Dual-function hemoglobin-encapsulating ZIF-8 nanoparticles: Oxygen transport capability and carbonic anhydrase-like activity.

Hemoglobin-based oxygen carriers (HBOCs) have emerged as a promising alternative to red blood cell (RBC) transfusions, addressing key limitations such as cold storage requirements, restricted availability of universal donor blood, and the need for cross-matching, which collectively hinder their immediate use in emergency settings. Despite their potential, existing HBOCs primarily focus on oxygen delivery while overlooking a crucial physiological function of native RBCs: carbon dioxide (CO2) transport, which is essential not only for gas exchange but also for acid-base homeostasis. In this study, we explore the potential of our previously developed hemoglobin (Hb)-loaded zeolitic imidazole framework-8 nanoparticles (Hb@ZIF-8 NPs) as a dual-function RBC substitute capable of both oxygen delivery and CO2 transport. ZIF-8, a metal-organic framework (MOF), was selected due to its high porosity, biocompatibility, and structural resemblance to the catalytic center of carbonic anhydrase (CA), the native enzyme responsible for catalyzing the reversible hydration of CO2 within RBCs. Our results show that, similarly to CA, Hb@ZIF-8 NPs synthesized with polyethylene glycol (PEG) as a seeding agent and varying Hb concentrations can catalyze the hydrolysis of p-nitrophenyl acetate (p-NPA) into p-nitrophenol (p-NP), which can be detected spectrophotometrically. Notably, enzyme kinetics analysis reveals that Hb@ZIF-8 NPs follow Michaelis-Menten kinetics, with coefficients of determination (R2) exceeding 0.99 in most cases, indicating strong adherence to enzyme behavior. The maximum reaction velocity (Vmax) decreased with PEG and increasing Hb content, likely due to reduced availability of the MOF's metal ion, which acts as the active catalytic sites. However, the Michaelis-Menten constant (Km) suggests that the presence of PEG and Hb enhances substrate affinity, possibly through interactions with p-NPA. The catalytic efficiency (Keff) was higher in Hb@ZIF-8 NPs with greater Hb content, suggesting improved enzymatic activity alongside oxygen transport. These findings establish Hb@ZIF-8 NPs as HBOCs also capable of mimicking CA esterase activity, reinforcing their dual role in oxygen and CO2 transport. This integration of oxygen-carrying capacity with CA-like functionality represents a significant advancement in the development of artificial RBCs, with potential clinical implications for improving systemic oxygenation and preventing acidosis in transfusion-limited or emergency settings.