Enhancement of cancer selectivity of doxorubicin by ultrasound-responsive gold nanoparticles

Among the new advances in cancer treatment, nanomedicine has emerged as a new multidisciplinary approach. This new field stems from the promise of new complementary ideas, such as selective cell targeting and drug delivery, which would open up new perspectives for anti-cancer drugs. Indeed, one of the most widely used chemotherapeutic compounds, doxorubicin (Doxo), has already made progress in the introduction of nanomedicine with the development of a liposomal formulation to improve the safety profile, but unfortunately not yet its efficacy against cancer cells. Therefore, this thesis project aims to investigate, using an in vitro model of a KB epidermoid carcinoma cell line overexpressing the folate receptor (FR), frequently overexpressed in a wide range of cancers, whether folic acid-conjugated gold nanoparticles are able to exert a targeted and timely release of Doxo via ultrasound (US) exposure. To do so, folate-coated gold nanoparticles loaded with Doxo (FDGs) have been developed in collaboration with Professor Stefano Salamaso from the University of Padova, Italy, and cells overexpressing FR (KBFR+) have been cultured. Moreover, FDGs were tuned for being responsive to low-intensity US exposure to obtain a cancer-selective and timely release of Doxo from FDGs. First, flow cytometry analysis has been carried out to confirm the expression of FR on KBFR+ cells and consequently, an uptake assay has been performed to investigate the FDG internalization into KBFR+. These experiments have shown an overexpression of FR in the KBFR+ cells compared to the KB wild-type cells and an increase in the uptake of FDGs in KBFR+ cells compared to the KB wild-type cells and MCF-7 cells, a breast cancer cell line that does not express FR at all. Since these data have confirmed that our FDG selectively targeted KBFR+, a cell proliferation assay was used to evaluate the effect of FDGs and Doxo alone on KBFR+ cells. Therefore, cells have been incubated with FDGs loading 2 μM Doxo or with Doxo alone at the same concentration. As expected, the cell proliferation assay showed a low cytotoxic effect in our KBFR+ cells under FDGs compared to Doxo alone. To elicit the FDG cytotoxicity by a remote-controlled stimulus, a low-intensity US treatment has been set up (1.505 MHz; 0.62 W/cm2; 51% Duty Cycle; 3 minutes) for the timely release of Doxo in KBFR+ cells. 5 The cell proliferation assay performed at 24 and 72 hours after US exposure showed statistically significant cytotoxicity when KBFR+ cells underwent FDG plus US exposure compared to KBFR+ cells, KBFR+ cells subjected only to FDG treatment and KBFR+ cells exposed to US only. To confirm the cytotoxic effect of Doxo released by FDGs under US exposure on KBFR+ cells, radical oxygen species (ROS) generation, cell death, and intracellular Doxo trafficking by fluorescence and confocal microscopy have been evaluated. ROS production, one of the main mechanisms of action of Doxo, and cell death analysis confirmed a statistically significant difference in cytotoxicity of Doxo on KBFR+ cells when FDG was exposed to US, along with an increase of ROS generation up to 50% and an increase in necrotic cells population of 44.7%, compared US and FDGs alone. Fluorescence and confocal microscopy investigations showed the cytosolic location of FDGs after their incubation in KBFR+ cells and a random nuclear translocation of Doxo to the nucleus after US exposure confirming the US-mediated timely release of Doxo from FDGs.