Hemoglobin vesicles, which encapsulated concentrated hemoglobin (Hb) with a bilayer of dipalmitoylphosphatidylcholine/cholesterol/palmitic acid, were prepared under various preparation conditions in order to decrease the number of bilayers (n) constructing the vesicle and increase the Hb concentration in the interior of the vesicle ([Hb]in). n decreased when the surface potential of the bilayer became more negative because of the electrostatic repulsion between the bilayers, while with a changing ζ-potential of Hb from positive to negative, [Hb]in showed a precipitous fall because of the electrostatic repulsion between Hb and the surface of the bilayer. A temperature decrease leads to a quality increase in the Hb vesicles ([Hb]/[lipid]) in spite of the [Hb]in decrease by the viscosity increase of the Hb solution. This is explained by the effective reduction of n due to the reduction in membrane fluidity and the protonation of Hb.

The intervesicular aggregation of phospholipid vesicles is induced by the addition of water-soluble polymers such as poly(ethylene glycol), dextran, etc. due to the interaction between the vesicular surface and the water-soluble polymers. The interaction can be expressed by the critical molecular weight (M c) of the water-soluble polymers for the aggregation of vesicles. The surface modification of vesicles with glycolipids (O ,O 5 -bis(octadecyl) N-maltooligonoyl-l-glutamate) accelerates the aggregation of vesicles induced by dextran; therefore, M c significantly decreased due to the surface modification. No dependence of phospholipid concentration and dextran concentration in an aqueous phase on the M c indicates that dextran does not act as a cross-linking agent among the vesicles. A clear dependence of the density of the saccharide chains on the vesicular surface on the M c suggests that dextran should adsorb on the surface of the vesicles by the interaction with the oligosaccharide chains on the surface and cause vesicular aggregation. A lower critical solution temperature was observed for this kind of interaction, and the critical temperature was controlled by changing the molecular weight of dextran.

The hemoglobin vesicle (HbV) has a cellular structure which encapsulates concentrated Hb in the inner aqueous phase of a phospholipid bilayer vesicle. Hb is gradually autoxidized to methemoglobin (metHb), which can not bind oxygen during oxygen transport under physiological conditions. In order to reduce metHb in HbV, we evaluated the reduction of metHb by electron transfer across the bilayer membrane of HbV from a reductant added to the outer aqueous phase. Water-soluble methylene blue (MB) and hydrophobic ubiquinone 10 (UQ) were selected as electron mediators. Under a nitrogen atmosphere, the addition of the reduced form nicotinamide-adenine dinucleotide (NADH) to the outer aqueous phase of UQ-incorporated HbV showed only a slow reduction rate for metHb. On the other hand, when MB and NADH were added under a nitrogen atmosphere to HbV containing 40% metHb, a rapid decrease in the metHb percentage was observed. The entire reaction was controlled by a reaction with NADH and MB in the outer aqueous phase. Under aerobic conditions, the decrease in the efficiency of the metHb reduction and rapid oxidation after reaching the minimal metHb percentage were observed. This was confirmed to be due to the influence of hydrogen peroxide; the decrease was prevented by the co-encapsulation of catalase.

Bis(2,-sre-glycero-3-phosphocholine (DODPC), constructing the bilayer membrane of a vesicle, was polymerized by UV irradiation. The freeze-dried vesicles were completely dissolved in chloroform to obtain cross-linked phospholipid polymer (polyDODPC). The polyDODPC consisted of high molecular weight components and oligomers. The polyDODPC was dispersed by sonication, and two size distributions of the reconstituted vesicles were confirmed, larger vesicles from high molecular weight polyDODPC and smaller vesicles from oligomers. When the polyDODPC vesicles of various sizes and unimodal size distribution were reconstituted by an extrusion method, the vesicles of the same size as the vesicles at polymerization showed excellent stability compared with the other sizes. Interestingly, the original size of the polyDODPC vesicles could be completely restored by a cholate dialysis method. It was also clarified that vesicles larger than the original size were reconstituted from the higher molecular weight component, and the smaller vesicles were from the oligomers. Furthermore, a linear phospholipid polymer obtained from l-palmitoyl-2-(2,4-octadecadienoyl)-sn-glycero-3-phosphocholine could not restore the original size. Those results indicate that the original size of the vesicles can be restored completely by all polyDODPCs having the original composition of large and small molecular weights and cross-linked structure.

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