Hemoglobin nanoparticles equipped with antioxidant coatings as a novel type of therapeutic oxygen carriers

The transfusion of red blood cells (RBCs) is a well-established clinical procedure for patients experiencing acute blood loss. However, there are several challenges associated with blood transfusions. These include the need for blood typing and matching, particular storage conditions, a short shelf-life, potential risks of virus transmission, and a global shortage in blood availability. As such, there is an imminent demand for the development of RBCs substitutes to overcome these challenges and supplement blood transfusions. Prominent advances in the development of hemoglobin (Hb)-based oxygen carriers (HBOCs) as RBC substitutes have been made in the past decades. Nonetheless, several shortcomings of the reported HBOCs still need to be addressed, including the preservation Hb’s functionality and achieve a high Hb content to obtain a high oxygen (O2) delivering capacity.
In the current thesis, these challenges have been addressed to obtain HBOCs not only with a high Hb content, but also with remarkable antioxidant properties that shield this Hb from autoxidation into non-functional methemoglobin (metHb). For this, a desolvation technique and different functional surface coating techniques were employed.
Desolvation techniques have previously been used for the fabrication of protein particles both in nano- and micro-sizes. Here, the assembly of nanoparticles (NPs) fully made of Hb (Hb-NPs) by the desolvation technique was demonstrated. For this, several desolvation-related parameters were optimized (i.e., Hb concentration, the ratio of organic phase/water phase, and sonication intensity/time) to obtain Hb-NPs with a size of ~568 nm and a polydispersity index of ~0.2. Next, a surface functionalization with a polydopamine (PDA) coating was performed to further stabilize the Hb-NPs and, more importantly, provide them with antioxidant properties. The resulting PDA-coated Hb-NPs (Hb/PDA-NPs) were able to scavenge reactive oxygen species such as superoxide radical anion and hydrogen peroxide. Most importantly, PDA’s antioxidant features protected the encapsulated Hb from autooxidation into metHb. Furthermore, the biocompatibility and ability of the Hb/PDA-NPs to reversibly bind O₂ were also demonstrated.
Next, for better preservation of Hb functionality within the Hb-NPs, process optimizations were performed. These included the desolvation being carried out at 4 °C and the use of a different metal phenolic network (MPN) coating consisting of tannic acid and iron (III) ions. The resulting MPN-coated Hb-NPs (MPN@Hb-NPs) were spherical NPs with a size of ~250 nm and a polydispersity index of ~0.1. The encapsulated Hb well maintained its O₂ delivery capacity in the MPN@Hb-NPs. Furthermore, these modified Hb-NPs could scavenge multiple reactive oxygen and nitrogen species due to the MPN coating. This was demonstrated also in the presence of donor RBCs, macrophages, and endothelial cells. Moreover, Hb protection by the antioxidant MPN coating was suggested by a decreased metHb conversion. Lastly, the MPN@Hb-NPs were also remarkably biocompatible, suggesting their great potential as HBOCs.
Overall, these reported HBOCs with an antioxidant surface coating and a high Hb content are a step forward towards the development of a new generation of improved HBOCs.