Analisi delle strategie basate sull'uso degli ultrasuoni per migliorare l'efficacia della doxorubicina nel trattamento del tumore ovarico

Ultrasound (US) is a mechanical wave, employed in diverse fields. Its safety and tissue penetrating ability has prompted the possibility of employing US-based strategies for therapeutic purposes. US exerts biological effects through mechanisms such as heat generation, acoustic streaming and cavitation. Therefore, US has been used for various purposes such as tumor ablation, sonoporation, sonophoresis and triggered drug release but one of the most attractive approach is the sonodynamic therapy (SDT). SDT is based on the use of US to activate a molecule, called sonosensitizer, enhancing its cytotoxicity. SDT derives from photodynamic therapy (PDT), that uses light instead of US but with a poor tissue penetrating ability. One molecule that can act both as sono- and photo-sensitizer is doxorubicin (doxo), a potent anticancer agent. Doxo is used in clinics for a huge variety of cancers, in particular it has a key role in the second line treatment of ovarian cancer (OC). However, the strategies employed so far in OC treatment have not yet been shown to have huge impact on its overall mortality. For this reason, many efforts in pharmacological research have been made in order to increase doxo efficacy in OC, at the same time abolishing its severe side effects. US-based strategies seem to be highly encouraging. In this thesis project the scientific literature has been analyzed to compare the results nowadays acquired on the possible uses of US for enhancing doxo efficacy. For instance, enhanced drug uptake has been achieved due to US-mediated cell membrane permeabilization (i.e., sonoporation). Moreover, many US-triggered drug delivery systems encapsulating doxo such as heat-sensitive liposomes and microbubbles has been developed and found to be promising. Interestingly, to address relapsing issues proper of OC it was hypothesized to increase the doxo-induced immunogenic cell death (ICD), a functionally distinct form of regulated cell death. ICD facilitates an adaptive immune response specific for dead cell-derived antigens that can lead to long lasting protective antitumor immunity. Therefore, laboratory experiments have been performed on human OC cell line A2780 to test if the activation of doxo by light or US is effective in maximizing the doxo-ICD. Hypericin (hyp), a natural well-known ICD inducer, has been used as yardstick, in comparison to doxo. The activity of doxo and hyp under light exposure was first investigated on A2780 cell proliferation, observing a statically significant decrease of cell proliferation over time. Specific biomarkers associated with ICD have been analyzed such as the exposure of calreticulin (CRT) at cell surface and the extracellular secretion of ATP. Light exposure of doxo and hyp was able to induce a statistically significant increase of extracellular secretion of ATP and CRT expression by cytofluorimetric analysis. Since SDT derives from PDT and US seems to have an immunomodulatory potential, these data support the investigation of US for maximizing doxo-ICD. Therefore, it would have been interesting to investigate ICD markers under US exposure of doxo and hyp. Along with this, the expression of genes involved in the ICD pathway (like CRT, LC3II and HMGB1) would have been analyzed by RT-PCR after light or US exposure of doxo and hyp. The data obtained have been compared to the results reported in the literature, showing that the use of US as an ICD-inducer should be very promising to increase doxo efficacy.