Hb-based Oxygen Carriers Research Articles

Comparative Evaluation of UV-Vis Spectroscopy-Based Approaches for Hemoglobin Quantification: Method Selection and Practical Insights.

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.

OMX: A NOVEL OXYGEN DELIVERY BIOTHERAPEUTIC IMPROVES OUTCOMES IN AN OVINE MODEL OF CONTROLLED HEMORRHAGIC SHOCK.

Hemorrhagic shock is a major source of morbidity and mortality worldwide. While whole blood or blood product transfusion is a first-line treatment, maintaining robust supplies presents significant logistical challenges, particularly in austere environments. OMX is a novel nonhemoglobin (Hb)-based oxygen carrier derived from the H-NOX (heme-nitric oxide/oxygen binding) protein family. Because of their engineered oxygen (O 2 ) affinities, OMX proteins only deliver O 2 to severely hypoxic tissues. Additionally, unlike Hb-based oxygen carriers, OMX proteins do not scavenge nitric oxide in the vasculature. To determine the safety and efficacy of OMX in supporting tissue oxygen delivery and cardiovascular function in a large animal model of controlled hemorrhage, 2-3-week-old lambs were anesthetized, intubated, and mechanically ventilated. Hypovolemic shock was induced by acute hemorrhage to obtain a 50% reduction over 30 min. Vehicle (n = 16) or 400 mg/kg OMX (n = 13) treatment was administered over 15 min. Hemodynamics, arterial blood gases, and laboratory values were monitored throughout the 6-h study. Comparisons between groups were made using t tests, Wilcoxon rank sum test, and Fisher's exact test. Survival was assessed using Kaplan-Meier curves and the log-rank test. We found that OMX was well-tolerated and significantly improved lactate and base deficit trends, and hemodynamic indices ( P < 0.05). Median survival time was greater in the OMX-treated group (4.7 vs. 6.0 h, P < 0.003), and overall survival was significantly increased in the OMX-treated group (25% vs. 85%, P = 0.004). We conclude that OMX is well-tolerated and improves metabolic, hemodynamic, and survival outcomes in an ovine model of controlled hemorrhagic shock.

Bio-inspired design approaches to artificial blood technology: Oxygen carriers

Bio-inspired design approaches to artificial blood technology: Oxygen carriers Allan Doctor, MD, Professor of Pediatrics at the University of Maryland School of Medicine, shares his expertise on bio-inspired design approaches to artificial blood technology: oxygen carriers. There is a need for an artificial oxygen (O2) carrier for use when banked blood is unavailable or undesirable. To date, efforts to develop Hb-based oxygen carriers (HBOCs) have failed successful translation, because of design flaws that do not preserve physiologic interactions of normal red blood cells (RBCs): First, HBOCs capture O2 in lungs but do not release O2 effectively to tissue, and second, HBOCs trap nitric oxide (NO), causing vasoconstriction – which critically limits blood flow, particularly in ischemic vascular beds; together, failure to both effectively release (to tissue) captured O2 and blood flow restriction undermines the potential benefit from otherwise improved O2 content.

Structural and functional alterations of polydopamine-coated hemoglobin: New insights for the development of successful oxygen carriers

One of the major factors that is currently hindering the development of hemoglobin (Hb)-based oxygen carriers (HBOCs) is the autoxidation of Hb into nonfunctional methemoglobin. Modification with polydopamine (PDA), which is a biocompatible free radical scavenger has shown the ability to protect Hb against oxidation. Due to its tremendous potential in the development of successful HBOCs, herein, we conduct a thorough evaluation of the effect of PDA on the stability, aggregation, structure and function of the underlying Hb. By UV–vis spectrometry we show that PDA can prevent Hb's aggregation while thermal denaturation studies indicate that, although PDA coating resulted in a lower midpoint transition temperature, it was also able to protect the protein from full denaturation. These results are further corroborated by differential scanning calorimetry. Circular dichroism reveals that PDA can promote changes in Hb's secondary structure while, by UV–vis spectroscopy, we show that PDA also interacts with the porphyrin complex located in Hb's hydrophobic pocket. Last but not least, affinity studies show that PDA-coated Hb has a higher capability for oxygen release. Such an effect is further enhanced at lower pH. Importantly, through molecular docking simulations we provide a plausible explanation for the observed experimental results.

