Encapsulation of hemoglobin in non-phospholipid vesicles.

ABSTRACT

The efficiency of encapsulating hemoglobin in non-phospholipid liposomes by rapidly mixing hemoglobin with lipids heated above their solid-liquid phase transition temperature was examined. Human hemoglobin was mixed at 55-60 degrees C with a lipid solution containing polyoxyethylene-2 cetyl ether and cholesterol (molar ratio, 31) at 60-65 degrees 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 approximately 300 nm to 10 microns. 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 aqueouslipid hydration ratio. Hemoglobin concentrations were varied from 1 to 10 nM (in heme). Aqueouslipid ratios of 81 and 41 were tested. Percent encapsulation varied from 13-30%, with the greatest efficiency, i.e., 30%, at a 41 hydration ratio of hemoglobinlipid at 5.6 mM hemoglobin.