Factors Influencing the in Vitro Stability of Artificial Red Blood Cells Based on Hemoglobin-Containing Liposomes

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Abstract
The effects of membrane phospholipid composition, surface charge and cholesterol content on the deteriorating interactions between hemoglobin (Hb) and phospholipid bilayers were studied. Hb was either encapsulated in multiamellar liposomes (hemosomes), or incubated with small unilamellar vesicles (SUV). Negatively charged phospholipids increased the rate of oxyHb decay in unsaturated lipid hemosomes. This effect was not linked to Hb-induced lipid peroxidation, since the latter process was inhibited in hemosomes with negative surface charge. Cholesterol decreased both the negative-charge elicited fall in oxyHb-level, and Hb-induced lipid peroxidation. In hemosomes prepared from synthetic, saturated phospholipids, negative surface charge (phosphatidic acid) elicited drastic denaturation (bleaching) of Hb, which effect was completely prevented by cholesterol. The experiments with SUV prepared from unsaturated lipids indicated intercalation of Hb into the bilayer due to hydrophobic interaction. This process was decreased by membrane cholesterol. Negative surface charge of the vesicles, through an electrostatic interaction with the positively charged heme, resulted in the displacement of heme relative to globin. This process was also decreased by cholesterol. With saturated, negatively charged SUV, the penetration of Hb into the bilayer was smaller, but the ionic interaction between the acidic lipids and the heme led to the detachment of the letter from globin. Cholesterol in such membrane increased the intercalation of Hb into the membrane, and at the same time completely prevented the loss of heme. The latter observations suggest that the fluid phase of the membrane favours the hydrophobic interaction with the protein, whereas the gel state promotes the partition of the heme into the bilayer. It is suggested that the effects of cholesterol are indirect, mediated by changes in membrane fluidity. By highlighting potentially harmful reactions between Hb and phospholipid bilayers, our findings may help the design of in-vitro stable hemosomcs.