Poly(ethylene glycol)-conjugation and deoxygenation enable long-term preservation of hemoglobin-vesicles as oxygen carriers in a liquid state

The stability of hemoglobin vesicles (HbV) as an oxygen infusion was tested during the storage for 1 year at 4, 23, and 40 °C. The surface of the HbV was modified with poly(ethylene glycol) (PEG), and the suspension was deoxygenated with nitrogen bubbling. The samples stored at 4 and 23 °C showed a stable dispersion state for 1 year, though the sample stored at 40 °C showed the precipitation and decomposition of vesicular components, a decrease in pH, and 4% leakage of total Hb after 1 year. The PEG chains on the vesicular surface stabilize the dispersion state and prevent the aggregation and fusion due to their steric hindrance. The original metHb content (ca. 3%) before the preservation gradually decreased to less than 1% in all the samples after 1 month due to the presence of homocysteine inside the vesicles which consumed the residual oxygen and gradually reduced the trace amount of metHb. The rate of metHb formation was strongly dependent on the partial pressure of oxygen, and no increase in metHb formation was observed due to the intrinsic stability of the deoxygenated Hb. Preservation at 4 and 23 °C slightly reduced P₅₀ (increased the oxygen affinity) from 38 Torr to 32 and 31 Torr, respectively. These results indicate the possibility that HbV suspension can be stored at room temperature for at least 1 year.