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 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. 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) 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.

Hemocompatibility of dextran-graft-polyacrylamide/zinc oxide nanosystems: hemolysis or eryptosis?

“…Data on the fluorescence was acquired using a BD FACS Canto™ II flow cytometer (Becton Dickinson, USA) and processed further with FlowJo™ (v10, BDBiosciences, USA) software. Three eryptosis parameters were quantified: forward scatter (FSC) signal (cell shrinkage), the percentage of annexin V-positive cells (PS exposure), and dichlorofluorescein (DCF) fluorescence (ROS generation) [39,41,42,54].…”

“…There is compelling evidence that hemolytic properties of NPs are dependent on size, shape, surface charge, porosity, or surface modifications of nanomaterials [34]. However, eryptosis, a Ca 2+ -dependent programmed cell death of red blood cells (RBCs), is emerging as an alternative and probably more sensitive marker of hemocompatibility for blood-contacting nanomaterials [38][39][40][41][42][43][44], which can be potentially used as a screening approach for newly synthesized nanomaterials. Moreover, eryptotic erythrocytes are reported to be less sensitive to NPs-induced hemolysis than the healthy ones [45].…”