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Hemoglobin (Hb)-based oxygen carriers are investigated as a potential alternative or supplement to regular blood transfusions, particularly in critical and life-threatening scenarios. These include situations like severe trauma in remote areas, battlefield conditions, instances where blood transfusion is not feasible due to compatibility concerns, or when patients decline transfusions based on religious beliefs. This study introduces a novel method utilizing poly(ethylene glycol) (PEG) to entrap Hb within ZIF-8 nanoparticles (i.e., Hb@ZIF-8 NPs). Through meticulous screening, we achieved Hb@ZIF-8 NPs with a record-high Hb concentration of 34 mg mL -1 . These NPs, sized at 168 nm, displayed exceptional properties: a remarkable 95 % oxyhemoglobin content, excellent encapsulation efficiency of 85 %, and resistance to Hb oxidation into methemoglobin (metHb). The addition of PEG emerged as a crucial factor amplifying Hb entrapment within ZIF-8, especially at higher Hb concentrations, reaching an unprecedented 34 mg mL -1 . Importantly, PEG exhibited a protective effect, preventing metHb conversion in Hb@ZIF-8 NPs at elevated Hb concentrations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The growing demand for effective alternatives to red blood cells (RBCs) has spurred significant research into hemoglobin (Hb)-based oxygen carriers (HBOCs). Accurate characterization of HBOCs-including Hb content, encapsulation efficiency, and yield-is crucial for ensuring effective oxygen delivery, economic viability, and the prevention of adverse effects caused by free Hb. However, the choice of quantification methods for HBOCs is often driven more by tradition than by a thorough assessment of available options. This study meticulously compares various UV-vis spectroscopy-based methods for Hb quantification, focusing on their efficacy in measuring Hb extracted from bovine RBCs across different concentration levels. The findings identify the sodium lauryl sulfate Hb method as the preferred choice due to its specificity, ease of use, cost-effectiveness, and safety, particularly when compared to cyanmethemoglobin-based methods. Additionally, the study discusses the suitability of these methods for HBOC characterization, emphasizing the importance of considering carrier components and potential interferences by analyzing the absorbance spectrum before selecting a method. Overall, this study provides valuable insights into the selection of accurate and reliable Hb quantification methods, which are essential for rigorous HBOC characterization and advancements in medical research.

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 (O 2 ) carriers (HBOCs) which make use of the main component of RBCs and the responsible protein for O 2 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 O 2 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 O 2 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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.