In vitro evaluation of multifunctional albumin nanoparticles for the sonochemotherapy of pancreatic cancer

Pancreatic cancer remains one of the most lethal forms of cancer with a 5-year survival less than 10%. Surgery, chemotherapy and radiotherapy are traditionally used to extend patient survival. Sonodynamic therapy (SDT) is an emerging approach for the selective treatment of solid tumors. In this approach, the interaction of ultrasound (US) with a sound-sensitive drug (sonosensitizer) induces cancer cell death by reactive oxygen species (ROS) generation. The low invasiveness and the deep tissue penetration make SDT effective in treating deep and large tumors. Many nanoparticles (NPs) have been designed for tumor-targeted delivery of sonosensitizers. Moreover, NPs can deliver simultaneously different therapeutic agents, thus promoting potential synergistic effects of chemotherapeutic drugs and sonosensitizers when exposed to US. Therefore, combining NPs and US in SDT might give interesting results compared to the conventional chemotherapeutic treatments. The aim of this thesis project was to evaluate on an in vitro 2D and 3D model of pancreatic cancer (BxPC-3) the therapeutic effect of SDT in combination with human serum albumin nanoparticles (Nab) loaded with the sonosensitizer IR-780 (S), the chemotherapeutic drug paclitaxel (P) and the immunomodulator drug indoximod (I). The effect of Nab loaded with noncytotoxic concentrations of the different therapeutic agents was investigated to assess a synergistic action between the US-activated sonosensitizer and the chemoimmunotherapeutic agents regarding the effects on cell proliferation, cell death and occurrence of specific damage-associated molecular patterns (DAMPs), like calreticulin (CRT) and high mobility group box 1 (HMGB1). In order to select the proper noncytotoxic concentrations and incubation time for SDT, uptake and cytotoxicity of Nab loaded with IR-780, paclitaxel and indoximod (Nab-SPI) were evaluated in BxPC-3 cells. A significant uptake of Nab-SPI was detected after 24 h of incubation (p<0.01) with a sonosensitizer concentration of 0.1 μg/mL. SDT with Nab-SPI determined a significant decrease of BxPC-3 cell proliferation after 72 h (p<0.01) along with a significant increase of apoptotic and necrotic cell death. In addition, a significant increase of CRT and HMGB1 was detected at 6 h (p<0.001) and 24 h (p<0.001) after SDT with Nab-SPI, respectively. According to the observed immunogenic cell death (ICD) induced by SDT, the activation of dendritic cells (DCs) was investigated to confirm an effective immune response against tumor cells. Thereby, peripheral blood mononuclear cells (PBMCs) were co-cultured with BxPC-3 cells, previously exposed to SDT with Nab-SPI, for 1, 12 and 24 h showing a significant activation after 1 h (p<0.001). Finally, to confirm the data observed in 2D cell cultures, BxPC-3 spheroids have been developed and studied extensively. The uptake of Nab loaded with IR-780 at 10 μg/mL in BxPC-3 spheroids increased from 24 to 72 h of incubation (p<0.001). After SDT with Nab-SPI, a reduction of the volume of treated spheroids was observed after 24 and 48 h, even if not statistically significant. Moreover, SDT-induced cell death in spheroids was investigated by PI fluorescence, observing a significant increase signal in those spheroids treated with Nab-SPI and US, compared to untreated spheroids (p<0.05). In conclusion, the sonochemotherapy achieved by US exposure of Nab-SPI was able to significantly promote in vitro tumor cell killing and ICD in BxPC-3.