Rare-earth orthovanadate nanoparticles trigger Ca 2+ -dependent eryptosis.

Introduction . Rare-earth orthovanadate nanoparticles (ReVO ₄ :Eu ³⁺ , 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 GdVO ₄ :Eu ³⁺ and LaVO ₄ :Eu ³⁺ 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 GdVO ₄ :Eu ³⁺ or LaVO ₄ :Eu ³⁺ 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 Ca ²⁺ levels via FLUO4 AM staining by flow cytometry. Internalization of europium-enabled luminescent GdVO ₄ :Eu ³⁺ and LaVO ₄ :Eu ³⁺ 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 Ca ²⁺ concentrations were revealed even at subtoxic concentrations of nanoparticles. LaVO ₄ :Eu ³⁺ nanoparticles increased intracellular calcium levels in a more pronounced way compared with GdVO ₄ :Eu ³⁺ nanoparticles. Our data disclose that the small-sized (15 nm) GdVO ₄ :Eu ³⁺ nanoparticles were internalized after a 24 h incubation, while the large-sized (∼30 nm) LaVO ₄ :Eu ³⁺ nanoparticles were localized preferentially around erythrocytes. Conclusions. Both internalized GdVO ₄ :Eu ³⁺ and non-internalized LaVO ₄ :Eu ³⁺ nanoparticles (80 mg l ^-1 ) promote eryptosis of erythrocytes after a 24 h exposure in vitro via Ca ²⁺ signaling without involvement of oxidative stress. Eryptosis is a promising model for assessing nanotoxicity.
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