Sonication Effectively Reduces Nanoparticle Size in Hemoglobin-Based Oxygen Carriers (HBOCs) Produced Through Coprecipitation: Implications for Red Blood Cell Substitutes

Engineered hemoglobin (Hb) particles are O2 delivery vehicles that may be used as red blood cell (RBC) substitutes when blood is unavailable. One promising method produces high-purity Hb particles through coprecipitation with a removable inorganic template that yields porous Hb particles; however, this method traditionally results in particle diameters too large for applications in transfusion medicine. The current study investigates the incorporation of a sonication operation during Hb particle synthesis to eliminate aggregates and decrease mean particle diameter. We report an improved synthesis method for Hb nanoparticles, having optimized reagent ratios for increased Hb entrapment. The highest entrapment was observed at an equimolar ratio of 0.65 M inorganic precursors and a Hb concentration of 7.5 mg/mL. Sonication drastically reduced Hb particle diameter from a range of several micrometers to a mean diameter of ∼250 nm. The benefits of increased sonication duration and intensity rapidly plateaued, being able to achieve an order of magnitude reduction in particle size after 5 min of moderate sonication. We evaluated the Hb nanoparticles for in vitro O2 transport to validate their potential in transfusion medicine applications, revealing an O2 offloading rate constant of 4.2 s–1, and O2 affinity (i.e., P 50) of 8.66 mmHg, which was comparable to previously reported Hb-based O2 carriers (HBOCs). Taken together, these data describe a synthesis method capable of reliably producing Hb nanoparticles of reduced size, without the presence of large particles or aggregates while maintaining O2 transport characteristics.