Degree of PEGylatation of Lumbricus terrestris Hemoglobin Improves Microcirculatory Blood Flow but Increases the Rate of Auto-Oxidation.

INTRODUCTION: The demand for red blood cells (RBCs) is on the rise due to the increasing diagnosis of chronic diseases such as sickle cell anemia, malaria, and thalassemia. Despite many commercial attempts, there are no U.S. FDA-approved artificial RBCs for use in humans. Existing RBC substitutes have employed various strategies to transport oxygen, extend the circulation time, and reduce organ toxicity, but none have replicated the natural protective mechanisms of RBCs, which prevent hemoglobin (Hb) dimerization and heme iron oxidation. Lumbricus terrestris (earthworm) erythrocruorin (LtEc) is a naturally occurring extracellular hemoglobin (Hb) with promising attributes: large molecular diameter (30 nm), high molecular weight (3.6 MDa), low auto-oxidation rate, and limited nitric oxide-scavenging properties. These characteristics make LtEc an ideal candidate as an RBC substitute. However, LtEc has a significant drawback, its short circulatory half-life. To address this issue, we explored thiol-mediated surface PEGylation of LtEc (PEG-LtEc) at varying polyethylene glycol (PEG) surface coverages. Increasing PEG surface coverage beyond 40% destabilizes LtEc into smaller subunits that are 1/12th the size of LtEc. Therefore, we evaluated two PEG surface coverage options: PEG-LtEc-0.2 (20% PEGylation) and PEG-LtEc-1.0 (100% PEGylation). METHODS: We conducted experiments using golden Syrian hamsters with dorsal window chambers and catheters to assess the efficacy of these solutions. We measured microvascular parameters, organ function, cerebral blood flow, circulation time, mean arterial pressure, heart rate, and blood gases and performed histology to screen for toxicity. CONCLUSION: Our findings indicate that both PEG-LtEc molecules offer significant benefits in restoring microvascular parameters, organ function, cerebral blood flow, and circulation time compared to LtEc alone. Notably, PEG-LtEc-1.0 showed superior microvascular perfusion, although it exhibited a higher rate of auto-oxidation compared to PEG-LtEc-0.2. These results underscore the advantages of PEGylation in terms of tissue perfusion and organ health while highlighting its limitations.

An ex vivo method to evaluate vasoactivity induced by hemoglobin-based oxygen carriers in resistance vessels.

Red blood cell substitutes offer a solution to the problem of blood shortage and side effects of blood transfusion. Hemoglobin-based oxygen carriers (HBOCs) are one of the promising substitutes for red blood cells. Vasoactivity, which refers to the side effect of HBOCs that causes vasoconstriction and subsequent hypertension, limits the clinical application of HBOCs. In this study, an ex vivo method for the evaluation of vasoactivity induced by HBOCs was established based on isolated rat mesenteric artery vessels and the DMT120CP system. The DMT120CP system, equipped with a flowmeter, permits the control of intravascular pressure, pressure gradient, and flow conditions with high accuracy, simulating the physiological conditions for isolated vessels. The concentration of noradrenaline was optimized to 1 × 10-6∼3 × 10-6 M. PEGylated bovine hemoglobin (PEG-bHb) was synthesized and perfused into the vessel for vasoactivity evaluation, with bHb as the positive control and PSS buffer solution as the negative control. PEG-bHb showed a hydration diameter of 15.5 ± 1.4 nm and a P50 value of 6.99 mmHg. PEG-bHb exhibited a colloid osmotic pressure of 64.1 mmHg and a viscosity of 1.73 cp at 40 mg/mL. The established vasoactivity evaluation method showed significant differences in samples (bHb or PEG-bHb) with different vasoactivity properties. The vasoconstriction percentage induced by PEG-bHb samples synthesized in different batches showed coefficients of variation less than 5%, indicating good applicability and repeatability. The established evaluation method can be applied to study the vasoactivity induction and elimination strategies, promoting the clinical application of HBOCs.

