Rare-earth orthovanadate nanoparticles trigger Ca2+-dependent eryptosis

Abstract

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^-1GdVO₄: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 (∼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 exposurein vitrovia Ca²⁺signaling without involvement of oxidative stress. Eryptosis is a promising model for assessing nanotoxicity.

Keywords: cell death; confocal laser scanning microscopy; erythrocytes; flow cytometry; nanotoxicity.