The effect of incubation conditions on the hemolytic properties of unmodified graphene oxide with various concentrations

Abstract

The hemolysis assay is a common method used to evaluate the in vitro biocompatibility of biomaterials, but presently there is inadequate understanding of the interactions between RBCs and biomaterials, especially from the view of the incubation conditions and sample concentration. In this report, we used unmodified graphene oxide (GO) as a biomaterial model and measured the hemolytic properties of GO at a series of concentrations under multiple conditions, including medium type, red blood cell (RBC) species, RBC concentration, storage duration of the RBCs, animal health status, centrifugal speed, exposure time and incubation temperature. Additionally, a calculation method for the hemolytic index was proposed so as to objectively evaluate the hemolysis properties, distinct from frequently used methods. Compared to the hemolytic results under these conditions, we found that the hemolytic properties of GO had huge differences, and false-negative or false-positive errors might be produced by inappropriate incubation conditions. In our opinion, the feasible conditions for unmodified GO are: isotonic glucose solution as the medium type, fresh and healthy rabbit RBCs as the test object, 1% hematocrit as the RBC concentration, 3000 rpm for 5 min as the centrifugation conditions, 6 h as the exposure time and physiological temperature (37 °C) as the incubation temperature.