A major challenge of recent decades is the fight against inoperable and radio-resistant cancers. Nowadays, one of the most serious cases is pancreatic cancer, because it very often has not symptoms that allow for a diagnosis in the early stages.1 Survival rate is extremely low and treatments such as surgery, radiotherapy and chemotherapy often are not effective and need to be combined to fight cancer. An important treatment for inoperable and radio-resistant tumours is hadrontherapy, which is more effective than other treatments such as radiotherapy, but still not widespread due to the lack of centres able to provide it. Hadrontherapy consists in irradiation with charged particles, such as protons or carbon ions. Its effectiveness is due to minimal side effects to healthy tissues. Furthermore, ions cause several double-strand breaks in the DNA of cancer cells, which are more difficult to be repaired.2 This tumour treating technique is increasingly studied and very often tested in combination with other therapies such as chemotherapy. Another interesting and innovative treatment is magnetic hyperthermia, which has been extensively investigated in the last years. This therapeutic technique, also called magnetic fluid hyperthermia (MFH), involves the local heating of tumour tissue by the administration of magnetic nanoparticles (MNP) that release heat after applying an alternating magnetic field.3 This therapy is not particularly effective if applied individually, but recent studies have analysed combined approaches with promising results.4,5 In the present work, one of the therapeutic combinations aimed at obtaining a better curative effect is proposed. It focuses on the combined action of hadrontherapy and magnetic hyperthermia with magnetite nanoparticles (Fe3O4). In particular, the research project in which the present work is included aims at investigating the combined effect of hadrontherapy with carbon ions and magnetic hyperthermia on cell cultures. This combination has not been analysed in the literature yet. The introductory chapter focuses on pancreatic cancer and notions of radiobiology, and then it describes hadrontherapy, magnetic nanoparticles and MFH. The following chapter describes the experimental part, i.e. an in vitro analysis of the combined action of carbon ion irradiation and magnetic hyperthermia as a possible treatment against pancreatic cancer. In addition, to test the effectiveness of the treatment, the experimental protocol was also repeated with photon radiation (X-rays). Combined therapy experiments were carried out on cell cultures of human pancreatic adenocarcinoma BxPC3 (ECACC code 93120816). The experimental analysis involved both a cell line characterisation (plating efficiency and doubling time) and a study of the properties of nanoparticles. The MNP were characterized for their magnetic properties by a SQUID magnetometer, while their dimensional distribution and their Zeta potential were estimated by dynamic light scattering (DLS). A morpho-structural analysis of magnetite nanoparticles was also carried out by a transmission electron microscope (TEM) and X-ray diffraction patterns were acquired to assess their purity and crystalline structure. Finally, their Specific Adsorption Rate (SAR) was experimentally assessed, defining their heating efficiency when subjected to an alternating magnetic field. After characterizing the particles, the magnetic hyperthermia protocol was optimised and then applied to cell cultures after irradiation with carbon ions at Centro Nazionale Adroterapia Oncologica (CNAO). After sample treatment, clonogenic assays were carried out to evaluate cell survival after each treatment combination. Experimental results have shown that magnetic hyperthermia, combined with both hadrontherapy and X-ray radiotherapy, has an additive positive effect in reducing cell survival. They confirm that these combined treatments are very promising therapeutic possibilities. Further studies will focus on optimising and implementing nanoparticles synthesis and MFH to test this promising, combined therapeutic approach in vivo.
