La terapia sonodinamica (SDT) è un trattamento che coinvolge una combinazione di ultrasuoni (US) a bassa intensità e agenti chimici noti come sonosensibilizzanti. La SDT si basa sulla terapia fotodinamica (PDT), dove la luce è usata per attivare il sensibilizzante al posto degli US. Il maggior inconveniente della PDT è la bassa penetrazione della luce nei tessuti, ovviato usando gli US. Il meccanismo d'azione della SDT rimane dibattuto e diversi studi suggeriscono l'influenza della natura del modello biologico, del sensibilizzante e dei parametri degli US. Da tenere in considerazione è la capacità della SDT di contrastare la multiresistenza delle cellule tumorali. Il Rosa Bengala (RB) e l'Ipericina (Hyp), conosciuti come fotosensibilizzanti, non hanno ancora un ruolo chiaro nella SDT. Lo scopo principale della tesi è quello di investigare la responsività del RB e dell'Hyp all'esposizione agli US delle cellule tumorali del colon umano (HT-29) fatte crescere in colture bi- e tridimensionali (2D e 3D), considerando, come riferimento, l'esposizione alla luce. Analisi sull'assorbimento cellulare, sulla localizzazione intracellulare e sull'IC50 sono state effettuate sulle HT-29 in coltura 2D, per valutare le concentrazioni ottimali di sensibilizzante e i tempi per effettuare esperimenti sono- e fotodinamici. Per valutare l'efficacia della SDT con il RB e l'Hyp, sono state effettuate le analisi di imaging sulla morte cellulare e di citometria a flusso sulla coltura 3D di HT-29. Gli sferoidi sono stati generati usando piastre ricoperte di agarosio, trattate con le stesse condizioni sperimentali delle colture 2D, considerando una maggiore concentrazione di sensibilizzanti. RB e Hyp hanno mostrato una distribuzione citoplasmatica a livello mitocondriale, trovando per l'Hyp grandi accumuli di sensibilizzante presso la membrana cellulare. Nelle colture 2D, l'esposizione fotodinamica delle HT-29 pre-incubate con il RB ha mostrato una diminuzione statisticamente significativa della crescita cellulare a 48 e 72 ore dopo il trattamento, mentre l'esposizione sonodinamica ha provocato soltanto un lieve decremento della crescita cellulare nelle stesse tempistiche. L'esposizione fotodinamica delle HT-29 pre-incubate con l'Hyp ha indotto una diminuzione significativa della crescita cellulare dopo 48 e 72 ore; similmente, l'esposizione sonodinamica ha provocato un decremento statisticamente significativo della crescita cellulare nelle stesse tempistiche. Gli sferoidi sottoposti alla PDT con il RB mostrano disgregazione completa delle strutture 3D con un aumento significativo della morte cellulare e dell'intensità luminosa dello ioduro di propidio (PI) a 48 ore dal trattamento. Un minore incremento statisticamente significativo della morte cellulare è stato osservato quando gli sferoidi sono stati pre-incubati con il RB e poi trattati con US. La PDT con l'Hyp ha indotto una leggera modifica della forma esterna dello sferoide con una lieve induzione della morte cellulare a 48 ore dal trattamento; la SDT con l'Hyp ha mostrato una crescita statisticamente significativa della morte cellulare. I dati rivelano una responsività significativa del RB e dell'Hyp all'esposizione agli US sebbene ne mostrino una minore all'esposizione alla luce. L'attivazione sonodinamica ha portato a differenti risultati in termini di morte cellulare che possono essere influenzati non solo dalle proprietà dei sensibilizzanti ma anche dall'organizzazione nelle colture 2D e 3D.