Nanomaterial-related hemoglobin-based oxygen carriers, with emphasis on liposome and nano-capsules, for biomedical applications: current status and future perspectives.

Oxygen is necessary for life and plays a key pivotal in maintaining normal physiological functions and treat of diseases. Hemoglobin-based oxygen carriers (HBOCs) have been studied and developed as a replacement for red blood cells (RBCs) in oxygen transport due to their similar oxygen-carrying capacities. However, applications of HBOCs are hindered by vasoactivity, oxidative toxicity, and a relatively short circulatory half-life. With advancements in nanotechnology, Hb encapsulation, absorption, bioconjugation, entrapment, and attachment to nanomaterials have been used to prepare nanomaterial-related HBOCs to address these challenges and pend their application in several biomedical and therapeutic contexts. This review focuses on the progress of this class of nanomaterial-related HBOCs in the fields of hemorrhagic shock, ischemic stroke, cancer, and wound healing, and speculates on future research directions. The advancements in nanomaterial-related HBOCs are expected to lead significant breakthroughs in blood substitutes, enabling their widespread use in the treatment of clinical diseases.

Novel high molecular weight polymerized hemoglobin in a non-obese model of cardiovascular and metabolic dysfunction.

The widespread adoption of high-calorie, high-fat, high-sucrose diets (HFHSD) has become a global health concern, particularly due to their association with cardiovascular diseases and metabolic disorders. These comorbidities increase susceptibility to severe outcomes from viral infections and trauma, with trauma-related incidents significantly contributing to global mortality rates. This context underscores the critical need for a reliable blood supply. Recent research has focused on high molecular weight (MW) polymerized human hemoglobin (PolyhHb) as a promising alternative to red blood cells (RBCs), showing encouraging outcomes in previous studies. Given the overlap of metabolic disorders and trauma-related health issues, it is crucial to assess the potential toxicity of PolyhHb transfusions, particularly in models that represent these vulnerable populations. This study evaluated the effects of PolyhHb exchange transfusion in guinea pigs that had developed metabolic disorders due to a 12-week HFHSD regimen. The guinea pigs, underwent a 20 % blood volume exchange transfusion with either PolyhHb or the lower molecular weight polymerized bovine hemoglobin, Oxyglobin. Results revealed that both PolyhHb and Oxyglobin transfusions led to liver damage, with a more pronounced effect observed in HFHSD-fed animals. Additionally, markers of cardiac dysfunction indicated signs of cardiac injury in both the HFHSD and normal diet groups following the Oxyglobin transfusion. This study highlights how pre-existing metabolic disorders can exacerbate the potential side effects of hemoglobin-based oxygen carriers (HBOCs). Importantly, the newer generation of high MW PolyhHb showed lower cardiac toxicity compared to the earlier generation low MW PolyhHb, known as Oxyglobin, even in models with pre-existing endothelial and metabolic challenges.

Transfusion management and hemoglobin-based oxygen carrier treatment in a patient with anti-Rh17 antibody.

BACKGROUND: A 54-year-old Hispanic OPos female with known history of anti-Rh17 antibodies was diagnosed with Philadelphia-Chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). Rh17, also known as Hr0, is a high-frequency antigen composed of several epitopes on the RhCE protein. Anti-Rh17 antibodies can be made by individuals with missing or varied C/c, E/e antigens. Anti-Rh17 antibodies are clinically significant given multiple case reports of hemolytic disease of the fetus and newborn (HDFN). Finding compatible units for patients with anti-Rh17 can be particularly difficult given that only 1 in 100,000 people are Rh17 negative. STUDY DESIGN AND METHODS: Search for compatible units was conducted by the American Rare Donor Program (ARDP) with no leads. After chemotherapy induction and despite erythropoiesis stimulating agent administration, the patient's hemoglobin continued to trend down to a nadir of 2.8 g/dL. Here we report transfusion of incompatible pRBC to this patient with critically symptomatic anemia. HBOC-201 (Hemopure) was obtained and administered under an emergency compassionate/expanded access designation from the Food and Drug Administration (FDA) under an emergency Investigational New Drug (IND) application. RESULTS AND DISCUSSION: Overall difficulties in this case included the challenge of finding compatible units, dilemma of transfusing incompatible units in a patient with severe anemia and obtaining alternatives to blood products. This case report demonstrates the successful use of HBOC-21 in treating life-threatening anemia.

