High Oxygen Affinity Polymerized Bovine Hemoglobin-Based Oxygen Carriers as Potential Artificial Blood Substitutes

Given the periodic shortages and safety concerns of blood, the development of an artificial blood substitute capable of adequately transporting oxygen without eliciting undue side effects would be quite beneficial. Acellular modified hemoglobin-based oxygen carriers with high oxygen affinity (P50 < 26 mmHg) are a new class of blood substitutes being explored. It has been theorized that these oxygen carriers may have less vasoactive side effects compared to their low oxygen affinity counterparts and may transport oxygen in a more physiological manner. In this study, a parallel synthetic approach was used to produce a small library of oxygen carriers by cross-linking/polymerizing bovine hemoglobin (Hb) with glutaraldehyde and ring-opened saccharides. The type and concentration of cross-linker and quenching agent, as well as reaction time, were all manipulated to yield oxygen carriers with various molecular weight distributions and oxygen binding properties. A high degree of Hb cross-linking with glutaraldehyde resulted in the synthesis of polymerized Hb (polyHb) dispersions with low P50s (< 26 mmHg), low Hill coefficients (< 2.3), and high methemoglobin (metHb) levels (> 10%). In this work, the high metHb level of glutaraldehyde polyHb dispersions was countered by reducing the polyHb dispersions post-reaction with ascorbic acid. Utilization of ring-opened saccharides as Hb cross-linking reagents resulted in polyHb dispersions with even lower P50s (~7-9 mmHg) compared to glutaraldehyde polyHb dispersions. In fact, o-methylglucopyranoside showed the most promise as an Hb cross-linking reagent and resulted in a higher degree of Hb cross-linking and lower metHb levels compared to other ring-opened saccharides. Polymerization of bovine Hb with glutaraldehyde resulted in decreased allosteric effects of pH and Cl- on the oxygen binding properties of polyHb dispersions, although it was not completely abolished. Modeling the oxygen transporting ability of these oxygen carriers in a human capillary showed that a moderately low P50 (such as 18 mmHg) oxygen carrier delivered oxygen more consistently over a wide range of hypoxic pO2 levels. In contrast, very low P50 (such as 6 or 13 mmHg) oxygen carriers specifically target oxygen delivery to tissues with low oxygen tensions, which are observed clinically in cases of severe hypoxia.