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

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 multilamellar 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 hemosomes.

Oxygen Carriers (“Blood Substitutes”)Raison d'Etre, Chemistry, and Some PhysiologyBlut ist ein ganz besondrer Saft

“…Its purpose is to reduce membrane permeability, improve liposome resistance to fusion and lysis, hinder Hb oxidation and denaturation, and reduce lipid peroxidation. The rigid cholesterol molecule inhibits both hydrophobic- and ionic-type interactions of phospholipid membranes with proteins, probably through changes in membrane fluidity, in line with cholesterol's condensing/stabilizing effect in unsaturated bilayers. , It reduces intercalation of Hb into the lipidic bilayer and displacement of heme . The presence of cholesterol in the formulation also prevents redistribution of cholesterol from RBC membranes to the liposome membrane, a process which renders RBCs osmotically fragile …”

“…934,973 It reduces intercalation of Hb into the lipidic bilayer and displacement of heme. 974 The presence of cholesterol in the formulation also prevents redistribution of cholesterol from RBC membranes to the liposome membrane, a process which renders RBCs osmotically fragile. 975 Small amounts of negatively charged phospholipids, such as PA, PE, dicetyl phosphate, dimyristoylphosphatidylglycerol, phosphatidylserine, phosphatidyl inositol, and PEG-PE 4.106d, have often been added to increase surface charge and inhibit liposome aggregation and fusion during storage.…”

Improved formulation of liposome-encapsulated hemoglobin with an anionic non-phospholipid

“…Such interactions result in a rapid uptake of LEH by the RES, and toxic effects manifested as vasoconstriction, pulmonary hypertension, dyspnea, etc. Anionic phospholipids also enhance the rate of hemoglobin oxidation and displace heme relative to globin [30]. It is possible to partially reduce the toxicity of anionic phospholipids by PEG modification of LEH surface [18].…”

Histopathological changes of rat brain after direct injection of Hb‐vesicles (artificial oxygen carriers) and neurological impact in an intracerebral hemorrhage model

“…The in vitro demonstration of HbV resistance to PLA 2 enzymatic lipolysis might support the notion that the HbV is also more resistant to PLA 2 in the brain and shows a lower rate of degradation than RBC. Moreover, it has been reported that DPPC, which comprises saturated acyl chains, is less subject to lipid peroxidation than the unsaturated phospholipids in biomembranes 43, 44…”