How Nitric Oxide Hindered the Search for Hemoglobin-Based Oxygen Carriers as Human Blood Substitutes.

The search for a clinically affordable substitute of human blood for transfusion is still an unmet need of modern society. More than 50 years of research on acellular hemoglobin (Hb)-based oxygen carriers (HBOC) have not yet produced a single formulation able to carry oxygen to hemorrhage-challenged tissues without compromising the body's functions. Of the several bottlenecks encountered, the high reactivity of acellular Hb with circulating nitric oxide (NO) is particularly arduous to overcome because of the NO-scavenging effect, which causes life-threatening side effects as vasoconstriction, inflammation, coagulopathies, and redox imbalance. The purpose of this manuscript is not to add a review of candidate HBOC formulations but to focus on the biochemical and physiological events that underly NO scavenging by acellular Hb. To this purpose, we examine the differential chemistry of the reaction of NO with erythrocyte and acellular Hb, the NO signaling paths in physiological and HBOC-challenged situations, and the protein engineering tools that are predicted to modulate the NO-scavenging effect. A better understanding of two mechanisms linked to the NO reactivity of acellular Hb, the nitrosylated Hb and the nitrite reductase hypotheses, may become essential to focus HBOC research toward clinical targets.

Neuroprotective effects of a hemoglobin-based oxygen carrier (stroma-free hemoglobin nanoparticle) on ischemia reperfusion injury

The application of hemoglobin (Hb)-based oxygen carriers (HBOCs) to the treatment of cerebral ischemia has been investigated. A cluster of 1 Hb and 3 human serum albumins (Hb-HSA3) was found to exert neuroprotective effects on ischemia/reperfusion injury. Stroma-free hemoglobin nanoparticles (SFHbNP), a subsequently developed HBOC consisting of a spherical polymerized stroma-free Hb core with a HSA shell, contains the natural antioxidant enzyme catalase and, thus, is expected to exert additive effects. We herein investigated whether SFHbNP exerted enhanced neuroprotective effects in a rat transient middle cerebral artery occlusion (tMCAO) model. Rats were subjected to 2-hour tMCAO and divided into the following 3 groups with the intravenous administration of the respective reagents: (1) phosphate-buffered saline (PBS), as a vehicle (2) Hb-HSA3, and (3) SFHbNP. After 24-hour reperfusion, infarct and edema volumes decreased in the order of the PBS, Hb-HSA3, and SFHbNP groups, with a significant difference (p < 0.05) between the PBS and SFHbNP groups. Similar reductions were observed in oxidative stress, leukocyte recruitment, and blood–brain barrier disruption in the order of the PBS, Hb-HSA3, and SFHbNP groups. In the early phase of reperfusion within 6 h, microvascular HBOC perfusion and cerebral blood flow were maintained at high levels during the reperfusion period in the Hb-HSA3 and SFHbNP groups. However, a difference was observed in tissue oxygen partial pressure levels, which significantly decreased after 6-hour reperfusion in the Hb-HSA3 group, but remained high in the SFHbNP group. A superior oxygen transport ability appears to be related to the enhanced neuroprotective effects of SFHbNP.

