Hemoglobin–Albumin Clusters as a Red Blood Cell Substitute

Abstract

Core–shell protein cluster comprising bovine hemoglobin (HbBv) in the core and human serum albumin (HSA) at the shell was created as an artificial O₂ carrier designed for use as a red blood cell (RBC) substitute. The protein cluster was prepared by covalent linkage between the Cys-34 residue of HSA and the surface Lys amino groups of HbBv using heterobifunctional cross-linker. The average HSA/HbBv ratio of one cluster was determined as 3.0 ± 0.2; therefore we indicated this hemoglobin–albumin cluster as HbBv–HSA ₃ . Human Hb A (HbA) can be also used for a core protein to synthesize HbA–HSA ₃ cluster. The isoelectric point of HbBv–HSA ₃ (pI = 5.1) was markedly lower than that of HbBv and almost identical to the value of HSA. SFM and TEM measurements revealed a triangular shape of HbBv–HSA₃. The complete 3D structure based on TEM data was reconstructed. The clusters showed moderately higher O₂ affinities than the native HbBv and HbA. Viscosity and blood cell counting measurements demonstrated that HbBv–HSA ₃ has good compatibility with whole blood. Intravenous administration of HbBv–HSA ₃ into anesthetized rats elicited no unfavorable increase in systemic blood pressure by vasoconstriction. The half-life of ¹²⁵I-labeled cluster in circulating blood is longer than that of HSA. All results indicate that HbBv–HSA ₃ has sufficient preclinical safety as an alternative material for RBC transfusion. Interestingly, clusters prepared under N₂ atmosphere showed low O₂ affinity resembling human RBC. Furthermore, the exterior HSA units possess a remarkable ability to bind antioxidant agent, such as Pt nanoparticle (PtNP). The peripheral HSA–PtNP shell prevents oxidation of the core HbBv, which enables the formation of an extremely stable O₂ complex even in H₂O₂ solution. This chapter reviews the synthesis, structure, O₂-binding property, and preclinical safety of hemoglobin–albumin cluster as a promising RBC substitute for practical use.