ZIF-8 Metal–Organic Framework Nanoparticles Loaded with Hemoglobin as a Potential Red Blood Cell Substitute

Hemoglobin (Hb)-based oxygen carriers (HBOCs) are being developed as artificial red blood cell (RBC) substitutes for use in transfusion medicine. Unfortunately, prior generations of HBOCs were not able to successfully minimize key side effects, including vasoconstriction, systemic hypertension, and oxidative tissue injury, which is primarily due to the extravasation of cell-free Hb from the vascular space into the tissue space. Therefore, to potentially reduce these side effects, we successfully encapsulated Hb within a zeolitic imidazolate framework (ZIF-8) to form ZIF-8-Hb nanoparticles (ZIF-8P-Hb). Both ZIF-8 and ZIF-8P-Hb nanoparticles were synthesized at a relatively high molar ratio of 2-methylimidazole:zinc, which resulted in a monodisperse nanoparticle size distribution. In addition, the flow conditions for tangential flow filtration-facilitated purification of the nanoparticles did not exert a strong effect on the nanoparticle size distribution. ZIF-8P-Hb nanoparticles exhibited high stability, ultrahigh Hb encapsulation efficiency, and a monodisperse size distribution. Additionally, ZIF-8P-Hb nanoparticles exhibited a ζ-potential of −11.2 ± 0.9 mV, demonstrating its potentially enhanced biocompatibility in comparison to bare ZIF-8 nanoparticles (40.7 ± 2.0 mV). More significantly, ZIF-8P-Hb nanoparticles exhibited significantly enhanced hydrothermal stability with negligible release of cell-free Hb. Furthermore, ZIF-8P-Hb displayed a significantly lower haptoglobin binding rate constant compared to cell-free Hb, indicating its potentially slower in vivo clearance in comparison to cell-free Hb. Moreover, we observed a relatively low level of hemolysis when ZIF-8P-Hb nanoparticles were incubated with RBCs (<5%), which demonstrates a suitable safety profile. To further optimize the ZIF-8P-Hb nanoparticle synthesis protocol, various procedural parameters were systematically investigated to evaluate their impact on the size distribution of ZIF-8 and ZIF-8P-Hb nanoparticles. Taken together, this work provides a comprehensive approach for synthesizing a monodisperse HBOC as a potential artificial RBC substitute.