Biocompatibility of the oxygen carrier polymerized human hemoglobin towards HepG2/C3A cells

Hemoglobin (Hb) based oxygen carriers (HBOCs) are designed to minimize the toxicity of extracellular Hb, while preserving its high oxygen-carrying capacity for oxygen delivery to cells. Polymerized human Hb (PolyHb) is a novel type of nanosized HBOC synthesized via glutaraldehyde-mediated crosslinking of free Hb, and which preserves the predominant quaternary state during the crosslinking reaction (low oxygen affinity tense (T) quaternary state PolyHb is synthesized at 0% Hb oxygen saturation, and high oxygen affinity relaxed (R) quaternary state PolyHb is synthesized at 100% Hb oxygen saturation). Major potential applications for PolyHbs, and HBOCs in general, include oxygenation of bioreactor systems containing large liver cell masses, and ex-vivo perfusion preservation of explanted liver grafts. The toxicity of these compounds toward liver cells must be evaluated before testing their use in these complex systems for oxygen delivery. Herein, we characterized the effect of PolyHbs on the hepatoma cell line HepG2/C3A, used as a model hepatocyte and as a cell line used in some investigational bioartificial liver support devices. HepG2/C3A cells were incubated in cell culture media containing PolyHbs or unmodified Hb at concentrations up to 50 mg/mL and for up to 6 days. PolyHbs were well tolerated at a dose of 10 mg/mL, with no significant decrease in cell viability; however, proliferation was inhibited as much as 10-fold after 6 days of exposure at 50 mg/mL. Secretion of albumin, and urea, as well as glucose and ammonia removal were measured in presence of 10 mg/mL of PolyHbs or unmodified Hb. In addition, methoxy- and ethoxy-resorufin deacetylase (MROD and EROD) activities, which reflect cytochrome P450 metabolism, were measured. R-state PolyHb displayed improved or intact activity in 3 out of 7 functions compared to unmodified Hb. T-state PolyHb displayed improved or intact activity in 4 out of 7 functions compared to unmodified Hb. Thus, PolyHbs, both in the R-state and T-state, are safer to use at a concentration of 10 mg/mL as compared to unmodified Hb in static culture liver-related applications.

Hemoglobin-stabilized gold nanoclusters displaying oxygen transport ability, self-antioxidation, auto-fluorescence properties and long-term storage potential.

The development of hemoglobin (Hb)-based oxygen carriers (HBOCs) holds a lot of potential to overcome important drawbacks of donor blood such as a short shelf life or the potential risk of infection. However, a crucial limitation of current HBOCs is the autoxidation of Hb into methemoglobin (metHb), which lacks oxygen-carrying capacity. Herein, we address this challenge by fabricating a Hb and gold nanoclusters (AuNCs) composite (Hb@AuNCs) which preserves the exceptional features of both systems. Specifically, the Hb@AuNCs retain the oxygen-transporting properties of Hb, while the AuNCs provide antioxidant functionality as shown by their ability to catalytically deplete harmful reactive oxygen species (ROS). Importantly, these ROS-scavenging properties translate into antioxidant protection by minimizing the autoxidation of Hb into non-functional metHb. Furthermore, the AuNCs render Hb@AuNCs with auto-fluorescence properties which could potentially allow them to be monitored once administered into the body. Last but not least, these three features (i.e., oxygen transport, antioxidant and fluorescence properties) are well maintained following storage as a freeze-dried product. Thus, overall, the as-prepared Hb@AuNCs hold the potential to be used as a multifunctional blood surrogate in the near future.

Hemoglobin‐Related Biomaterials and their Applications

Hemoglobin (Hb) is a physiological oxygen‐transport metalloprotein present in the red blood cells. With the in‐depth study, many functions of Hb are developed, and kinds of Hb‐related biomaterials are applied for biomedical therapeutics in the past decades. Herein, the recent advances of Hb‐related biomaterials and their applications, including the clinical progress of Hb‐based oxygen carriers (HBOCs) as blood substitutes, and the applications in the field of tumor therapy, wound healing, and anti‐inflammation therapy through utilizing its function of multiple gas delivery (O2, CO, NO), peroxidase‐like catalysis, and targeting macrophage, are summarized. With the rapid development of science and technology, more versatile Hb‐related biomaterials are designed in the future for other applications.

