Encapsulation of Hb into unsaturated lipid vesicles and γ‐ray polymerization

A carbonyl hemoglobin (HbCO) solution was stirred with a mixed powder of polymerizable 1,2‐bis(2,4‐octadecadienoyl)‐sn‐glycero‐3‐phosphocholine (DODPC), cholesterol and stearic acid (7/7/2 by mol). The mixture was extruded through polycarbonate membrane filters (final pore size = 0.2 μm Ø). The average diameter of the resulting vesicles was 203 ± 39 nm. The [Hb]/[Lipid] ratio (the weight ratio of Hb in vesicle to lipid) increased with the Hb concentration, and decreased with the NaCl concentration. A maximum [Hb]/[lipid] ratio was observed at pH 6.9, which was the same as the isoelectric point of Hb. The vesicles were stabilized by γ‐irradiation (⁶⁰Co) because the bilayer lipids bound each other to yield polyphospholipids. Denaturation of Hb by γ‐irradiation was not detected. These polyphospholipid vesicles encapsulating Hb were stable even against the freeze–thaw cycles and the freeze‐drying procedure.