In vivo evaluation of electron mediators for the reduction of methemoglobin encapsulated in liposomes using electron energies produced by red blood cell glycolysis

Earlier studies have clarified that NADH and NADPH, re-energized repeatedly by red blood cell (RBC) glycolysis, can be used in extracellular chemical reactions, where electron energies are extracted by electron mediators, such as methylene blue (MB). The electron mediators, which are reduced by NAD(P)H, permeate both the membranes of RBC and phospholipid bilayer of liposomes encapsulating haemoglobin (Hb-vesicles, HbV) and reduce autoxidized ferric methemoglobin (metHb) in HbV to ferrous Hb. Moreover, in vitro screening study clarified some other potential electron mediators with comparable capacity to reduce metHb. Given this background, eight of these compounds: MB, 1,9-dimethyl MB, azure A, azure B (AB), azure, toluidine blue, brilliant cresyl blue and toluylene blue, were evaluated in both in vitro and in vivo studies in this work. Compared with MB as a reference, in vitro experiments demonstrated that most compounds caused effective metHb reduction of HbV in the presence of RBC. However, in vivo experiments of bolus injection of autoxidized HbV to rats (10mL HbV/kg body weight) followed by injection of the dye (1.53mL/kg body weight, 2.6mM) led to some differences from in vitro results. Effective metHb reduction was found for the combination of AB. To evaluate AB effectiveness further, a haemorrhagic shock and resuscitation model was used, where the rats were resuscitated with HbV. When the level of metHb increased to 50%, a dye solution was injected. Again, AB caused sufficient reduction of metHb. Through these in vivo experiments, this study clarified that AB is a suitable electron mediator to prolong the functional lifetime of HbV. [GRAPHICS] .