The Assembly and Characterization of Hemoglobin-based Oxygen Carriers with Antioxidant Properties

For the body to carry out basic metabolic activities, there is a constant need for the supply of oxygen towards its tissues and cells. Loss of oxygen supply can be caused by blood diseases such as hemophilia, ischemia, and chronic anemia or due to blood loss during traumatic injury and surgery. To replenish the oxygen carrying capacity within the body, the transfusion of donor whole blood or isolated red blood cells (RBCs) is a well-established clinical procedure.

However, the usage of donor RBCs comes with drawbacks such as the need for typing and cross-matching, limited availability, and short shelf life with specific storage requirements. Therefore, research efforts have been dedicated to fabricating hemoglobin (Hb)-based oxygen carriers (HBOCs), which are able to replace or complement standard transfusion procedures.

Hb is the main component of RBCs and is responsible for ~98% of blood’s oxygen carrying capacity. However, stroma-free Hb cannot be administered directly as blood substitute due to various adverse effects related to the protein’s dissociation, extravasation into the smooth muscle tissue, and saturation of the Hb-removal pathways.

To overcome the Hb’s toxicity while preserving its functionality, Hb is either chemically modified or entrapped within an encapsulation platform. In this PhD thesis, we employed a polymeric-based nanocarrier and Hb was either encapsulated in its core or adsorbed onto its surface without the need to chemically modifying the protein.

Next, the Hb’s functionality has to be protected against the oxidation of Hb into non-functional methemoglobin, a process that occurs over time and is accelerated by the presence of reactive oxygen species (ROS). Thus, to inhibit or retard the oxidation of Hb, an antioxidant system is incorporated. The incorporation of nanozymes was assessed, which are a type of nanoparticles (NPs) that display enzymatic activity.

Specifically, cerium oxide (CeO2)-NPs were employed, which have shown superoxide dismutase-like and catalase-like activity. Polydopamine (PDA) was furthermore explored to facilitate surface modification and to protect the Hb structure and functionality. Finally, to provide the nanocarrier system with stealth properties, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and RBC membrane (RBC-M) coatings were employed.

PEG is regarded as the golden standard to obtain stealth coating and has been widely explored. Alternatively, RBC-M is novel biological-derived stealth coating, which employs the RBC’s surface properties to achieve long circulation. First, HBOC with antioxidant properties were prepared by depositing PDA-coated Hb (HbPDA), CeO2-NPs, and PLL-g-PEG onto a poly(lactide-co-glycolide) (PLGA)-NP core.

The assembly was thoroughly assessed by evaluating the functionality of the different components prior and after deposition onto the nanocarrier system. It was shown the HbPDA had preserved protein structure and functionality, the CeO2-NPs were able to deplete ROS, and the PLL-g-PEG reduced the protein adsorption and cell association/uptake.

Furthermore, the complete HBOC system was shown to be biocompatible, hemocompatible, and was able to deplete ROS for at least five cycles. Next, the PLGA-NPs were evaluated by their ability to entrap Hb within their core, thereby increasing the Hb content of the HBOCs. Hb-loaded PLGA-NPs (PLGAHb-NPs) were fabricated using the double emulsification solvent evaporation method, where the effect of Hb, PLGA, and emulsifier concentration was assessed.

Optimal fabrication parameters were chosen to prepare functional PLGAHb-NPs with improved Hb-content and a good size distribution. Finally, to improve the stealth properties of the HBOC, the usage of RBC-M as a stealth coating was investigated. The PLGAHb-NPs were used as NP cores to deposit CeO2-NPs onto, followed by RBC-M coating.

The PLGAHb-NPs were shown to reversibly bind and release oxygen, while the ROS scavenging and storage stability of the CeO2-NPs were further assessed in comparison to the native enzymes. For the RBC-M deposition, three methodologies, namely the sonication, extrusion, and combination method were assessed and the RBC-M’s ability to reduce protein adsorption was demonstrated.

It was furthermore shown that the RBC-M did not affect the underlaying compounds, yielding RBC-M coated nanocarriers with retained oxygen carrying capacity and ROS depletion ability, thus making the as-prepared nanocarriers potent HBOCs with antioxidant properties.