Modulation of O ₂ Affinity and Enzymatic Activity of Core‒Shell Structured Hemoglobin Nanoparticles

Hemoglobin‐based O 2 carriers (HBOCs) are promising substitutes for red blood cells (RBCs), but their performance depends critically on optimized O 2 affinity and stability of oxygenated hemoglobin (oxy Hb). In this study, we report the precise modulation of both O 2 affinity and enzymatic (antioxidant) activity in core–shell structured stroma‐free Hb nanoparticles (X–SFHbNPs, X = human serum albumin (HSA) or poly(2‐ethyl‐2‐oxazoline) (POx)). Low‐affinity nanoparticles (X–SFHbNP L s) were prepared from deoxy Hb (T‐state), and circular dichroism (CD) analysis indicated that the core Hb retains a T‐state‐like structure even under oxygenated conditions. Blending high‐ and low‐affinity nanoparticles enabled fine tuning of O 2 affinity. POx–SFHbNP preserved the intrinsic catalase (Cat), superoxide dismutase (SOD), and carbonic anhydrase (CA) activities of the parent SFHb. Moreover, POx–Hb(Cat/SOD/CA)NPs, prepared by incorporating elevated levels of these enzymes into purified Hb, exhibited enhanced enzymatic activities. Both X–SFHbNPs and X–Hb(Cat/SOD/CA)NPs showed excellent resistance to oxidation, with negligible metHb formation after 2 h in aqueous H 2 O 2 . These multifunctional Hb nanoparticles provide a versatile platform for advanced HBOCs with tunable O 2 transport and antioxidant capabilities. Furthermore, X‒SFHbNPs showed reduced binding affinities toward haptoglobin, a Hb‐binding plasma protein.