Metal-Phenolic Networks as Broad-Spectrum Antioxidant Coatings for Hemoglobin Nanoparticles Working as Oxygen Carriers

Timely administration of donor red blood cells (RBCs) is a crucial and life-saving procedure to restore tissue oxygenation in patients suffering from acute blood loss. However, important drawbacks of using allogenic RBCs are their limited availability and portability, specific storage requirements, short shelf life, or the need for blood type matching. These limitations result in serious logistical challenges that make the transfusion of donor RBCs difficult in extreme life-threatening situations prior to hospital admission. Thus, the engineering of hemoglobin (Hb) nanoparticles (Hb-NPs), which are free from the aforementioned limitations, has emerged as a promising strategy to create RBC substitutes to be used when donor blood is not available. Despite the tremendous progress achieved in recent years, many challenges still need to be overcome. For example, it is still difficult to create Hb-NPs with a high Hb content while also preventing the autoxidation of Hb into nonfunctional methemoglobin (metHb). Herein, the fabrication of small, solid Hb-NPs with an antioxidant coating is reported. By desolvation precipitation, Hb-NPs with an average hydrodynamic diameter of ∼250 nm and a polydispersity index of ∼0.2 are fabricated. A metal-phenolic network (MPN) layer consisting of a phenolic ligand (i.e., tannic acid) cross-linked through iron(III) ions is deposited onto the Hb-NPs surface to render antioxidant protection. The resulting MPN-coated Hb-NPs (MPN@Hb-NPs) maintain the ability of the encapsulated Hb to reversibly bind and release oxygen. The antioxidant properties are demonstrated, showing that MPN@Hb-NPs can effectively scavenge multiple reactive oxygen and nitrogen species, both in solution and in the presence of human RBCs and two relevant cell lines, namely, macrophages and endothelial cells. Importantly, these outstanding antioxidant properties resulting from the MPN translate into decreased metHb conversion. Finally, the newly reported MPN@Hb-NPs are also biocompatible, as shown by hemolysis rate and cell viability studies and can be used to protect the cells from oxidative damage. All in all, we have identified a novel strategy to minimize heme-mediated oxidative reactions that could potentially bring this new generation of Hb-based oxygen carriers a step closer to the clinic.

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.

Tangential flow filtration facilitated fractionation and PEGylation of low and high-molecular weight polymerized hemoglobins and their biophysical properties.

Various types of hemoglobin (Hb)-based oxygen carriers (HBOCs) have been developed as red blood cell substitutes for treating blood loss when blood is not available. Among those HBOCs, glutaraldehyde polymerized Hbs have attracted significant attention due to their facile synthetic route, and ability to expand the blood volume and deliver oxygen. Hemopure®, Oxyglobin®, and PolyHeme® are the most well-known commercially developed glutaraldehyde polymerized Hbs. Unfortunately, only Oxyglobin® was approved by the FDA for veterinary use in the United States, while Hemopure® and PolyHeme® failed phase III clinical trials due to their ability to extravasate from the blood volume into the tissue space which facilitated nitric oxide scavenging and tissue deposition of iron, which elicited vasoconstriction, hypertension and oxidative tissue injury. Fortunately, conjugation of poly (ethylene glycol) (PEG) on the surface of Hb is capable of reducing the vasoactivity of Hb by creating a hydration layer surrounding the Hb molecule, which increases its hydrodynamic diameter and reduces tissue extravasation. Several commercial PEGylated Hbs (MP4®, Sanguinate®, Euro-PEG-Hb) have been developed for clinical use with a longer circulatory half-life and improved safety compared to Hb. However, all of these commercial products exhibited relatively high oxygen affinity compared to Hb, which limited their clinical use. To dually address the limitations of prior generations of polymerized and PEGylated Hbs, this current study describes the PEGylation of polymerized bovine Hb (PEG-PolybHb) in both the tense (T) and relaxed (R) quaternary state via thiol-maleimide chemistry to produce an HBOC with low or high oxygen affinity. The biophysical properties of PEG-PolybHb were measured and compared with those of commercial polymerized and PEGylated HBOCs. T-state PEG-PolybHb possessed higher hydrodynamic volume and P50 than previous generations of commercial PEGylated Hbs. Both T- and R-state PEG-PolybHb exhibited significantly lower haptoglobin binding rates than the precursor PolybHb, indicating potentially reduced clearance by CD163 + monocytes and macrophages. Thus, T-state PEG-PolybHb is expected to function as a promising HBOC due to its low oxygen affinity and enhanced stealth properties afforded by the PEG hydration shell.