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.

Recognition mechanisms of hemoglobin particles by monocytes - CD163 may just be one.

Hemoglobin-based oxygen carriers (HBOCs) as blood substitutes are one of the great hopes of modern transfusion and emergency medicine. After the major safety-relevant challenges of the last decades seem to be largely overcome, current developments have in common that they are affected by degradation and excretion at an early stage in test organisms. Several possible mechanisms that may be responsible for this are discussed in the literature. One of them is CD163, the receptor of the complex of haptoglobin (Hp) and hemoglobin (Hb). The receptor has been shown in various studies to have a direct affinity for Hb in the absence of Hp. Thus, it seems reasonable that CD163 could possibly also bind Hb within HBOCs and cause phagocytosis of the particles. In this work we investigated the role of CD163 in the uptake of our hemoglobin sub-micron particles (HbMPs) in monocytes and additionally screened for alternative ways of particle recognition by monocytes. In our experiments, blockade of CD163 by specific monoclonal antibodies proved to partly inhibit HbMP uptake by monocytes. It appears, however, that several other phagocytosis pathways for HbMPs might exist, independent of CD163 and also Hb.

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.

Structural optimization and prospect of constructing hemoglobin oxygen carriers based on hemoglobin.

The current global shortage of organ resources, the imbalance in donor-recipient demand and the increasing number of high-risk donors make organ preservation a necessity to consider appropriate storage options. The current method of use often has risks such as blood group mismatch, short shelf life, and susceptibility. HBOCs have positive effects such as anti-apoptotic, anti-inflammatory, antioxidant and anti-proliferative, which have significant advantages in organ storage. Therefore, it is the common pursuit of researchers to design and synthesize HBOCs with safety, ideal oxygen-carrying capacity, easy storage, etc. that are widely applicable and optimal for different organs. There has been a recent advancement in understanding HBOCs mechanisms, which is discussed in this review.

Solid-Phase Polymerization Using Anion-Exchange Resin Can Almost Completely Crosslink Hemoglobin to Prepare Hemoglobin-Based Oxygen Carriers.

Introduction: A limitation of hemoglobin-based oxygen carriers (HBOCs) as oxygen therapeutics is unpolymerized hemoglobin, which induces vasoconstriction leading to hypertension. The removal of unpolymerized hemoglobin from polymerized hemoglobin (PolyHb) is complex, expensive, and time-consuming. Methods: Herein, we developed a method to completely polymerize hemoglobin almost without unpolymerized hemoglobin. Hemoglobin was adsorbed on the anion-exchange resin Q Sepharose Fast Flow or DEAE Sepharose Fast Flow, and acetal, a crosslinker prepared from glutaraldehyde and ethylene glycol, was employed to polymerize the hemoglobin. The polymerization conditions, including reaction time, pH, resin type, and molar ratios of glutaraldehyde to ethylene glycol and hemoglobin to acetal, were optimized. The blood pressure and blood gas of mice injected with PolyHb were monitored as well. Results: The optimal polymerization condition of PolyHb was when the molar ratio of glutaraldehyde to ethylene glycol was 1:20, and the molar ratio of 10 mg/mL hemoglobin adsorbed on anion-exchange resin to glutaraldehyde was 1:300 for 60 min. Under optimized reactive conditions, hemoglobin was almost completely polymerized, with <1% hemoglobin remaining unpolymerized, and the molecular weight of PolyHb was more centrally distributed. Furthermore, hypertension was not induced in mice by PolyHb, and there were also no pathological changes observed in arterial oxygen, blood gas, electrolytes, and some metabolic indicators. Conclusion: The findings of this study indicate that the use of solid-phase polymerization and acetal is a highly effective and innovative approach to HBOCs, resulting in the almost completely polymerized hemoglobin. These results offer promising implications for the development of new methods for preparing HBOCs.

