Encapsulation of Hemoglobin in Non-Phospholipid Vesicles

The efficiency of encapsulating hemoglobin in non-phospholipid liposomes by rapidly mixing hemoglobin with lipids heated above their solidliquid phase transition temperature was examined. Human hemoglobin was mixed at 55-60°C with a lipid solution containing polyoxyethylene-2 cetyl ether and cholesterol (molar ratio, 3:1) at 60-65°C. Repeated mixing was carried out through a high-shear orifice, followed by rapid cooling and additional mixing. Lipid vesicles were heterogeneous in size, with diameters from ∼300 nm to 10 μm. The non-encapsulated aqueous phase was removed by centrifugation, and total hemoglobin was determined spectrophotometrically. Encapsulation efficiency was calculated as the percentage of hemoglobin associated with the liposome phase (i.e., encapsulated) as a function of hemoglobin concentration and the aqueous:lipid hydration ratio. Hemoglobin concentrations were varied from 1 to 10 mM (in heme). Aqueous:lipid ratios of 8:1 and 4:1 were tested. Percent encapsulation varied from 13-30%, with the greatest efficiency, i.e., 30%, at a 4:1 hydration ratio of hemoglobin:lipid at 5.6 mM hemoglobin.