Potential electron mediators to extract electron energies of RBC glycolysis for prolonged in vivo functional lifetime of hemoglobin vesicles.

Developing a functional blood substitute as an alternative to donated blood for clinical use is believed to relieve present and future blood shortages, and to reduce the risks of infection and blood type mismatching. Hemoglobin vesicle (HbV) encapsulates a purified and concentrated human-derived Hb solution in a phospholipid vesicle (liposome). The in vivo safety and efficacy of HbV as a transfusion alternative have been clarified. Auto-oxidation of ferrous Hb in HbV gradually increases the level of ferric methemoglobin (metHb) and impairs the oxygen transport capabilities. The extension of the functional half-life of HbV has recently been proposed using an electron mediator, methylene blue (MB), which acts as a shuttle between red blood cells (RBC) and HbV. MB transfers electron energies of NAD(P)H, produced by RBC glycolysis, to metHb in HbV. Work presented here focuses on screening of 15 potential electron mediators, with appropriate redox potential and water solubility, for electron transfer from RBC to HbV. The results are assessed with regard to the chemical properties of the candidates. The compounds examined in this study were dimethyl methylene blue (DMB), methylene green, azure A, azure B, azure C, toluidine blue (TDB), thionin acetate, phenazine methosulfate, brilliant cresyl blue, cresyl violet, gallocyanine, toluylene blue, indigo carmine, indigotetrasulfonate, and MB. Six candidates were found to be unsuitable because of their insufficient diffusion across membranes, or overly high or nonexistent reactivity with relevant biomolecules. However, 9 displayed favorable metHb reduction. Among the suitable candidates, phenothiazines DMB and TDB exhibited effectiveness like MB did. In comparison to MB, they showed faster reduction by electron-donating NAD(P)H, coupled with showing a lower rate of reoxidation in the presence of molecular oxygen. Ascertaining the best electron mediator can provide a pathway for extending the lifetime and efficiency of potential blood substitutes.