Exploiting multifunctional nanoparticles for enhancing the chemo-sonodynamic cancer cell death on an in vitro breast cancer model

Sonodynamic therapy (SDT) has emerged as a promising approach for cancer therapy. SDT exploits ultrasound (US) to trigger a sonosensitizer in order to transfer energy to nearby oxygen molecules, which subsequently generate reactive oxygen species (ROS), leading to further cytotoxicity for therapeutic purposes. SDT has negligible invasiveness and deep tissue penetration, being able to target lesion zones with enhanced precision, hence leaving normal tissue undamaged. Increasing evidence has revealed that SDT and certain antineoplastic drugs show the ability to switch apoptosis process towards immunogenic cell death (ICD), boosting the host immune response and eradicating primary tumour growth. The aim of this thesis project is to investigate on an in vitro model of human breast cancer (MCF-7) cells the effect of a combination therapy in which human serum albumin (HSA) nanoparticles containing sonosensitizer IR-780, paclitaxel (PTX) and indoximod (D-1MT), were exposed to US stimulation. Specifically, combination therapy can be considered as first-line treatment of numerous malignancies to improve clinical outcomes thanks to synergistic drug actions and to prevention of drug resistance onset. Moreover, emerging evidence suggests that the combination between immune adjuvant drugs and other cancer therapies (e.g., chemotherapy, photodynamic therapy, SDT) provides a significant boost in antitumor outcome. Indeed, a central focus was to investigate if this sonodynamic treatment was able to determine an immunogenic cell death (ICD) activation. PTX and D-1MT were selected as widely used antitumor drugs against breast cancer, the first as a microtubule-stabilizing drug and the second as a competitive inhibitor of IDO1 with an immune checkpoint inhibitory activity. In particular, noncytotoxic concentrations of drugs, loaded into HSA nanoparticles, were used to assess a synergistic action between SDT and chemo-immunotherapy in term of effects on cell proliferation, ROS production, cell death and appearance of specific damage-associated molecular patterns (DAMPs), like calreticulin (CRT) and high mobility group box 1 (HMGB1). In order to select the proper noncytotoxic sonosensitizer concentration and incubation time for SDT, sensitizer cytotoxicity and uptake were firstly evaluated. A synergistic activity of PTX and D-1MT on MCF-7 cell proliferation was observed at 48 (p < 0.01) and 72 h (p < 0.001) when both drugs, loaded together with the sonosensitizer into HSA nanoparticles, underwent to US stimulation. However, this approach was not able to determine a significant cell modulation towards ICD. On the other side, when MCF-7 cells underwent first to the sonodynamic stimulation of sensitizer loaded into HSA nanoparticles, followed by a 24 h incubation with PTX and D-1MT loaded into HSA nanoparticles, a significant decrease in cell proliferation over time (p < 0.001), along with a significant increase of cell death at 36 h after the treatment and an important ROS production were observed. Moreover, a significant increase in the occurrence of CRT 14 h after the treatment and HMGB1 48 h after the treatment were appreciated (p < 0.05 and p < 0.01, respectively). Therefore, the temporal division of SDT process from the chemo-immunotherapy approach showed to be more effective in killing cancer cells and displaying ICD markers. In conclusion, the strategy to combine SDT with PTX and D-1MT loaded into HSA nanoparticles can be considered as an effective app