Haemoglobin-vesicles as artificial oxygen carriers: present situation and future visions.

During the long history of development of haemoglobin (Hb)-based O2 carriers (HBOCs), many side effects of Hb molecules have become apparent. They imply the physiological importance of the cellular structure of red blood cells. Hb-vesicles (HbV) are artificial O2 carriers that encapsulate concentrated Hb solution with a thin lipid membrane. We have overcome the intrinsic issues of the suspension of HbV as a molecular assembly, such as stability for storage and in blood circulation, blood compatibility and prompt degradation in the reticuloendothelial system. Animal tests clarified the efficacy of HbV as a transfusion alternative and the possibility for other clinical applications. The results of ongoing HbV research make us confident in advancing further development of HbV, with the expectation of its eventual realization.

Distribution of heme oxygenase isoforms in rat liver. Topographic basis for carbon monoxide-mediated microvascular relaxation.

Carbon monoxide (CO) derived from heme oxygenase has recently been shown to play a role in controlling hepatobiliary function, but intrahepatic distribution of the enzyme is unknown. We examined distribution of two kinds of the heme oxygenase isoforms (HO-1 and HO-2) in rat liver immunohistochemically using monoclonal antibodies. The results showed that distribution of the two isoforms had distinct topographic patterns: HO-1, an inducible isoform, was observed only in Kupffer cells, while HO-2, a constitutive form, distributed to parenchymal cells, but not to Kupffer cells. Both isoforms were undetectable in hepatic stellate cells and sinusoidal endothelial cells. Of the two isoforms, HO-2 in the parenchymal cell rather than HO-1 in the Kupffer cell, appears to play a major role in regulation of microvascular tone. In the perfused liver, administration of HbO2, a CO-trapping reagent that can diffuse across the fenestrated endothelium into the space of Disse, elicited a marked sinusoidal constriction, while administration of a liposome-encapsulated Hb that cannot enter the space had no effect on the microvascular tone. These results suggest that CO evolved by HO-2 in the parenchymal cells, and, released to the extrasinusoidal space, served as the physiological relaxant for hepatic sinusoids.

Dissociation of aggregated ferroheme complexes and protoporphyrin IX by water-soluble polymers.

The ferroheme-pyridine complex, ferroheme and protoporphyrin IX form the aggregates by the hydrophobic interaction in aqueous solutions. We found by spectrophotometric and fluorometric measurements that the aggregates dissociated into the monomers by the addition of water-soluble polymers, such as, poly(ethyleneoxide), poly(vinylalcohol), poly(vinylpyrrolidone) and poly(styrene sulfonate). The dissociation by the polymers proceeded as their hydrophobicities increased. The aggregated ferroheme was effectively dissociated by the copolymers of 4-vinylpyridine which were water-soluble polymer-ligands.

1H-NMR study of the effect of synthetic polymers on the fluidity, transition temperature and fusion of dipalmitoyl phosphatidylcholine small vesicles.

The interaction of water-soluble polymers with dipalmitoyl phosphatidylcholine small vesicles and the effect on vesicle fusion were studied by means of 1H-NMR spectrometry. The motion of dipalmitoyl phosphatidylcholine molecules decreased on interaction with the polymers and was detected as a change in the signal intensity. The interaction behavior of polymers is very sensitive to the chemical structure of the applied polymers. Poly(styrene sulfonic acid) and poly(ethylene glycol) decreased the motion of the choline methyl group, predominantly through coulombic and hydrophobic interaction forces, respectively. For example, in the case of the poly(styrene sulfonic acid)-containing system, the signal intensity of the choline methyl group was decreased about 15% while those of the hydrophobic methylene and terminal methyl groups were scarcely decreased by the addition of polymer to a final concentration of 4.0 x 10(-2) unit mol/l. These polymers are considered to interact with the surface of the vesicle membrane. On the other hand, poly(L-glutamic acid) and poly(N-vinyl-2-pyrrolidone) decreased the signal intensities of not only the choline methyl group, but also those of the hydrophobic methylene and terminal methyl groups. This result suggests that part of these polymers might be incorporated into the hydrophobic region of the vesicle membrane. Addition of the non-ionic polymers inhibited vesicle fusion considerably. This effect was explained by the stabilization of dipalmitoyl phosphatidylcholine vesicles by complexation with these polymers.

Cooperative reactions of poly-L-lysine-heme complex with molecular oxygen, carbon monoxide, or cyanide ion.

The interaction of the alpha-helical poly-L-lysine-heme complex with molecular oxygen, carbon monoxide, or cyanide ion was studied. Binding equilibrium curve and activation parameters for the reactions were determined. Sigmoid responses were observed for the absorption of molecular oxygen or carbon monoxide by the complex and the cooperative parameter was found to be 2.1. This indicated a cooperative interaction between hemes situated on a cylindrical alpha-helix of poly-L-lysine. But those of other polymer-ligand-heme complexes were 1.0. The cooperative reaction mechanism, in which an alpha-helical poly-L-lysine plays an important role, was suggested.

