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Hemoglobin Vesicle
Hemoglobin-vesicles (HbV) are the cellular particles which encapsulate a purified and concentrated Hb solution within a phospholipid bilayer membrane. The process of preparing the HbV particles has required vast fundamental knowledge of macromolecular and supramolecular science. Significant efforts have been made to improve the production process of HbV under the completely sterile conditions and to design a pilot plant for this product (Fig. 1).
Surface modification of the HbV particles with polyethyleneglycol (PEG) inhibits intervesicular aggregation and fusion, thus maintaining the homogeneous dispersion state. This is an important technique for the preservation of HbV at room temperature over two years. Physicochemical properties such as viscosity, colloid osmotic pressure, oxygen affinity, and circulation time can be easily adjusted with appropriate amounts of additives in the inner and outer aqueous phases of the particles. Preclinical studies of resuscitation from hemorrhagic shock, extreme hemodilution, extracorporeal circulation, liquid ventilation, etc., have revealed the sufficient oxygen transporting ability of HbV. (Div. General Thoracic Sur. Dept. Surg.,Keio Univ.)
A red blood cell (8 µm) has the ability to deform to a parachute-like structure to penetrate through a narrow capillary (4 µm). It is difficult to artificially mimic this deformability for the present, so that the particle diameter of an oxygen carrier should inevitably be smaller than the capillary diameter. However, the acellular Hb modifications such as intramolecular crosslinked or polymerized Hbs, which have been extensively studied in the US and EU, are so small (less than 50 nm) that they tend to extravasate faster and show a shorter circulation time. It should be emphasized that the acellular Hbs induce a significant side effect, hypertension, as a result of vasoconstriction which has been suggested to be due to the binding of NO and/or CO as endogenous vasorelaxation factors. On the other hand, our HbV has a larger particle diameter of about 250 nm, so that it does not extravasate easily and remains in the blood circulation for a longer time. Actually, no hypertension nor vasoconstriction is confirmed in the rat liver perfusion model (Dept. Biochem., Keio Univ.) and the hamster dorsal skinfold window model (Dept. Bioeng., UC San Diego) (Fig. 2).
The reticuloendothelial system (RES, or mononuclear phagocytic system) plays an important role in host defense functions such as protective elimination of foreign materials such as viruses, bacteria, and bacterial endotoxins, and metabolic decomposition composites in the blood. It has been confirmed by an autoradiogram that the HbV particles, after finishing O2 transport, are finally captured by phagocytes in the RES mainly in the liver, spleen and bone marrow, and the particles are metabolized promptly (Univ. Texas, San Antonio). Even though the phagocytic activity shows transient changes, a histopathological examination with anti-human Hb antibody staining and transmission electron microscopy clearly demonstrated the complete disappearance of the HbV particles within 7 days and the deposited metabolites such as iron and lipids within 14 days (Fig. 3, Keio Univ. Dept. Phathol.)
