità di liposomi, mantenendo al contempo un’adeguata vitalità cellulare. Tuttavia, è emersa la necessità di ottimizzare le strategie di incapsulazione per migliorare l’efficacia dei sistemi di rilascio liposomiale nella terapia oncologica.
Nanocarrier Liposomiali in Elettrochemioterapia: una nuova strategia per l'ottimizzazione del trattamento
VALENTINO, SARA
2023/2024
Abstract
Reversible electroporation (R-EP) utilizes electric pulses to induce the formation of transient pores in the cell membrane, facilitating the uptake of hydrophilic molecules, peptides, and nucleic acids. This mechanism forms the basis of electrochemotherapy (ECT), a therapeutic approach designed to enhance chemotherapy drug uptake in cancer cells via EP. Currently, ECT following the ESOPE standard operating procedure (SOP) is employed in the treatment of skin and mucosal cancers, improving the uptake of bleomycin or cisplatin. However, drug uptake remains poorly selective, and systemic toxic effects are still present. To address these limitations, the use of nanocarriers in ECT treatment has been proposed to further enhance therapeutic outcomes. In this study, liposomes composed of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) cholesterol and the cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) were prepared using a microfluidic technique and subsequently characterized. Liposomes were selected due to their phospholipid bilayer structure, which closely resembles that of cell membranes, making them responsive to the electric field applied during EP. FaDu cells, a hypopharingeal head and neck squamous cell carcinoma (HNSCC) line, were used as an in vitro 2D model. To evaluate intracellular delivery efficiency, liposomes were loaded with propidium iodide (PI), a fluorescent hydrophilic marker used as a model molecule and tested in combination with EP. Liposomes were characterized in terms of size (nm ± SD), polidispersity index (PDI), encapsulation efficiency (EE%) and cell viability. Additionally, fluorescein-labeled liposomes were utilized to evaluate the uptake by cancer cells following the EP process at different voltages (0V, 160V, 200V, and 250V), using DHPE (N-(Fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt). This study demonstrates the feasibility of employing liposomal nanosystems in combination with electroporation to enhance drug uptake in FaDu cells. Through EP, a higher number of liposomes was detected while maintaining suitable cell viability. However, the need to optimize encapsulation efficiency strategies to improve the efficacy of liposome-based delivery systems in cancer therapy was highlighted and requires further investigation.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14239/28708