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-HSA3. Human Hb A (HbA) can be also used for a core protein to synthesize HbA-HSA3 cluster. The isoelectric point of HbBv-HSA3 (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-HSA3 has good compatibility with whole blood. Intravenous administration of HbBv-HSA3 into anesthetized rats elicited no unfavorable increase in systemic blood pressure by vasoconstriction. The half-life of 125 I-labeled cluster in circulating blood is longer than that of HSA. All results indicate that HbBv-HSA3 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.