Advancing multifunctional hemoglobin-based oxygen carriers with enhanced stealth and antioxidant properties

Oxygen delivery is fundamental to tissue survival and is almost exclusively mediated by hemoglobin (Hb), a protein within red blood cells. In clinical situations involving acute blood loss, trauma or limited access to donor blood, impaired oxygen transport can have serious consequences and be associated with high mortality. Early oxygen delivery in emergency situations can be highly beneficial, but access to donor blood is often limited outside hospital settings. Although blood transfusions remain lifesaving procedures, their use is restricted by short storage lifetime and additional safety-related processing requirements, highlighting the need for alternative oxygen delivery methods. Efforts to address this need began decades ago with the design and development of Hb-based oxygen carriers (HBOCs). Beyond their original role as blood substitutes, HBOCs have gained increasing interest as oxygen delivery systems for hypoxic and ischemic-related pathologies, including cancer, ischemic stroke and wound healing. However, several challenges must be addressed to maximize the potential of these oxygen delivery systems.

In this thesis, metal–organic frameworks (MOFs) were explored as carriers for the development of multifunctional HBOCs that combine efficient oxygen delivery with antioxidant activity and reduced immune recognition. In addition, preliminary scale-up efforts were conducted to address production requirements associated with future in vivo studies and translational development.

First, zeolitic imidazole framework-90 (ZIF-90) nanoparticles (NPs) were developed as carriers for Hb via in situ encapsulation under mild synthesis conditions. The resulting Hb-loaded ZIF-90 NPs (Hb@ZIF-90 NPs) exhibited moderate Hb loadings, high drug loading efficiency, near-neutral surface charge and altered oxygen-binding behavior. Specifically, encapsulated Hb exhibited increased oxygen affinity, associated with enhanced oxygen release under low oxygen partial pressures, which is advantageous for applications involving severe hypoxia, such as ischemic tissues.

To further enhance functionality, cerium oxide- (CeO₂) and platinum- (Pt) based nanozymes (NZs) were incorporated onto the surface of Hb@ZIF-90 NPs to provide strong reactive oxygen species (ROS)-scavenging activity. The NZs exhibited pronounced superoxide dismutase- (SOD) and catalase-like activity, resulting in multifunctional systems that combined effective antioxidant activity with largely preserved oxygen transport. Dual CeO₂/Pt-NZ functionalization further enhanced SOD-like activity without additional improvement in oxygen delivery, indicating that while NZ incorporation enabled effective ROS scavenging compared to uncoated systems, it did not necessarily prevent Hb oxidation under conditions of oxidative stress. At elevated ROS levels, higher CeO₂-NZ concentrations resulted in reduced oxygen release, likely due to increased Hb oxidation arising from redox interactions with the heme iron of Hb. These findings highlight the need for controlled NZ loading and spatial separation between NZs and Hb to maintain oxygen delivery while enabling efficient ROS scavenging activity.

To improve circulation time and reduce immune recognition, previously optimized Hb-loaded ZIF-8 NPs (Hb@ZIF-8 NPs) were functionalized with a covalently bound human serum albumin (HSA) layer, yielding the HSA-coated Hb@ZIF-8 NPs (Hb@ZIF-8/HSA NPs). This surface modification, followed by crosslinking with glutaraldehyde, enhanced formulation stability in physiologically relevant media and reduced opsonin adsorption. Compared to conventional polymer coatings, HSA functionalization provided superior stealth properties while preserving cooperative oxygen-binding and release behavior, highlighting its potential to improve HBOC efficacy in vivo.

Finally, this thesis addresses the challenges associated with scalable production of MOF-based HBOCs. A laboratory-scale-up of Hb@ZIF-8/HSA NPs was performed to generate sufficient material for an initial in vivo evaluation in a hemorrhagic shock model, and a preliminary pilot-scale manufacturing strategy was proposed, incorporating continuous processing and a preliminary cost analysis, providing a realistic pathway toward larger-scale production.

Overall, this work establishes a strong foundation for the advancement of MOF-based HBOCs that integrate oxygen delivery, antioxidant functionality and stealth properties, while initiating early investigations into scalability, thereby moving MOF-based HBOCs one step forward in their overall development.