Synthesis of bioactive hemoglobin-based oxygen carrier nanoparticles via metal-phenolic complexation.

The transfusion of donor red blood cells (RBCs) is seriously hampered by important drawbacks that include limited availability and portability, the requirement of being stored in refrigerated conditions, a short shelf life or the need for RBC group typing and crossmatching. Thus, hemoglobin (Hb)-based oxygen (O2) carriers (HBOCs) which make use of the main component of RBCs and the responsible protein for O2 transport, hold a lot of promise in modern transfusion and emergency medicine. Despite the great progress achieved, it is still difficult to create HBOCs with a high Hb content to attain the high O2 demands of our body. Herein a metal-phenolic self-assembly approach that can be conducted in water and in one step to prepare nanoparticles (NPs) fully made of Hb (Hb-NPs) is presented. In particular, by combining Hb with polyethylene glycol, tannic acid (TA) and manganese ions, spherical Hb-NPs with a uniform size around 350-525 nm are obtained. The functionality of the Hb-NPs is preserved as shown by their ability to bind and release O2 over multiple rounds. The binding mechanism of TA and Hb is thoroughly investigated by UV-vis absorption and fluorescence spectroscopy. The binding site number, apparent binding constant at two different temperatures and the corresponding thermodynamic parameters are identified. The results demonstrate that the TA-Hb interaction takes place through a static mechanism in a spontaneous process as shown by the decrease in Gibbs free energy. The associated increase in entropy suggests that the TA-Hb binding is dominated by hydrophobic interactions.

Metal-organic framework-based oxygen carriers with antioxidant activity resulting from the incorporation of gold nanozymes.

Blood transfusions are a life-saving procedure since they can preserve the body's oxygen levels in patients suffering from acute trauma, undergoing surgery, receiving chemotherapy or affected by severe blood disorders. Due to the central role of hemoglobin (Hb) in oxygen transport, so-called Hb-based oxygen carriers (HBOCs) are currently being developed for situations where donor blood is not available. In this context, an important challenge that needs to be addressed is the oxidation of Hb into methemoglobin (metHb), which is unable to bind and release oxygen. While several research groups have considered the incorporation of antioxidant enzymes to create HBOCs with minimal metHb conversion, the use of biological enzymes has important limitations related to their high cost, potential immunogenicity or low stability in vivo. Thus, nanomaterials with enzyme-like properties (i.e., nanozymes (NZs)) have emerged as a promising alternative. Amongst the different NZs, gold (Au)-based metallic nanoparticles are widely used for biomedical applications due to their biocompatibility and multi-enzyme mimicking abilities. Thus, in this work, we incorporate Au-based NZs into a type of HBOC previously reported by our group (i.e., Hb-loaded metal-organic framework (MOF)-based nanocarriers (NCs)) and investigate their antioxidant properties. Specifically, we prepare MOF-NCs loaded with Au-based NZs and demonstrate their ability to catalytically deplete over multiple rounds of two prominent reactive oxygen species (ROS) that exacerbate Hb's autoxidation (i.e., hydrogen peroxide and the superoxide radical). Importantly, following loading with Hb, we show how these ROS-scavenging properties translate into a decrease in metHb content. All in all, these results highlight the potential of NZs to create novel HBOCs with antioxidant protection which may find applications as a blood substitute in the future.

Hemoglobin-based oxygen carriers camouflaged with membranes extracted from red blood cells: Optimization and assessment of functionality.

Despite being an indispensable clinical procedure, the transfusion of donor blood has important limitations including a short shelf-life, limited availability and specific storage requirements. Therefore, a lot of effort has been devoted to developing hemoglobin (Hb)-based oxygen carriers (HBOCs) that are able to replace or complement standard blood transfusions, especially in extreme life-threatening situations. Herein, we employed a Hb-loaded poly(lactide-co-glycolide) core which was subsequently coated with nanozymes to protect the encapsulated Hb from oxidation by reactive oxygen species. To render HBOCs with long circulation in the vasculature, which is a crucial requirement to achieve the high oxygen demands of our organism, the carrier was coated with a red blood cell-derived membrane. Three coating methods were explored and evaluated by their ability to repel the deposition of proteins and minimize their uptake by an endothelial cell line. Preservation of the oxygen carrying capacity of the membrane-coated carrier was demonstrated by an oxygen-binding and releasing assay and, the functionality resulting from the entrapped nanozymes, was shown by means of superoxide radical anion and hydrogen peroxide depletion assays. All in all, we have demonstrated the potential of the membrane-coated nanocarriers as novel oxygen carrying systems with both antioxidant and stealth properties.

Optimization of Hemoglobin Encapsulation within PLGA Nanoparticles and Their Investigation as Potential Oxygen Carriers.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) display the excellent oxygen-carrying properties of red blood cells, while overcoming some of the limitations of donor blood. Various encapsulation platforms have been explored to prepare HBOCs which aim to avoid or minimize the adverse effects caused by the administration of free Hb. Herein, we entrapped Hb within a poly(lactide-co-glycolide) (PLGA) core, prepared by the double emulsion solvent evaporation method. We study the effect of the concentrations of Hb, PLGA, and emulsifier on the size, polydispersity (PDI), loading capacity (LC), and entrapment efficiency (EE) of the resulting Hb-loaded PLGA nanoparticles (HbNPs). Next, the ability of the HbNPs to reversibly bind and release oxygen was thoroughly evaluated. When needed, trehalose, a well-known protein stabilizer that has never been explored for the fabrication of HBOCs, was incorporated to preserve Hb's functionality. The optimized formulation had a size of 344 nm, a PDI of 0.172, a LC of 26.9%, and an EE of 40.7%. The HbNPs were imaged by microscopy and were further characterized by FTIR and CD spectroscopy to assess their chemical composition and structure. Finally, the ability of the encapsulated Hb to bind and release oxygen over several rounds was demonstrated, showing the preservation of its functionality.

Stepping stones to the future of haemoglobin-based blood products: clinical, preclinical and innovative examples.

There is an enormous demand for blood transfusions in daily clinical practices since blood products, especially red blood cells (RBCs), can significantly improve survival. However, donor-derived RBCs have important limitations as a result of their insufficient availability, the need for typing and cross-matching, short shelf-life or risk of pathogenic contamination. Thus, as a result of the unique oxygen-transport ability of hemoglobin (Hb), Hb-based oxygen carriers (HBOCs) have attracted a lot of attention for the development of RBC surrogates able to provide tissue oxygenation. Here, we highlight the progress in the development of HBOCs, focusing on different examples that have undergone exhaustive pre-clinical and clinical evaluation. In addition, we also provide a comprehensive review of very recent and innovative examples to aid in the development of the next generation of blood substitutes.