Un’importante sfida degli ultimi decenni è la lotta contro i tumori inoperabili e radioresistenti. Uno dei più gravi e attualmente in aumento è il cancro al pancreas, che molto spesso non comporta sintomi che permettano di individuarlo nei primi stadi.1 La sopravvivenza a questa tipologia di tumore è estremamente bassa ed i trattamenti come la chirurgia, la radioterapia e la chemioterapia spesso non risultano efficaci ed è necessario combinarli. Un importante trattamento per i tumori inoperabili e radioresistenti è l’adroterapia, che risulta essere più efficace di altre cure come la radioterapia, ma ancora poco diffusa a causa della carenza di centri in grado di erogarla. L’efficacia dell’irraggiamento con particelle cariche come protoni o ioni carbonio che caratterizza l’adroterapia è dovuta al fatto che si minimizzano gli effetti collaterali ai tessuti sani e gli ioni causano numerose doppie rotture del filamento del DNA delle cellule tumorali, che risultano più difficilmente riparabili.2 Tale tecnica di trattamento del tumore è sempre più studiata e molto spesso testata in combinazione con altre terapie, come ad esempio la chemioterapia. Un altro interessante ed innovativo trattamento è l’ipertermia, in particolare quella magnetica. L’ipertermia magneto-fluida (MFH) prevede il riscaldamento locale del tessuto tumorale a seguito della somministrazione di nanoparticelle magnetiche (MNP), che produrranno calore una volta perturbate con un campo magnetico alternato.3 Tuttavia, questa terapia non risulta particolarmente efficace se applicata singolarmente per trattare la zona tumorale, ma recenti studi hanno analizzato approcci combinati con risultati promettenti.4,5 Una delle combinazioni terapeutiche atte ad ottenere un migliore effetto curativo è proposta in questo lavoro di tesi e verte sull’azione combinata di adroterapia e ipertermia magnetica con nanoparticelle di magnetite (Fe3O4). In particolare, il progetto di ricerca in cui si inserisce il presente lavoro si prefigge di investigare l’effetto combinato di adroterapia con ioni carbonio e ipertermia magnetica su colture cellulari, la cui descrizione non è ancora stata affrontata in letteratura. Il capitolo introduttivo del presente elaborato si focalizza sul tumore al pancreas e nozioni di radiobiologia, descrivendo poi l’adroterapia, le nanoparticelle magnetiche e la MFH. Il capitolo seguente descrive la parte sperimentale che ha previsto un’analisi in vitro dell’azione combinata di adroterapia con ioni carbonio e ipertermia magnetica come possibile trattamento contro il tumore al pancreas. Inoltre, per verificare l’efficacia del trattamento, il protocollo sperimentale è stato ripetuto anche con l’irraggiamento con fotoni (raggi X). Gli esperimenti di terapia combinata sono stati effettuati su cellule di adenocarcinoma pancreatico umano BxPC3 (codice ECACC 93120816). L’indagine sperimentale ha previsto la caratterizzazione della linea cellulare (efficienza di piastramento e tempo di duplicazione) ed uno studio delle proprietà delle nanoparticelle. In particolare, le nanoparticelle sono state analizzate al magnetometro SQUID per valutarne le caratteristiche magnetiche, mentre il dynamic light scattering (DLS) ha permesso di stimarne la distribuzione dimensionale ed il potenziale Z. È stata inoltre svolta un’analisi morfo-strutturale delle nanoparticelle al microscopio elettronico a trasmissione (TEM) e ne sono stati acquisiti i pattern di diffrazione ai raggi X per valutare la purezza e la struttura cristallina. Infine, è stato valutato lo Specific Adsorption Rate (SAR) che definisce l’efficienza di riscaldamento delle nanoparticelle se sottoposte ad un campo magnetico alternato. Una volta note le caratteristiche delle particelle, si è ottimizzato il protocollo di ipertermia magnetica che è stato poi utilizzato al Centro Nazionale Adroterapia Oncologica (CNAO) dopo l’irraggiamento con ioni carbonio. Dopo il trattamento, sono stati svolti saggi clonogenici per valutare la sopravvivenza cellulare alle differenti modalità terapeutiche. I risultati sperimentali hanno evidenziato che l’ipertermia magnetica, combinata con l’adroterapia o con la radioterapia, ha un effetto positivo additivo nel ridurre la sopravvivenza cellulare. Ciò conferma che questi trattamenti combinati risultano essere possibilità terapeutiche molto promettenti. Ulteriori studi verteranno sull’ottimizzazione e l’implementazione del processo di sintesi delle nanoparticelle e di MFH finalizzati a testare in vivo questo promettente approccio terapeutico combinato.