Biophysical properties of tense quaternary state polymerized human hemoglobins bracketed between 500kDa and 0.2μm in size.

Polymerized hemoglobin (Hb)-based oxygen carriers (HBOCs) are a scalable and cost-effective red blood cell (RBC) substitute. However, previous generations of commercial polymerized HBOCs elicited oxidative tissue injury in vivo due to the presence of low molecular weight polymeric Hb species (<500 kDa) and cell-free Hb (64 kDa). Polymerized human Hb (PolyhHb) locked in the tense quaternary state (T-state) exhibits great promise to meet clinical needs where past polymerized HBOCs failed. This work shows that separation of T-state PolyhHb via a two-stage tangential flow filtration (TFF) purification train such that the Hb polymers are bracketed between 500 kDa and 0.2μm creates a uniform polymer size and largely eliminates the Hb species which elicit deleterious side effects in vivo. Biophysical characterization of these materials demonstrates their potential effectiveness as an RBC substitute and verifies the low percentage of low molecular weight Hb polymers and cell-free Hb. Size exclusion chromatography confirms that T-state PolyhHb can be consistently produced in a size range between 500 kDa and 0.2μm. Furthermore, the average molecular weight of all PolyhHb species produced is one or two orders of magnitude larger than that of the commercial polymerized HBOCs Hemolink and Oxyglobin, respectively. Haptoglobin binding kinetics confirms that two-stage TFF processing of PolyhHb reliably removes cell-free Hb and low molecular weight polymeric Hb species. T-state PolyhHbs demonstrate lower auto-oxidation rates compared to unmodified Hb and prior generations of commercial polymerized HBOCs. These results demonstrate T-state PolyhHb's feasibility as a next-generation polymerized HBOC for potential use in transfusion medicine.

Artificial oxygen carriers, from nanometer- to micrometer-sized particles, made of hemoglobin composites substituting for red blood cells

Blood transfusions are regarded as the most well-known and frequently performed cell transplantations. Although current transfusion systems are sophisticated, they cannot be freed from the inherent difficulties that include infection, short shelf life, and blood type mismatching. Artificial oxygen carriers produced using hemoglobin (Hb) are designated as Hb-based oxygen carriers (HBOCs), which are anticipated for use as biomaterials that have potential to resolve issues of transfusion by a radical paradigm shift. Various HBOCs, nanometer-sized to micrometer-sized bioparticles having an oxygen-carrying function, are developed for use as substitutes for red blood cells (RBCs). This paper presents an overview of the classification of HBOCs with reference to their histories, preparations, structures, functions, and in vitro and in vivo properties. Additionally, we give a more detailed introduction of our academic studies of liposome encapsulated Hb, designated as Hb-vesicles (HbV), which mimic the physiologically important corpuscular structure of RBCs. This review outlines perennial efforts and approaches to mimic RBC functions through chemical, genetic, and encapsulation techniques. It will provide important insights into the eventual realization of an alternative for RBC transfusion.