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.

Translational Research of Hemoglobin Vesicles as a Transfusion Alternative.

Clinical situations arise in which blood for transfusion becomes scarce or unavailable. Considerable demand for a transfusion alternative persists because of various difficulties posed by blood donation and transfusion systems. Hemoglobin-vesicles (Hb- V) are artificial oxygen carriers being developed for use as a transfusion alternative. Just as biomembranes of red blood cells (RBCs) do, phospholipid vesicles (liposomes) for Hb encapsulation can protect the human body from the toxic effects of molecular Hb. The main HbV component, Hb, is obtained from discarded human donated blood. Therefore, HbV can be categorized as a biologic agent targeting oxygen for peripheral tissues. The purification procedure strictly eliminates the possibility of viral contamination. It also removes all concomitant unstable enzymes present in RBC for utmost safety from infection. The deoxygenated HbVs, which are storable for over the years at ambient temperature, can function as an alternative to blood transfusion for resuscitation from hemorrhagic shock and O2 therapeutics. Moreover, a recent study clarified beneficial effects for anti- oxidation and anti-inflammation by carbon monoxide (CO)-bound HbVs. Autoxidation of HbV (HbO2 → metHb + O2 -.) is unavoidable after intravenous administration. Co-injection of methylene blue can extract the intraerythrocytic glycolytic electron energy effectively and reduce metHb. Other phenothiazine dyes can also function as electron mediators to improve the functional life span of HbV. This review paper summarizes recent progress of the research and development of HbV, aimed at clinical applications.

Oxygen Delivery Approaches to Augment Cell Survival After Myocardial Infarction: Progress and Challenges.

Myocardial infarction (MI), triggered by blockage of a coronary artery, remains the most common cause of death worldwide. After MI, the capability of providing sufficient blood and oxygen significantly decreases in the heart. This event leads to depletion of oxygen from cardiac tissue and consequently leads to massive cardiac cell death due to hypoxemia. Over the past few decades, many studies have been carried out to discover acceptable approaches to treat MI. However, very few have addressed the crucial role of efficient oxygen delivery to the injured heart. Thus, various strategies were developed to increase the delivery of oxygen to cardiac tissue and improve its function. Here, we have given an overall discussion of the oxygen delivery mechanisms and how the current technologies are employed to treat patients suffering from MI, including a comprehensive view on three major technical approaches such as oxygen therapy, hemoglobin-based oxygen carriers (HBOCs), and oxygen-releasing biomaterials (ORBs). Although oxygen therapy and HBOCs have shown promising results in several animal and clinical studies, they still have a few drawbacks which limit their effectiveness. More recent studies have investigated the efficacy of ORBs which may play a key role in the future of oxygenation of cardiac tissue. In addition, a summary of conducted studies under each approach and the remaining challenges of these methods are discussed.

From hemoglobin allostery to hemoglobin-based oxygen carriers.

Hemoglobin (Hb) plays its vital role through structural and functional properties evolutionarily optimized to work within red blood cells, i.e., the tetrameric assembly, well-defined oxygen affinity, positive cooperativity, and heterotropic allosteric regulation by protons, chloride and 2,3-diphosphoglycerate. Outside red blood cells, the Hb tetramer dissociates into dimers, which exhibit high oxygen affinity and neither cooperativity nor allosteric regulation. They are prone to extravasate, thus scavenging endothelial NO and causing hypertension, and cause nephrotoxicity. In addition, they are more prone to autoxidation, generating radicals. The need to overcome the adverse effects associated with cell-free Hb has always been a major hurdle in the development of substitutes of allogeneic blood transfusions for all clinical situations where blood is unavailable or cannot be used due to, for example, religious objections. This class of therapeutics, indicated as hemoglobin-based oxygen carriers (HBOCs), is formed by genetically and/or chemically modified Hbs. Many efforts were devoted to the exploitation of the wealth of biochemical and biophysical information available on Hb structure, function, and dynamics to design safe HBOCs, overcoming the negative effects of free plasma Hb. Unfortunately, so far, no HBOC has been approved by FDA and EMA, except for compassionate use. However, the unmet clinical needs that triggered intensive investigations more than fifty years ago are still awaiting an answer. Recently, HBOCs "repositioning" has led to their successful application in organ perfusion fluids.

