Nanoparticles loaded with meso-tetrakis (4-sulfonatophenyl) porphyrin enhance efficacy of sonodynamic therapy in an in vitro cancer model

F Foglietta1, R Canaparo1, M Ballestri2, G Sotgiu2, A Guerrini2, G Varchi2, E Imbalzano1, R Frairia3, L Serpe1. 1Università degli Studi di Torino, Drug Science and Technology, 10125, Italy, 2CNR, Istituto per la Sintesi Organica e la Fotoreattività, 40129, Italy, 3università degli Studi di Torino, Clinical Pathophysiology, 10126 10, Italy

Ultrasound, which has been conventionally used for diagnostic imaging in medicine, is now being extensively studied for a large number of biomedical applications because it can affect tissue through a variety of mechanisms. It has been reported that some chemical compounds known as sonosensitizers, such as the non-toxic hematoporphyrins, can be activated by ultrasound and kill cancer cells. Ultrasound-induced inertial cavitation may cause an energy transfer which is able to cause electronic excitation in the sonosensitizer resulting in the generation of a variety of free radicals. This innovative anti-cancer approach has been termed sonodynamic therapy. In this study we have investigated new highly lypophilic poly-methyl methacrylate core-shell nanoparticles (NP) engineered in order to load meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS) as sonosensitizing agent. The sonodynamic treatment (SDT) with high energy shock waves (HESW), generated by a piezoelectric device (Piezoson 100, Wolf), at the energy flux density of 0,43 mJ/mm2 for 500 shots (4 shots/sec), was investigated on a human neuroblastoma cell line (SH-SY5Y) pre-incubated for 12 h with 100 μg/ml of TPPS loaded nanoparticles (TPPS-NP). In particular we evaluated the in vitro sonodynamic effects of TPPS-NP on SH-SY5Y cells with respect to free TPPS and unloaded NP. SH-SY5Y cells were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air and cultured in a 1 : 1 mixture of Ham’s F12 and Eagle’s minimum essential medium supplemented with L-glutamine, penicillin and streptomycin, and 10% foetal calf serum. After the SDT cell growth was evaluated by WST-1 assay at 24, 48 and 72 h; reactive oxygen species (ROS) production by dichlorofluorescein flow cytometric analysis at 1, 5, 30, 60 and 180 min; mRNA expression by real time RT-PCR at 24 h; cell death by flow cytometric analysis with SYTOX Green and APC-Annexin V at 48 h. Data are expressed as mean ± sem of three separate experiments and statistical comparisons between treatment groups were performed with analysis of variance (two-way ANOVA) and the threshold of significance was calculated by the Bonferroni’s test. 48 h after the SDT a significant decrease in cell growth was observed in cells exposed to TPPS loaded NP (0.304 ± 0.009; p<0.001) compared to untreated cells (0.644 ± 0.003), cells exposed only to unloaded NP (0.504 ± 0.006) and cells exposed only to free TPPS (0.595 ± 0.002). Moreover, 3 h after SDT a 25 fold increase in ROS production with respect to control cells (p<0.01) was observed in cells exposed to TPPS-NP. In the mRNA expression profile of the panel of genes studied it must be point out a significant enhanced mRNA expression of the pro-apoptotic BAD (p<0.01) and pro-oxidant NQO1 (p<0.01) genes in cells exposed to TPPS-NP compared to control cells at 24 h after SDT. Cell death analysis by flow cytometry revealed a significant increase in necrosis (p<0.05) and apoptosis (p<0.01) in cells exposed to TPPS-NP compared to control cells at 48 h after SDT. Therefore, since the sonosensitizers are essential for the success of this new anti-cancer approach, we believe that combine these nanoparticles with TPPS, might be able to improve the effect of sonodynamic treatment.