Rare-earth orthovanadate nanoparticles trigger Ca²⁺-dependent eryptosis

Introduction. Rare-earth orthovanadate nanoparticles (ReVO4:Eu3+, Re = Gd, Y or La) are promising agents for photodynamic therapy of cancer due to their modifiable redox properties. However, their toxicity limits their application. Objective. The aim of this research was to elucidate pro-eryptotic effects of GdVO4:Eu3+ and LaVO4:Eu3+ nanoparticles with identification of underlying mechanisms of eryptosis induction and to determine their pharmacological potential in eryptosis-related diseases. Methods. Blood samples (n = 9) were incubated for 24 h with 0-10-20-40-80 mg l(-1) GdVO4:Eu3+ or LaVO4:Eu3+ nanoparticles, washed and used to prepare erythrocyte suspensions to analyze the cell membrane scrambling (annexin-V-FITC staining), cell shrinkage (forward scatter signaling), reactive oxygen species (ROS) generation through 2 ',7 '-dichlorodihydrofluorescein diacetate (H2DCFDA) staining and intracellular Ca2+ levels via FLUO4 AM staining by flow cytometry. Internalization of europium-enabled luminescent GdVO4:Eu3+ and LaVO4:Eu3+ nanoparticles was assessed by confocal laser scanning microscopy. Results. Both nanoparticles triggered eryptosis at concentrations of 80 mg l(-1). ROS-mediated mechanisms were not involved in rare-earth orthovanadate nanoparticles-induced eryptosis. Elevated cytosolic Ca2+ concentrations were revealed even at subtoxic concentrations of nanoparticles. LaVO4:Eu3+ nanoparticles increased intracellular calcium levels in a more pronounced way compared with GdVO4:Eu3+ nanoparticles. Our data disclose that the small-sized (15 nm) GdVO4:Eu3+ nanoparticles were internalized after a 24 h incubation, while the large-sized (similar to 30 nm) LaVO4:Eu3+ nanoparticles were localized preferentially around erythrocytes. Conclusions. Both internalized GdVO4:Eu3+ and non-internalized LaVO4:Eu3+ nanoparticles (80 mg l(-1)) promote eryptosis of erythrocytes after a 24 h exposure in vitro via Ca2+ signaling without involvement of oxidative stress. Eryptosis is a promising model for assessing nanotoxicity.