Preparation of haemoglobin vesicles
HbVs were prepared as previously described16,17,23. Briefly, Hb was purified from outdated donor human blood provided by the Japanese Red Cross Society. The encapsulated carbonylhemoglobin (HbCO, 38 g/dL) contained 5.9 mM pyridoxal 5’-phosphate (Sigma Chemical, Saint Louis, USA) as an allosteric effector for regulating oxygen affinity. The lipid bilayer was a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine; cholesterol; and 1,5-bis-O-hexadecyl-N-succinyl-L-glutamate at a molar ratio of 5:4:0.9 and 1,2-distearoyl-sn-glycero-3-phosphatidyl-ethanolamine-N-PEG (0.3 mol%). HbV particles were suspended in a physiological salt solution. Nitrogen gas was bubbled through the solution for storage. The properties of HbV used in this experiment are shown in Table S1. HbV was sufficiently retained in the blood (half-life: 47-72 hours)24. Before the experiments, the HbV solution was mixed with a 25% human serum albumin (Benesis, Osaka, Japan) to adjust the albumin concentration of the vesicle-suspension medium to 5 g/dL. Under these conditions, the colloid osmotic pressure of the suspension was maintained stable at approximately 20 mmHg24. HbVs were stored in deoxygenated glass vials at 4°C for at least 10-12 months and used for all experiments as the HbV test solution.