Dextran-Graft-Polyacrylamide/Zinc Oxide Nanosystem does not Trigger Eryptosis 

Hemocompatibility of nanomaterials is an important step in the assessment of their safety due to the inevitable interactions of nanomaterials with blood upon their administration. A nanosystem composed of dextran-graft-polyacrylamide (D-PAA) and ZnO nanoparticles synthesized using zinc acetate as a precursor for nanoparticles (D-PAA/ZnO NPs (-OAc)) has been reported to show anti-cancer effects. However, its hemocompatibility is not fully tested. Eryptosis (Erythrocyte apoptosis or Red blood cell programmed death) is a type of apoptosis that occurs in injured erythrocytes (RBCs) due to various factors including hyperosmolarity, oxidative stress, energy depletion, heavy metals exposure or xenobiotics. Like apoptosis, eryptosis is characterized by cell shrinkage, membrane blebbing, activation of proteases, and phosphatidylserine exposure at the outer membrane leaflet. To test eryptosis parameters as an effective strategy to assess the hemocompatibility of newly synthesized nanomaterials. Erythrocytes incubated with D-PAA/ZnO NPs (-OAc) nanocomplex at concentrations 0-100-200-400 mg / L for 24 h were stained with Annexin V-FITC and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) with further flow cytometric analysis. Here We found that D-PAA/ZnO NPs (-OAc) nanocomplex does not trigger eryptosis, i.e. a suicidal regulated cell death of erythrocytes. In particular, incubation of the nanocomplex with blood (hematocrit 0.4%) for 24 h at concentrations 0-100-200-400 mg / L does not promote cell shrinkage (forward scatter signaling), phosphatidylserine externalization (Annexin V-FITC staining) or reactive oxygen species (ROS) generation (H2DCFDA staining), which are known to be hallmarks of eryptosis. Thus, our findings indicate that eryptosis is not triggered by D-PAA/ZnO NPs (-OAc) nanocomplex at concentrations demonstrating anti-cancer activity contributing to experimental evidence concerning the low toxicity of this nanocomplex. Furthermore, we believe that eryptosis can be widely used as a sensitive and reliable marker of nanomaterial hemocompatibility.