Human polymerized hemoglobin as an alternative to blood in resuscitation from hemorrhagic shock

Hemoglobin (Hb) based oxygen carriers (HBOCs) have been developed as an alternative to blood. However, previous generations of HBOCs failed clinical trials due to toxicological and safety issues. High molecular weight (MW) polymerized human Hb (PolyhHb) has been recently tested with results that demonstrate reduced vasoconstriction and hypertension. Recently, it was observed that guinea pigs (GP) are a more clinically relevant model to test the safety of PolyHb due to a lack of ascorbic acid production, as humans present the same characteristics. Therefore, the present study aimed to evaluate the efficacy and safety of resuscitation from hemorrhagic shock (HS) with fresh blood, 2 weeks stored blood, and PolyhHb in a GP model. GP weighing between 300-400 were anesthetized. The carotid artery and jugular vein were catheterized. Hemorrhagic Shock (HS) was induced by 40 % blood volume (BV) withdrawal from the carotid artery (BV estimated as 7.5% of body weight) and the hypovolemic state was maintained for 50 minutes. After the 50 minutes into HS, GP were divided into three study groups, and 25% of their blood volume was reinfused with Fresh Blood (FB), Stored Blood (SB), or PolyhHb (n=6 animals/group). Mean arterial pressure (MAP), heart rate (HR), blood gasses, and hematocrit (Hct) were measured 15 minutes after resuscitation (R1), and animals were allowed to recover from anesthesia. Moreover, measurements were taken 2 hours (R2) and 24 hours (R3) after resuscitation, after R3 animals were euthanized and blood and tissues harvested. Markers of organs function and damage were evaluated. All groups decreased MAP equally during shock. At R3 MAP was equal between groups. HR was not different among groups. Hct decreased due to HS. FB presented higher Hct at R1 compared to the other groups, and Hct remained higher during R2 and R3. SB presented higher Hct than PolyhHb at R1 and R3. PolyhHb presented a lower total Hb than FB and SB at R3. There were no differences observed between groups in terms of pH, pO2, or pCO2 throughout the protocol. Lactate increased equally between groups, and lactate recovered to baseline for all groups after reperfusion. At R3 AST, ALP, and liver CXCL1 were increased on the SB group, but no differences were found on ALT, SOD, MDA, indicating that SB caused liver damage. SB and PolyhHb groups increased serum creatinine but no differences were found for BUN and UNgal. Serum IL-6 and CXCL1 were not different between groups, but SB presented higher IL-10 compared to all the other groups. Moreover, SB increased catecholamines and cardiac troponin at R3 suggesting cardiac damage. These results suggest that PolyhHb was as effective in recovering guinea pigs from HS as FB and SB, moreover, SB caused some degree of liver and cardiac damage, and increased catecholamines when PolyhHB seems to not have these same side effects. Therefore, these results are promising and encourage the use of this new generation of PolyhHb as an alternative when blood is not available. Furthermore, more experiments should be done to ensure PolyhHb safety before moving forward to a clinical trial.

Metal-organic framework-based oxygen carriers with antioxidant protection as a result of a polydopamine coating.

Rapid haemorrhage control to restore tissue oxygenation is essential in order to improve survival following traumatic injury. To this end, the current clinical standard relies on the timely administration of donor blood. However, limited availability and portability, special storage requirements, the need for blood type matching and risks of disease transmission result in severe logistical challenges, impeding the use of donor blood in pre-hospital scenarios. Therefore, great effort has been devoted to the development of haemoglobin (Hb)-based oxygen carriers (HBOCs), which could be used as a "bridge" to maintain tissue oxygenation until hospital admission. HBOCs hold the potential to diminish the deleterious effects of acute bleeding and associated mortality rates. We recently presented a novel HBOC, consisting of Hb-loaded metal organic framework (MOF)-based nanoparticles (NPs) (MOFHb-NPs), and demonstrated its ability to reversibly bind and release oxygen. However, a long standing challenge when developing HBOCs is that, over time, Hb oxidizes to non-functional methaemoglobin (metHb). Herein, we address this challenge by modifying the surface of the as-prepared MOFHb-NPs with an antioxidant polydopamine (PDA) coating. The conditions promoting the greatest PDA deposition are first optimized. Next, the ability of the resulting PDA-coated MOFHb-NPs to scavenge important reactive oxygen species is demonstrated both in a test tube and in the presence of two relevant cell lines (i.e., macrophages and endothelial cells). Importantly, this antioxidant protection translates into minimal metHb conversion.

