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Hemoglobin vesicles (Hb-V) are artificial red blood cells encapsulating highly concentrated hemoglobin (Hb) in liposomes comprising phospholipids, cholesterol, negatively charged lipids, and polyethylene glycol (PEG)-conjugated phospholipids. Safety and efficacy of Hb-V as a transfusion alternative have been extensively studied. For this study, we prepared Hb-V using the kneading method with a rotation-revolution mixer as an alternative to the conventional extrusion method. We optimized the kneading operation parameters to obtain Hb-V with a high yield. Results show that the Hb encapsulation efficiency was increased dramatically up to 74.2%, which is higher than that of the extrusion method (20%) because the kneading method enabled mixing of a highly concentrated carbonylhemoglobin (HbCO) solution (40 g/dL) and a considerably large amount of powdered lipids in only 10 min. The high viscosity of the Hb-lipid mixture paste (ca. 10 3 -10 5 cP) favorably induces frictional heat by kneading and increases the paste temperature (ca. 60 °C), which facilitates lipid dispersion and liposome formation. During the kneading operation using a thermostable HbCO solution, Hb denaturation was prevented. Hb-V prepared using this method showed no marked changes in particle sizes, Hb denaturation, or Hb leakage from liposomes during two years of long-term storage-stability tests. Collectively, these results demonstrate that the kneading method using a rotation-revolution mixer shows good potential as a new method to produce Hb-V.

Preparation of Artificial Red Blood Cells (Hemoglobin Vesicles) Using the Rotation–Revolution Mixer for High Encapsulation Efficiency