Sonodynamic therapy (SDT) is a promising non-invasive treatment that involves a combination of low-intensity ultrasound (US) and chemical agents known as sonosensitizers. SDT is based on photodynamic therapy (PDT), where light is used to activate the sensitizer instead of US, leading to reactive oxygen species (ROS) generation and cell death. PDT main drawback is represented by the poor tissue light penetration, that can be overcome by using US that is known to penetrate more deeply into tissues. SDT mechanism of action still remains under debate and several studies suggested that it might be influenced by the nature of biological model, the sensitizer and the US parameters involved. Furthermore, an interesting aspect to be elucidate is the ability of SDT to counteract the multidrug resistance of cancer cells. Rose Bengal (RB) and Hypericin (Hyp) are well-known photosensitizers but their role in SDT is still unclear. The main aim of this work is to investigate the responsiveness of RB and Hyp to US exposure on a human colon cancer cell line (HT-29) grown into two- and three-dimensional (2D and 3D) cultures, taking light exposure as a reference. To evaluate the optimal sensitizer concentrations and time to perform sonodynamic and photodynamic experiments, cellular uptake, intracellular localization and IC50 analyses were performed on HT-29 cells in 2D culture. Then, to evaluate the efficacy of SDT with RB and Hyp, cell death imaging and flow cytometry analyses were performed on HT-29 cells in 3D culture. HT-29 3D spheroids were generated by using agarose coated plates and were treated with the same experimental conditions used in 2D culture but considering a higher sensitizer concentration. Both sensitizers showed a cytoplasmic distribution at mitochondrial level, even if in the case of Hyp, sensitizer accumulations were found close to cell membrane. On 2D culture, photodynamic exposure of RB pre-incubated HT-29 cells showed a statistically significant decrease of cell growth 48 and 72 hours after the treatment, while sonodynamic exposure of RB pre-incubated HT-29 cells provoked only a slight decrease of cell growth at the same time points. Photodynamic exposure of Hyp pre-incubated HT-29 cells induced a significant decrease of cell growth 48 and 72 hours after the treatment and similarly sonodynamic exposure of Hyp pre-incubated cells provoked a statistically significant decrease of cell growth at the same time points. Spheroids that underwent to PDT with RB showed a complete disaggregation of 3D structures, along with a significant increase of dead cells and a bright propidium iodide (PI) intensity 48 hours after the treatment. On the other hand, a lower statistically significant increase of dead cells was observed when spheroids were previously incubated with RB and then treated with US. PDT with Hyp was able to induce a slight external modification of spheroid shape along with a slight induction of cell death 48 hours after the treatment and interestingly, when HT-29 spheroids were pre-treated with Hyp and then exposed to US, a higher statistically significant increase of cell death was observed. Our data show a significant responsiveness of RB and Hyp to US exposure although to a lesser extent than to light exposure. Moreover, the sonodynamic activation leads to different results in terms of cancer cell death that may be influenced not only by the properties of the sensitizer but also by the cell organization into 2D or 3D cultures.
Valutazione in vitro dell'attività sonodinamica di Rosa Bengala e Ipericina in colture tridimensionali di cellule tumorali del colon umano
SANTACROCE, ALESSIA MARIA
2019/2020
Abstract
Sonodynamic therapy (SDT) is a promising non-invasive treatment that involves a combination of low-intensity ultrasound (US) and chemical agents known as sonosensitizers. SDT is based on photodynamic therapy (PDT), where light is used to activate the sensitizer instead of US, leading to reactive oxygen species (ROS) generation and cell death. PDT main drawback is represented by the poor tissue light penetration, that can be overcome by using US that is known to penetrate more deeply into tissues. SDT mechanism of action still remains under debate and several studies suggested that it might be influenced by the nature of biological model, the sensitizer and the US parameters involved. Furthermore, an interesting aspect to be elucidate is the ability of SDT to counteract the multidrug resistance of cancer cells. Rose Bengal (RB) and Hypericin (Hyp) are well-known photosensitizers but their role in SDT is still unclear. The main aim of this work is to investigate the responsiveness of RB and Hyp to US exposure on a human colon cancer cell line (HT-29) grown into two- and three-dimensional (2D and 3D) cultures, taking light exposure as a reference. To evaluate the optimal sensitizer concentrations and time to perform sonodynamic and photodynamic experiments, cellular uptake, intracellular localization and IC50 analyses were performed on HT-29 cells in 2D culture. Then, to evaluate the efficacy of SDT with RB and Hyp, cell death imaging and flow cytometry analyses were performed on HT-29 cells in 3D culture. HT-29 3D spheroids were generated by using agarose coated plates and were treated with the same experimental conditions used in 2D culture but considering a higher sensitizer concentration. Both sensitizers showed a cytoplasmic distribution at mitochondrial level, even if in the case of Hyp, sensitizer accumulations were found close to cell membrane. On 2D culture, photodynamic exposure of RB pre-incubated HT-29 cells showed a statistically significant decrease of cell growth 48 and 72 hours after the treatment, while sonodynamic exposure of RB pre-incubated HT-29 cells provoked only a slight decrease of cell growth at the same time points. Photodynamic exposure of Hyp pre-incubated HT-29 cells induced a significant decrease of cell growth 48 and 72 hours after the treatment and similarly sonodynamic exposure of Hyp pre-incubated cells provoked a statistically significant decrease of cell growth at the same time points. Spheroids that underwent to PDT with RB showed a complete disaggregation of 3D structures, along with a significant increase of dead cells and a bright propidium iodide (PI) intensity 48 hours after the treatment. On the other hand, a lower statistically significant increase of dead cells was observed when spheroids were previously incubated with RB and then treated with US. PDT with Hyp was able to induce a slight external modification of spheroid shape along with a slight induction of cell death 48 hours after the treatment and interestingly, when HT-29 spheroids were pre-treated with Hyp and then exposed to US, a higher statistically significant increase of cell death was observed. Our data show a significant responsiveness of RB and Hyp to US exposure although to a lesser extent than to light exposure. Moreover, the sonodynamic activation leads to different results in terms of cancer cell death that may be influenced not only by the properties of the sensitizer but also by the cell organization into 2D or 3D cultures.| File | Dimensione | Formato | |
|---|---|---|---|
|
873969_tesifinale_alessia_santacroce.pdf
non disponibili
Tipologia:
Altro materiale allegato
Dimensione
1.9 MB
Formato
Adobe PDF
|
1.9 MB | Adobe PDF |
I documenti in UNITESI sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
https://hdl.handle.net/20.500.14240/29237