Scalable desolvation synthesis, storage stability, and comprehensive biophysical characterization of human hemoglobin nanoparticles as a potential hemoglobin‐based oxygen carrier

Hemoglobin (Hb)-based oxygen carriers (HBOCs) are materials that exploit Hb's native oxygen transport while avoiding the adverse side-effects associated with the exposure of the cell-free, unmodified Hb tetramer (i.e. vasoconstriction, systemic hypertension, and oxidative tissue injury) in circulation. There are many synthetic routes to generate HBOCs including chemical polymerization, polymer surface conjugation, and protein encapsulation that meet the material and O₂ demands of different biomedical applications (e.g. emergency transfusion medicine, ex vivo organ perfusion, tumor oxygenation, etc.) especially when red blood cells may not be immediately available or desirable. There is currently no FDA approved HBOC in the US, therefore leaving room in the field for further HBOC design and optimization. An ideal HBOC should effectively bind and release O₂, have a scalable synthesis to meet material demand, and limit batch-to-batch variability to yield consistent, uniform material. This work demonstrates the successful synthesis of human Hb nanoparticles using a desolvation technique (hHb-dNPs) as a new HBOC candidate. hHb-dNPs demonstrated a monodisperse size distribution between 130 and 150 nm with spherical morphology, a negative surface charge, and hemocompatibility. hHb-dNPs exhibited high O₂ affinity with decreased cooperativity similar to that of relaxed-state polymerized Hb. The methodology demonstrated scalability from 5 to 500 mL starting volume, producing products with similar biophysical properties regardless of the scale. hHb-dNPs demonstrated structural and biophysical stability for a month at 4°C and -80°C with various cryoprotectants, with the optimal combination being hHb-dNPs co-stored at -80°C with HSA. Considering these results, hHb-dNPs are a promising potential next generation HBOC.