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

In this study, blood compatibility of ZnO nanoparticles-nanocomplex (D-PAA/ZnONPs(SO42-)) synthesized in situ into dextran-graft-polyacrylamide (D-PAA) using zinc oxide nanoparticles produced from zinc sulphate (D-PAA/ZnONPs(SO42-)) as a precursor was tested using hemolysis, osmotic fragility and eryptosis assays.
Materials and methods. Dose-dependent ability to induce eryptosis was assessed following 24 h incubation at concentrations of 0-800 mg / L analyzing hallmarks of eryptosis (cell shrinkage and phosphatidylserine externalization), as well as reactive oxygen species (ROS) generation. Hemolysis was detected spectrophotometrically based on hemoglobin release following exposure to the D-PAA/ZnONPs(SO42-) nanocomplex. Osmotic fragility test (OFT) involved detection of hemolysis of red blood cells exposed to 0.2% saline solution following incubation with the D-PAA/ZnONPs(SO42-) nanocomplex. Additional incubation of the nanocomplex in the presence or absence of either ascorbic acid or EGTA was used to reveal the implication of oxidative stress- or Ca2+-mediated mechanisms in D-PAA/ZnONPs(SO42-) nanocomplex-induced erythrotoxicity.
Results. Hemocompatibility assessment of the D-PAA/ZnONPs(SO42-) nanocomplex revealed that it induced hemolysis and reduced resistance of erythrocytes to osmotic stress at concentrations of above 400 and 200 mg / L, respectively. Oxidative stress- or Ca2+-mediated mechanisms were not involved in D-PAA/ZnONPs(SO42-) nanocomplex-induced hemolysis. Strikingly, the D-PAA/ZnONPs(SO42-) nanocomplex did not promote cell membrane scrambling, cell shrinkage and oxidative stress in red blood cells following the direct exposure for 24 h. Thus, the D-PAA/ZnONPs(SO42-) nanocomplex did not induce eryptosis in vitro. Eryptosis is generally considered to occur earlier than hemolysis in response to stress in order to prevent hemolytic cell death. Counterintuitively, our data suggest that hemolysis can be triggered by nanomaterials prior to eryptosis indicating that eryptosis and hemolysis assays should be used in combination for testing blood compatibility of nanomaterials. 
Conclusions. The D-PAA/ZnONPs(SO42-) nanocomplex has a good hemocompatibility profile at low concentrations. Hemocompatibility testing in nanotoxicology should include both eryptosis and hemolysis assays. 
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