Complexes between synthetic polymer ligands and ferri-delta and ferro-protoporphyrin IX.

The complexes of synthetic polymer ligands, i.e. poly-L-lysine, poly-4-vinyl-pyridine, poly-N-vinyl-2-methylimidazole and the higher branched polyethyleneimine, with ferri- or ferro-protoporphyrin IX were studied from the standpoint of polymer ligand effects by comparison with those of their monomeric model ligand complexes and poly-gamma-benzyl-L-glutamate containing an imidazole nucleus at the chain end. The coordination numbers and formation constants were determined optically and their structures were also estimated. The coordination number of a poly-L-lysine complex was two, but those of other polymer ligand complexes were one. One of the polymer effects, which was indicated by the large formation constants of the polymer complexes, was caused by the increment of the local ligand concentration around the polymer chain. Another was caused by the conformational effect of an alpha-helical structure in the poly-L-lysine complexes. The interaction of a poly-L-lysine-heme complex with molecular oxygen was also studied. An observed pseudo-allosteric phenomenon may be due to the specific structure of a poly-L-lysine complex which is different from those of other polymer ligand complexes.

Liposomal heme as oxygen carrier under semi-physiological conditions. Orientation study of heme embedded in a phospholipid bilayer by an electrooptical method.

The meso-tetra(alpha,alpha,alpha,alpha(o-pivalamidophenyl]porphinato iron-mono(1-lauryl-2-methylimidazole) complex embedded in the bilayer of dimyristoylphosphatidylcholine (liposomal heme) binds molecular oxygen reversibly at pH 7 and 37 degrees C. Orientation of the iron porphyrin complex in the phospholipid bilayer was studied by electric birefringence and dichroism. It was observed that both the phospholipid bibilayer of liposome and the porphyrin plane are oriented nearly in parallel to the electric field. Therefore the angle between the porphyrin plane and the bilayer is considered to be practically small.

Lipid microsphere containing lipophilic heme: preparation and oxygen transportation under physiological conditions.

Lipophilic heme (1-laurylimidazole-ligated 5,10,15,20-tetrakis(alpha, alpha, alpha, alpha-o- pivalamidophenyl)porphinatoiron(II) complex) is solubilized in lipid (triglyceride) at high concentrations and emulsified with a phospholipid in physiological salt solution, giving a deeply red-colored suspension of lipid microspheres (approx. 250 nm in diameter). The heme forms an oxygen adduct in a similar manner as oxyhemoglobin and the lipid microspheres take up and release oxygen reversibly at 37 degrees C in the aqueous medium. The oxygen-transporting ability is comparable with that of the red blood cell. Intravenous injection of the heme/lipid microsphere solution to rabbits demonstrates that it transports oxygen even in vivo and that it is cleared from the blood stream with a half-life time of approx. 1 h.

Fluorescence polarization study on the increase of membrane fluidity of human erythrocyte ghosts induced by synthetic water-soluble polymers.

The effect of water-soluble polymers on the membrane fluidity of human erythrocyte ghosts was investigated and was compared with that of concanavalin A by means of the fluorescence polarization technique. 8-Anilino-1-naphthalene sulfonic acid sodium salt and 1,6-diphenyl-1,3,5-hexatriene were used as probe molecules. The membrane fluidity was increased by the addition of polycations with concentrations of less than 2 x 10(-3) wt% 60 min after mixing. The fluidity changes were affected by the chemical structure (hydrophobicity, charge density, etc.) of polycations. Thus, the membrane fluidity increased markedly with increasing charge density on the chain backbone of polycations. On the other hand, nonionic polymers such as poly(ethylene glycol) and poly(N-vinyl-2-pyrrolidone) changed the membrane fluidity in a biphasic manner. That is, the fluidity of human erythrocyte ghost was temporarily increased and then decrease. For example, 20 wt.% of poly(ethylene glycol) gave a maximum fluidity 15 min after mixing with erythrocyte ghosts. A similar fluidity change was observed by adding concanavalin A. Such fluidity changes were not observed when lipid bilayer vesicles were used instead of cell membranes. These results suggested that the increase of membrane fluidity resulted from the intramembraneous aggregation of membrane-bound proteins which was induced by the added polymers. Cell agglutination was also induced by the addition of a large amount of polymers. This agglutination was considered to be due to the intermembraneous aggregation of membrane-bound proteins.

Interactions of functionalized polymeric liposomes having a porphinato-iron complex with some biological cells and components in vitro.

The hemocompatibility of functionalized polymeric liposome particles (diameter: 20-32 nm), which have a synthetic porphinato-iron complex in their polymerized bilayers and can carry oxygen, was studied in vitro. The ultramicroparticles did not induce hemolysis, platelet aggregation and plasma coagulation directly and were stable against hydrolysis by phospholipases A2 and D.