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.

Tracking Research on Hemoglobin-Based Oxygen Carriers: A Scientometric Analysis and In-Depth Review.

Numerous studies on the formulation and clinical applications of novel hemoglobin-based oxygen carriers (HBOCs) are reported in the scientific literature. However, there are fewer scientometric analysis related to HBOCs. Here, we illustrate recent studies on HBOCs using both a scientometric analysis approach and a scope review method. We used the former to investigate research on HBOCs from 1991 to 2022, exploring the current hotspots and research trends, and then we comprehensively analyzed the relationship between concepts based on the keyword analysis. The evolution of research fields, knowledge structures, and research topics in which HBOCs located are revealed by scientometric analysis. The elucidation of type, acting mechanism, potential clinical practice, and adverse effects of HBOCs helps to clarify the prospects of this biological agent. Scientometrics analyzed 1034 publications in this research field, and these findings provide a promising roadmap for further study.

Research Progress in Oxygen Carrier Design and Application.

Ischemia or hypoxia can lead to pathological changes in the metabolism and function of tissues and then lead to various diseases. Timely and effective blood resuscitation or improvement of hypoxia is very important for the treatment of diseases. However, there is a need to develop stable, nontoxic, and immunologically inert oxygen carriers due to limitations such as blood shortages, different blood types, and the risk of transmitting infections. With the development of various technologies, oxygen carriers based on hemoglobin and perfluorocarbon have been widely studied in recent years. This paper reviews the development and application of hemoglobin and perfluorocarbon oxygen carriers. The design of oxygen carriers was analyzed, and their application as blood substitutes or oxygen carriers in various hypoxic diseases was discussed. Finally, the characteristics and future research of ideal oxygen carriers were prospected to provide reference for follow-up research.

Artificial Blood Development Implications for Military Medicine.

Massive hemorrhaging remains the most common cause of preventable battlefield deaths. Blood used for trauma care requires a robust donation network, capacity for long-term storage, and extensive and accurate testing. Bioengineering technologies could offer a remedy to these constraints in the form of blood substitutes-fluids that could be transfused into patients to provide oxygen, carry away waste, and aid in coagulation-that would be used in prolonged casualty care and in far-forward settings, overcoming the obstacles of distance and time. The different molecular properties of red blood cells (RBCs), blood substitutes, and platelet replacements contribute to their respective utilities, and each type is currently represented in ongoing clinical trials. Hemoglobin oxygen carriers (HBOCs) are the most advanced RBC replacements, many of which are currently being evaluated in clinical trials in the United States and other countries. Despite recent advancements, challenges remaining in the development of blood alternatives include stability, oxygen capacity, and compatibility. The continued research and investment in new technologies has the potential to significantly benefit the treatment of life-threatening emergency injuries, both on the battlefield and in the civilian sector. In this review, we discuss military blood-management practices and military-specific uses of individual blood components, as well as describe and analyze several artificial blood products that could be options for future battlefield use.

Selective Attachment of Polyethylene Glycol to Hemerythrin for Potential Use in Blood Substitutes.

Due to its ability to reversibly bind O2, alongside a relatively low redox reactivity and a limited cytotoxicity, the oxygen-carrying protein hemerythrin has been considered as an alternative to hemoglobin in preparing blood substitutes. In order to increase the hydrodynamic volume and lower antigenicity, two site-directed variants, H82C and K92C, were engineered that contained a single cysteine residue on the surface of each hemerythrin octamer for the specific attachment of polyethylene glycol (PEG). A sulfhydryl-reactive PEGylation reagent with a 51.9 Å spacer arm was used for selective cysteine derivatization. The mutants were characterized by UV-vis spectroscopy, size-exclusion chromatography, oxygen affinity, and autooxidation rate measurements. The H82C variant showed altered oligomeric behavior compared to the wild-type and was unstable in the met form. The PEGylated K92C variant is reasonably stable, displays an oxygen affinity similar to that of the wild-type, and shows an increased rate of autoxidation; the latter disadvantage may be counteracted by further chemical modifications.

In vitro study on the effect of fibrinogen γ-chain peptide-coated ADP-encapsulated liposomes on postcardiopulmonary bypass coagulopathy using patient blood.