Effect of HSRP1 oxygen-carrying solution on early perfusion in rabbits with controlled severe hemorrhagic anemia / 中国输血杂志

【Objective】 To observe the effect of glutaraldehyde polymerized bovine hemoglobin injection (code: HSRP1) oxygen-carrying fluid on early perfusion of severe hemorrhagic anemia in rabbits. 【Methods】 The rabbit model of controlled severe hemorrhagic anemia was established. Twelve modeled rabbits were divided into glutaraldehyde polymerized bovine hemoglobin injection (code: HSRP1) group and sodium lactate ringer′s injection (LR) group, with 6 rabbits in each group(half male and half female). HSPR1 group and LR group were treated with HSRP1 and LR, respectively. The survival rate of experimental rabbits was observed, and the indexes of hemodynamics, venous blood gas, plasma hemoglobin, base surplus, lactic acid and bicarbonate were measured before and after blood loss, as well as each point within 24 h after infusion. 【Results】 The survival rate of HSRP1 group was significantly different from that of LR group (P<0.05); After exsanguination, the mean arterial pressure of each group was significantly different from that before exsanguination (P<0.05), but there was no significant difference between HSPR1 group and LR group after infusion; In the second stage of perfusion, the blood lactate concentration and base excess in the HSRP1 group were significantly different from those in the LR group at each time point (P<0.05), at 2 h after perfusion, the respiratory rate started to differ significantly from that of the LR group (P<0.05), heart rate was significantly different from LR group at 4 h after perfusion(P<0.05); There were no significant differences between HSRP1 group and LR group in plasma venous oxygen partial pressure, venous oxygen saturation and plasma hemoglobin at all time points. 【Conclusion】 HSPR1 is used for severe traumatic hemorrhagic shock in rabbits, and can improve the survival rate of experimental rabbits by providing oxygen to hypoxic tissues and correcting anaerobic metabolism. As a new oxygen-carrying fluid, HSPR1 can correct the oxygen supply balance of patients with severe hemorrhagicanemia in early stage.

Hemoglobin-Based Oxygen Carriers: Potential Applications in Solid Organ Preservation.

Ameliorating graft injury induced by ischemia and hypoxia, expanding the donor pool, and improving graft quality and recipient prognosis are still goals pursued by the transplant community. The preservation of organs during this process from donor to recipient is critical to the prognosis of both the graft and the recipient. At present, static cold storage, which is most widely used in clinical practice, not only reduces cell metabolism and oxygen demand through low temperature but also prevents cell edema and resists apoptosis through the application of traditional preservation solutions, but these do not improve hypoxia and increase oxygenation of the donor organ. In recent years, improving the ischemia and hypoxia of grafts during preservation and repairing the quality of marginal donor organs have been of great concern. Hemoglobin-based oxygen carriers (HBOCs) are "made of" natural hemoglobins that were originally developed as blood substitutes but have been extended to a variety of hypoxic clinical situations due to their ability to release oxygen. Compared with traditional preservation protocols, the addition of HBOCs to traditional preservation protocols provides more oxygen to organs to meet their energy metabolic needs, prolong preservation time, reduce ischemia-reperfusion injury to grafts, improve graft quality, and even increase the number of transplantable donors. The focus of the present study was to review the potential applications of HBOCs in solid organ preservation and provide new approaches to understanding the mechanism of the promising strategies for organ preservation.

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.

Therapeutic Potential of Hemoglobin Derived from the Marine Worm Arenicola marina (M101): A Literature Review of a Breakthrough Innovation.

