Valutazione in vitro della clorofilla come agente responsivo agli ultrasuoni per il trattamento sonodinamico del tumore prostatico

Prostate cancer remains the 2° leading cause of death in the USA and the 6 leading cause of death among men worldwide,the limits of the treatments used have led to research towards non-invasive and selective approaches.The development of novel treatment paradigms,such as SDT,might improve future treatment prospects.SDT is an anticancer approach under investigation that combines low intensity ultrasound with a sonosensitizer (non-cytotoxic compound) to achieve a therapeutic effect.SDT is based on PDT,but US has the advantage of better tissue penetration than light.Many of the photosensitizes are excellent sonosensitizers,the mechanism by which the sensitizer is activated to achieve the cytotoxic effect is not fully understood.It is generally accepted that site specific toxicity is caused by the production of ROS,induced by cavitation phenomenon.Chlorophyll(Chl)is a well-known photosensitizer can be activated by light to generate ROS.Chl degrades much faster than other sensitizers and the abundance of this pigment in nature encourage to select it as sonosensitizer in cancer treatment.The use of Chl as a sonosensitizer has been hypothesized thanks to its ability to produce ROS under appropriate US exposure.Chl is not yet so deeply exploited,and now there is great interest in searching for new sonosensitizers to increase efficacy whilst reducing side effects.The aim of this work is to investigate the anticancer activity of Chl as a sonosensitizer under US exposure on prostate cancer cell line.Chl has been considered under different formulations:Chl as free drug or Chl loaded into liposomes(Chl-LP),solid lipid nanoparticles(Chl-SLN)and polymeric nanoparticles(Chl-PLGA).The SDT effects of Chl have been studied in vitro on 2D PC-3 human prostate cancer cell culture and on 3D Du145 prostate spheroids.The biological characterization of Chl as cytotoxicity, cell uptake and cell localization has been studied.The physical characterization of the different Chl formulations have also been taken into consideration.To assess a synergistic effect between Chl and US,effects of sonodynamic treatment on PC-3 cells have been evaluated on cell proliferation,ROS production and cell death.To select the non-cytotoxic Chl concentration and incubation time for the sonodynamic treatment, cytotoxicity and cellular uptake have been evaluated resulting 5µM after 6h of incubation for Chl-LP or Chl-SLN and after 24h of incubation for Chl or Chl-PLGA.The SDT on PC-3 cells caused the highest significant reduction of cell proliferation with Chl-PLGA and US exposure (p<0.001) compared to untreated cells,along with a significant ROS production over time and an increase of apoptotic and necrotic cells.2D cell culture cannot replicate real microenvironment and cell behaviors in vivo, therefore the SDT has also been evaluated on Du145 spheroids,a 3D model that recreates a more realistic cellular microenvironment.A significant decrease of around 60%, compare to the control condition,in Du145 spheroid volume was observed after the treatment with Chl-PLGA(1.1x108±2.8x107 and 4.2x107±2.9x107, respectively).In conclusion,Chl can act as a sonosensitizer agent and its activity can be influenced by its formulation.The different sonodynamic activities observed according to the various formulations might be ascribed to their different cellular uptake and might be related to the interaction of the intracellular energy locally generated by US with the nanoparticle structure.