Analisi in vitro dell'azione combinata di adroterapia e ipertermia magnetica per il trattamento del tumore al pancreas
Cavaiani, Marco
2019/2020
Abstract
A major challenge of recent decades is the fight against inoperable and radio-resistant cancers. Nowadays, one of the most serious cases is pancreatic cancer, because it very often has not symptoms that allow for a diagnosis in the early stages.1 Survival rate is extremely low and treatments such as surgery, radiotherapy and chemotherapy often are not effective and need to be combined to fight cancer. An important treatment for inoperable and radio-resistant tumours is hadrontherapy, which is more effective than other treatments such as radiotherapy, but still not widespread due to the lack of centres able to provide it. Hadrontherapy consists in irradiation with charged particles, such as protons or carbon ions. Its effectiveness is due to minimal side effects to healthy tissues. Furthermore, ions cause several double-strand breaks in the DNA of cancer cells, which are more difficult to be repaired.2 This tumour treating technique is increasingly studied and very often tested in combination with other therapies such as chemotherapy. Another interesting and innovative treatment is magnetic hyperthermia, which has been extensively investigated in the last years. This therapeutic technique, also called magnetic fluid hyperthermia (MFH), involves the local heating of tumour tissue by the administration of magnetic nanoparticles (MNP) that release heat after applying an alternating magnetic field.3 This therapy is not particularly effective if applied individually, but recent studies have analysed combined approaches with promising results.4,5 In the present work, one of the therapeutic combinations aimed at obtaining a better curative effect is proposed. It focuses on the combined action of hadrontherapy and magnetic hyperthermia with magnetite nanoparticles (Fe3O4). In particular, the research project in which the present work is included aims at investigating the combined effect of hadrontherapy with carbon ions and magnetic hyperthermia on cell cultures. This combination has not been analysed in the literature yet. The introductory chapter focuses on pancreatic cancer and notions of radiobiology, and then it describes hadrontherapy, magnetic nanoparticles and MFH. The following chapter describes the experimental part, i.e. an in vitro analysis of the combined action of carbon ion irradiation and magnetic hyperthermia as a possible treatment against pancreatic cancer. In addition, to test the effectiveness of the treatment, the experimental protocol was also repeated with photon radiation (X-rays). Combined therapy experiments were carried out on cell cultures of human pancreatic adenocarcinoma BxPC3 (ECACC code 93120816). The experimental analysis involved both a cell line characterisation (plating efficiency and doubling time) and a study of the properties of nanoparticles. The MNP were characterized for their magnetic properties by a SQUID magnetometer, while their dimensional distribution and their Zeta potential were estimated by dynamic light scattering (DLS). A morpho-structural analysis of magnetite nanoparticles was also carried out by a transmission electron microscope (TEM) and X-ray diffraction patterns were acquired to assess their purity and crystalline structure. Finally, their Specific Adsorption Rate (SAR) was experimentally assessed, defining their heating efficiency when subjected to an alternating magnetic field. After characterizing the particles, the magnetic hyperthermia protocol was optimised and then applied to cell cultures after irradiation with carbon ions at Centro Nazionale Adroterapia Oncologica (CNAO). After sample treatment, clonogenic assays were carried out to evaluate cell survival after each treatment combination. Experimental results have shown that magnetic hyperthermia, combined with both hadrontherapy and X-ray radiotherapy, has an additive positive effect in reducing cell survival. They confirm that these combined treatments are very promising therapeutic possibilities. Further studies will focus on optimising and implementing nanoparticles synthesis and MFH to test this promising, combined therapeutic approach in vivo.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/167203