Oxygen (O2) is indispensable for aerobic respiration and cellular metabolism. In case of injury, reactive oxygen species are produced, causing oxidative stress, which triggers cell damaging chemical mediators leading to ischemic reperfusion injuries (IRI). Sufficient tissue oxygenation is necessary for optimal wound healing. In this context, several hemoglobin-based oxygen carriers have been developed and tested, especially as graft preservatives for transplant procedures. However, most of the commercially available O2 carriers increase oxidative stress and show some adverse effects. Interestingly, the hemoglobin derived from the marine lugworm Arenicola marina (M101) has been presented as an efficient therapeutic O2 carrier with potential anti-inflammatory, anti-bacterial, and antioxidant properties. Furthermore, it has demonstrated promise as a supplement to conventional organ preservatives by reducing IRI. This review summarizes the properties and various applications of M101. M101 is an innovative oxygen carrier with several beneficial therapeutic properties, and further research must be carried out to determine its efficacy in the management of different pathologies.

Ferrous hemoglobin and hemoglobin-based oxygen carriers acting as a peroxidase can inhibit oxidative damage to endothelial cells caused by hydrogen peroxide.

Oxidative damage caused by the ferryl hemoglobin is one of the major clinical adverse reactions of hemoglobin-based oxygen carriers (HBOCs), while the production of reactive oxygen species in a pathological state can oxidize hemoglobin (HbFe2+ ) to ferryl Hb, which can then enter the pseudoperoxidase cycle, making hemoglobin highly toxic. In this study, we found that ferrous hemoglobin and polymerized porcine hemoglobin (one of the HBOCs) have the peroxidase activity different from the pseudoperoxidase activity of ferric hemoglobin. Ferrous hemoglobin can catalyze the reaction of tyrosine (Tyr) with hydrogen peroxide. In addition, the results also indicated that ferrous hemoglobin and pPolyHb have a strong inhibitory effect on the pseudoperoxidase activity of ferric hemoglobin. Therefore, hydrogen peroxide was consumed in a large amount, which greatly prevented hemoglobin from becoming oxidized and entering the pseudoperoxidase cycle, thus inhibiting ferryl Hb toxicity. We further cultured human umbilical vein endothelial cells and monitored cell morphology, viability, cell cycle, apoptosis, lactate dehydrogenase (LDH) release, and malondialdehydes (MDAs) formation when incubated with H2 O2 , Tyr, and HbFe2+ . HbFe2+ and pPolyHb reduced cell cycle arrest, apoptosis, LDH release, and MDA formation. These results showed that reducing oxidative damage induced by H2 O2 and converted hemoglobin from a molecule that is toxic to one that inhibits oxidative damage, suggesting a new strategy for development of a safer HBOCs.

Determination of Methemoglobin in Hemoglobin Submicron Particles Using NMR Relaxometry.

Methemoglobin (MetHb) is a hemoglobin (Hb) derivative with the heme iron in ferric state (Fe3+), unable to deliver oxygen. Quantification of methemoglobin is a very important diagnostic parameter in hypoxia. Recently, novel hemoglobin microparticles (Hb-MP) with a narrow size distribution around 700 nm, consisting of cross-linked Hb were proposed as artificial oxygen carriers. The cross-linking of Hb by glutaraldehyde (GA) generates a certain amount of MetHb. Due to the strong light scattering, quantitative determination of MetHb in Hb-MP suspensions by common spectrophotometry is not possible. Here, we demonstrate that 1H2O NMR relaxometry is a perfect tool for direct measurement of total Hb and MetHb concentrations in Hb-MP samples. The longitudinal relaxation rate 1/T1 shows a linear increase with increasing MetHb concentration, whereas the transverse relaxation rate 1/T2 linearly increases with the total Hb concentration. In both linear regressions the determination coefficient (R2) is higher than 0.99. The method does not require time-consuming pretreatment or digestion of the particles and is not impaired by light scattering. Therefore, it can be established as the method of choice for the quality control of Hb-MP and similar hemoglobin-based oxygen carriers in the future.