Synthesis of Hemoglobin-Based Oxygen Carrier Nanoparticles By Desolvation Precipitation.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) present an alternative to red blood cells (RBCs) when blood is not available. However, the most widely used synthesis techniques have fundamental flaws, which may have contributed toward disappointing clinical application. Polymerized Hb contains a heterogeneous distribution of particle size and shape, while Hb encapsulation inside liposomes results in high lipid burden and low Hb content. Meanwhile, there are a variety of other nanoparticle synthetic techniques which, having found success as drug delivery vehicles, may be well suited to function as an HBOC. We synthesized desolvated Hb nanoparticles (Hb-dNPs) with diameters of approximately 250 nm by the controlled precipitation of Hb with ethanol. Oxidized dextran was found to be an effective surface stabilizing agent that maintained particle integrity. In vitro biophysical characterization showed a high-affinity oxygen delivery profile (P50 = 7.72 mm Hg), suggesting a potential for therapeutic use and opening a new avenue for HBOC research.

Glutaraldehyde-Polymerized Hemoglobin: In Search of Improved Performance as Oxygen Carrier in Hemorrhage Models.

Hemoglobin- (Hb-) based oxygen carriers (HBOC) have for several decades been explored for treatment of hemorrhage. In our previous top-up tests, HBOC with lower in vitro prooxidant reactivity (incorporating a peroxidase or serum albumin to this end) showed a measurable but small improvement of oxidative stress-related parameters. Here, such HBOCs are tested in a hemorrhage set-up; ovine hemoglobin is also tested for the first time in such a setting, based on in vitro data showing its improved performance versus bovine Hb against oxidative and nitrosative stress agents. Indeed, ovine Hb performs better than bovine Hb in terms of survival rates, arterial tension, immunology, and histology. On the other hand, unlike in the top-up models, where the nonheme peroxidase rubrerythrin as well as bovine serum albumin copolymerized with Hb were shown to improve the performance of HBOC, in the present hemorrhage models rubrerythrin fails dramatically as HBOC ingredient (with a distinct immunological reaction), whereas serum albumin appears not feasible if its source is a different species (i.e., bovine serum albumin fares distinctly worse than rat serum albumin, in HBOC transfusions in rats). An effect of the matrix in which the HBOCs are dissolved (PBS versus gelofusine versus plasma) is noted.

A triply modified human adult hemoglobin with low oxygen affinity, rapid autoxidation and high tetramer stability

The hypoxic environment of tumor may retard the efficacy of tumor therapeutic agents. Hemoglobin (Hb)-based oxygen carriers (HBOCs) could overcome the hypoxia of tumor by the oxygen delivery. However, typical HBOCs may not provide sufficient oxygen for their low oxygen transferring efficiency (OTE). In order to increase the OTE, human adult Hb (HbA) was subjected to triple modifications, i.e., αα-fumaryl crosslink at Lys-99(α), carboxymethylation at Val-1(α) and 8-arm PEG-based polymerization. Crosslink at Lys-99(α) and carboxymethylation at Val-1(α) synergistically led to a T-like quaternary structure of HbA. The dual modification significantly increased the partial oxygen pressure at 50% saturation (P50) of HbA from 14.8 mmHg to 34.6 mmHg and OTE from 9.1% to 33.1%. Eight-arm PEG-based polymerization slightly decreased the P50 of the Hb derivative to 27.8 mmHg and OTE to 30.5%. However, it can enlarge the molecular size of HbA and then prolong the serum duration of HbA. The triple modifications synergistically increased the autoxidation rate of Hb, which promote the production of reactive oxygen species (ROS). Therefore, the sufficient oxygen delivery and substantial production of ROS by the triply modified Hb may provide a potential strategy for tumor therapy.