Optimizing the Hemoglobin-Based Oxygen Carrier Molecular Size to Balance Vascular Function and Hemodynamic Responses after Transfusion.

Hemorrhagic shock remains a leading cause of mortality due to impaired oxygen delivery following severe blood loss. Hemoglobin-based oxygen carriers (HBOCs) are promising alternatives to red blood cell transfusion; however, their molecular size critically governs nitric oxide (NO) scavenging, vascular reactivity, and overall safety. Here, we systematically evaluated acellular human hemoglobin (hHb) and four polymerized hemoglobin formulations with progressively increasing molecular weights (PolyhHb-10, ∼1080 kDa; PolyhHb-12, ∼1200 kDa; PolyhHb-15, ∼1490 kDa; and PolyhHb-16, ∼1570 kDa) to define size-dependent determinants of vascular function. Ex vivo, isolated mesenteric arterioles from New Zealand white rabbits were perfused with HBOC-whole blood mixtures (1:0, 1:1, and 1:3) across physiologically relevant flow rates to quantify shear-mediated vasodilation and endothelial function. The acetylcholine challenge was used to probe NO bioavailability. In vivo validation was performed in Golden Syrian hamsters using a dorsal window chamber model to assess microvascular hemodynamics, including the vessel diameter, blood flow, and functional capillary density. PolyhHb-12 consistently preserved endothelial-dependent vasodilation and exhibited minimal NO scavenging across conditions. In contrast, larger polymers (PolyhHb-15 and PolyhHb-16) showed paradoxically increased NO scavenging despite improved capillary perfusion, indicating a trade-off between microcirculatory flow enhancement and endothelial signaling. In vivo, increasing the molecular size yielded diminishing returns in overall hemodynamic stability. Together, these findings identify an intermediate molecular size as optimal for balancing rheological properties, NO bioavailability, and vascular function. PolyhHb-12 emerges as a lead candidate, supporting the design of next-generation HBOCs with improved safety and efficacy for hemorrhagic shock resuscitation.