ROS production and their influence on the cellular antioxidative system in human erythrocytes incubated with daunorubicin and glutaraldehyde

Abstract

This study examined the effects of daunorubicin and glutaraldehyde on some parameters of erythrocytes. The aim of the article was to present the results of research aiming to identify the level of glutaraldehyde at which the hemoglobin oxidation, externalization of phosphatidylserine and the changes in the viability (hemolysis) of erythrocytes are not statistically significant and therefore this level of glutaraldehyde can be used for the drug carriers' preparation. Glutaraldehyde was used as a crosslinking agent to enhance the uptake of the drug within red blood cells and to prevent its leakage from the cells. Fluorescence microscopy, flow cytometry and fluorimetric measurements confirmed higher levels of the drug in glutaraldehyde-treated human erythrocytes. Unfortunately, substantial damage to the red blood cells was also noted. DNR increased oxidative processes in the cell, which in turn led to an increase in the reactive oxygen species (ROS) level. When the red blood cells were also treated with glutaraldehyde, ROS production was significantly higher. We also observed loss of both the reduced and the total glutathione. Moreover the decreased activity of glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD) was also observed. As hemoglobin, the erythrocytes' main component plays an essential role in the erythrocytes, the level of its oxidized form (metHb) in the erythrocytes and the phosphatidylserine exposure on the erythrocyte surface were also investigated. When higher concentrations of glutaraldehyde (0.0025-0.005%) were used for the uptake of DNR the elevated level of metHb was observed. Only at 0.0005% the level of oxidized form of Hb was within the physiological level and at that level the increase in the exposure of phosphatidylserine at the cell surface was not observed to be statistically significant. Moreover the percent of released hemoglobin was less than 1%. Based on these results it was concluded that glutaraldehyde can be used as a cross-linker between the drug (DNR) and the erythrocytes only at low concentration of about 0.0005%.