A core-shell protein cluster comprising hemoglobin and human serum albumins, hemoglobin-albumin cluster (Hb-HSA 3), was designed and synthesized for use as an artificial O 2 carrier and red blood cell (RBC) substitute. For initial preclinical safety evaluation of the Hb-HSA 3 solution, we observed blood compatibility in vitro, physiological responses after exchange transfusion into rats and blood circulation lifetime in dogs. Dilution of human whole blood with Hb-HSA 3 showed an appropriate decrease in blood cell number, proportional to the mixing volume ratio. Time courses in the circulation parameters and blood gas parameters after 20% exchange transfusion with Hb-HSA 3 in anesthetized rats were almost identical to those observed in a sham group (without infusion) and an HSA group (with HSA administration) for 6 h. Serum biochemical tests of the withdrawn blood indicated safety of the protein cluster. Furthermore, fluorescent Hb-HSA 3 was infused into beagle dogs to assess blood retention. Fluorescence measurements of the blood samples enabled us to ascertain the cluster half-life within the intravascular space. Histopathologic inspections of the vital organs imply no abnormality in tissues. All these results indicate sufficient initial preclinical safety of Hb-HSA 3 as an alternative material for use in RBC transfusion.

There is no blood bank for pet animals. Consequently, veterinarians themselves must obtain “blood” for transfusion therapy. Among the blood components, serum albumin and red blood cells (RBCs) are particularly important to save lives. This paper reports the synthesis, structure, and properties of artificial blood for the exclusive use of dogs. First, recombinant canine serum albumin (rCSA) was produced using genetic engineering with Pichia yeast. The proteins showed identical features to those of the native CSA derived from canine plasma. Furthermore, we ascertained the crystal structure of rCSA at 3.2 Å resolution. Pure rCSA can be used widely for numerous clinical and pharmaceutical applications. Second, hemoglobin wrapped covalently with rCSA, hemoglobin–albumin cluster (Hb-rCSA3), was synthesized as an artificial O2-carrier for the RBC substitute. This cluster possesses satisfactorily negative surface net charge (pI = 4.7), which supports enfolding of the Hb core by rCSA shells. The anti-CSA antibody recognized the rCSA exterior quantitatively. The O2-binding affinity was high (P50 = 9 Torr) compared to that of the native Hb. The Hb-rCSA3 cluster is anticipated for use as an alternative material for RBC transfusion, and as an O2 therapeutic reagent that can be exploited in various veterinary medicine situations.

A hemoglobin wrapped covalently by three human serum albumins, a Hb-HSA3 cluster, is an artificial O2-carrier with the potential to function as a red blood cell substitute. This paper describes the synthesis and O2-binding properties of new hemoglobin‒albumin clusters (i) bearing four HSA units at the periphery (Hb-HSA4, large-size variant) and (ii) containing an intramolecularly crosslinked Hb in the center (XLHb-HSA3, high O2-affinity variant). Dynamic light scattering measurements revealed that the Hb-HSA4 diameter is greater than that of either Hb-HSA3 or XLHb-HSA3. The XLHb-HSA3 showed moderately high O2-affinity compared to the others because of the chemical linkage between the Cys-93(β) residues in Hb. Furthermore, the blood circulation behavior of 125I-labeled clusters was investigated by assay of blood retention and tissue distribution after intravenous administration into anesthetized rats. The XLHb-HSA3 was metabolized faster than Hb-HSA3 and Hb-HSA4. Results suggest that the molecular structure of the protein cluster is a factor that can influence in vivo circulation behavior.

Dual-protein clusters comprising haemoglobin and recombinant feline serum albumins as a red blood cell substitute for exclusive use in cats.

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