Potential Electron Mediators to Extract Electron Energies of RBC Glycolysis for Prolonged in Vivo Functional Lifetime of Hemoglobin Vesicles

Abstract

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.

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

“…Because MB has been approved by the FDA as an antidote for methemoglobinemia [18], potential substances have been compared with MB to evaluate their efficacy. Results from both in vitro and in vivo studies support results described in an earlier report, which clarified that several different cationic organic dyes can contribute to similar electron-mediating properties as MB and thereby can reduce metHb [17].…”

“…In the present investigation, eight dyes were evaluated: MB, DMB, TDB, AA, AB, AC, TLB and BCB. Because of similarity in molecular structure and physiochemical properties, such as reaction rates with NADH/ NADPH, incorporation rate into HbVs and RBC, reoxidation rate and redox potential, the electron pathway is presumably similar to that of MB, as shown in Table 1 [17]. Because MB has been approved by the FDA as an antidote for methemoglobinemia [18], potential substances have been compared with MB to evaluate their efficacy.…”

“…In vivo level of metHb (%) at 3 h ( Figure 2 Data of the calculated second-order rate constants for each dye with NAD(P)H, incorporation ratio to vesicles and RBC membranes, reoxidation rate with O 2 , and redox potential (E 0 0 ) were obtained from Kettisen et al [17]. Data of in vitro and in vivo levels of metHb were taken from Figures 1 and 2, respectively.…”

“…MBH entering the HbV functions as an electron mediator between NADH/NADPH in RBCs and metHb compartmentalized inside the vesicles [16]. Earlier studies have verified that other cationic organic dyes (phenothiazines, phenazines, phenoxazines, indamine) are also useful in such redox reactions, and are thereby able to participate in the reduction of metHb [17]. The present study was undertaken to clarify whether the suggested potential electron mediators (Scheme 1) can prolong the functional lifetime of HbVs, as evaluated by in vivo studies.…”

Nanovesicular liposome-encapsulated hemoglobin (LEH) prevents multi-organ injuries in a rat model of hemorrhagic shock

“…Compared to these acellular hemoglobin-based products, encapsulated hemoglobin can provide longer circulation persistence without any vasoactivity and renal toxicity (Awasthi et al, 2006; Cabrales et al, 2012). Moreover, LEH can also carry hemoglobin stabilizers and allosteric modifiers of oxygen affinity, which can be co-encapsulated, to enhance the functional efficacy of the product (Kettisen et al, 2015). LEH resuscitation has been shown to maintain the hemodynamics and microvascular circulation after acute blood loss (Sakai et al, 2004), and improve cerebral oxygenation (Kaneda et al, 2014; Kawaguchi et al, 2014) in pre-clinical models.…”