Comprehensive biophysical and biochemical characterization of polymerized dopamine coated hemoglobin based oxygen carrier

Hemoglobin (Hb)-based oxygen carriers (HBOCs) are promising red blood cell (RBC) substitutes, when blood is unavailable. However, HBOCs can generate reactive oxygen species (ROS) species leading to oxidative stress, lipid peroxidation, DNA damage, and cell death. To address these issues, researchers have explored coating Hb and HBOCs with polymerized dopamine (PDA), to impart antioxidant properties via radical scavenging. This study builds upon previous PDA-coated Hb (PDA-hHb-1) research by quantifying HBOC biophysical properties and proposing two new PDA coating methods: 1) first binding carbon monoxide (CO) to human Hb (hHb) prior to PDA-coating to reduce metHb formation (PDA-hHb-13), and 2) forming PDA nanoparticles (NPs) before coating them with HbCO (PDA-hHb-15). After CO removal, PDA-hHb-13 and PDA-hHb-15 exhibited lower metHb levels (< 10 %) compared to PDA-hHb-1 (~13 %). PDA-hHb-15 had a reduced auto-oxidation rate constant compared to PDA-hHb-13, though both exceeded the precursor hHb. Functionally, PDA-coated hHbs retained similar oxygen offloading kinetics, with PDA-hHb-13 showing a lower oxygen affinity. PDA-coated hHbs exhibited enhanced ferric reducing and radical scavenging activity, but also increased ferryl species formation in the presence of H2O2, likely due to reduced catalase activity. Overall, PDA-coated Hb variants show promise as HBOCs with enhanced antioxidant properties, though further optimization is needed to balance stability and oxidative resilience.