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.