Liposome encapsulated hemoglobin: long-term storage stability and in vivo characterization

Liposome Encapsulated Hemoglobin (LEH) has been the focus of research and development at the Naval Research Laboratory in an effort to find a viable oxygen-carrying resuscitative fluid. Previous reports from our laboratory have shown that LEH binds and releases oxygen in a manner similar to red blood cells, and that it can sustain life when red cell hematocrits are decreased to critical levels. We have also reported on LEH with regards to preparative methods, scale-up feasibility, toxicity, hemodynamics, hemoglobin P50 modification by coencapsulation of organic phosphates, liposomal surface modification, and storage strategies. In this report, the issue of LEH efficacy following long-term storage in the dry state will be addressed. We have shown that hemoglobin, liposomes, and LEH may be successfully lyophilized and rehydrated to viable states. The modification of the LEH formulation by addition of the carbohydrate trehalose results in the successful lyophilization and storage of LEH. In vitro characterization of LEH stored in the dry state for up to six months includes measurement of oxygen-carrying capacity, liposome size retention, methemoglobin production, and the intraliposomal hemoglobin concentration. The in vivo studies report on physiological parameters such as circulation persistence, blood chemistry, and pathological examination in mice.