Physical properties of hemoglobin vesicles as red cell substitutes

Hemoglobin vesicles (HbV) as red cell substitutes were prepared from a purified carbonylhemoglobin (HbCO) solution and a lipid mixture composed of phospholipids, cholesterol, and α-tocopherol. The diameter was controlled to 251 ± 87 nm using an extrusion method; the vesicles penetrated through the membrane filters with regulated pore sizes. After the ligand exchanging reaction (HbCO→HbO₂), the oxygen affinity (P₅₀) of HbV was 32 Torr, which was controlled with the coencapsulation of pyridoxal 5′-phosphate. The rate of metHb formation in HbV was nonenzymatically reduced with the coencapsulation of DL-homocysteine. The Hb concentration of the HbV suspension, which was dispersed in a phosphate buffered saline solution (pH 7.4), was controlled at 10 g/dL. At this concentration, the total lipid concentration was 6.2 g/dL and the viscosity, 2.6 cP (230 s^-1), was lower than that of the blood (4.4 cP). The HbV suspension showed a typical non-Newtonian flow for a particle dispersion and agreed well with the Casson model. The viscosity at shear rates lower than 23 s^-1 showed a maximum with increasing the mixing ratio of human blood, plasma, or albumin, while no maximum was observed for the mixture with washed red blood cells. The aggregates of HbV are formed by interaction with plasma proteins, including albumin, while the aggregates reversibly dissociate at higher shear rate.