BACKGROUND: Fibrinogen γ-chain peptide-coated, adenosine 5'-diphosphate (ADP)-encapsulated liposomes (H12-ADP-liposomes) are potent hemostatic adjuvants that promote platelet thrombi formation at bleeding sites. Although we have reported the efficacy of these liposomes in a rabbit model of cardiopulmonary bypass coagulopathy, we are yet to address the possibility of their hypercoagulative potential, especially in human beings. OBJECTIVES: Considering its future clinical applications, we herein investigated the safety of using H12-ADP-liposomes in vitro using blood samples from patients who had received platelet transfusion after cardiopulmonary bypass surgeries. METHODS: Ten patients receiving platelet transfusions after cardiopulmonary bypass surgery were enrolled. Blood samples were collected at the following 3 points: at the time of incision, at the end of the cardiopulmonary bypass, and immediately after platelet transfusion. After incubating the samples with H12-ADP-liposomes or phosphate-buffered saline (PBS, as a control), blood coagulation, platelet activation, and platelet-leukocyte aggregate formation were evaluated. RESULTS: Patients' blood incubated with H12-ADP-liposomes did not differ from that incubated with PBS in coagulation ability, degree of platelet activation, and platelet-leukocyte aggregation at any of the time points. CONCLUSION: H12-ADP-liposomes did not cause abnormal coagulation, platelet activation, or platelet-leukocyte aggregation in the blood of patients who received platelet transfusion after a cardiopulmonary bypass. These results suggest that H12-ADP-liposomes could likely be safely used in these patients, providing hemostasis at the bleeding sites without causing considerable adverse reactions. Future studies are needed to ensure robust safety in human beings.

Construction and Evaluation of Zeolitic Imidazolate Framework-Encapsulated Hemoglobin Microparticles as Oxygen Carriers.

Artificial oxygen carriers, such as favorably hemoglobin-based oxygen carriers, have received considerable attention due to some drawbacks of human donor blood. Among all oxygen carriers, the metal organic framework (MOF) exhibits excellent oxygen-carrying capacity due to its good encapsulation efficiency and competitive biocompatibility. Recently, zeolitic imidazolate frameworks (ZIFs) with unique structure have attracted much attention due to their outstanding solvothermal stability. Notably, ZIF-8, the prototypical ZIF, has been utilized to load hemoglobin (Hb) as a potential blood substitute. In this work, another ZIF material, which possesses a high oxygen binding/release capability, suitable safety profile, high stability, and efficiency as a potential oxygen carrier, was used to encapsulate Hb in an environment-friendly condition.

Examination of central nervous system by functional observation battery after massive intravenous infusion of carbon monoxide-bound and oxygen-bound hemoglobin vesicles in rats.

Carbon monoxide (CO) is known as a toxic gas inducing "CO poisoning", which acutely affects the central nervous system (CNS) and which persistently affects brain functions depending on the exposure time and CO concentration. By contrast, in pathological rodent models, intravenous infusion of CO-bound hemoglobin vesicles (CO-HbV) has shown various beneficial effects such as anti-oxidative and anti-inflammatory reactions. This study assessed effects of CO-HbV infusion on CNS using a functional observation battery, sensory reflexes, grip strength, and landing foot splay measurements. The test fluids were CO-HbV and O2-bound HbV (O2-HbV) suspended in saline ([Hb] â€‹= â€‹10 â€‹g/dL), and saline alone for comparison. The rats received either 16 or 32 â€‹mL/kg of fluid intravenously at 1.5 â€‹mL/min/kg. Observations were made before infusion, and at 5 â€‹min, 4, 8, 24, 48 and 72 â€‹h after infusion. Massive doses of 16 and 32 â€‹mL/kg respectively corresponded to about 29 and 57% of the whole circulating blood volume (56 â€‹mL/kg). No toxicological effect was observed in any measurement item for any group in comparison to the control saline infusion group. Histopathological examination of hippocampal tissue at 14 days after infusion showed the number of necrotic cells to be minimal. Results obtained from rats in this experiment suggest that the massive intravenous infusion of CO-HbV yields beneficial anti-oxidative and anti-inflammatory effects without showing CO-poisoning-related symptoms of CNS damage.

Oxidation reactions of cellular and acellular hemoglobins: Implications for human health.

Oxygen reversibly binds to the redox active iron, a transition metal in human Hemoglobin (Hb), which subsequently undergoes oxidation in air. This process is akin to iron rusting in non-biological systems. This results in the formation of non-oxygen carrying methemoglobin (ferric) (Fe3+) and reactive oxygen species (ROS). In circulating red blood cells (RBCs), Hb remains largely in the ferrous functional form (HbF2+) throughout the RBC's lifespan due to the presence of effective enzymatic and non-enzymatic proteins that keep the levels of metHb to a minimum (1%-3%). In biological systems Hb is viewed as a Fenton reagent where oxidative toxicity is attributed to the formation of a highly reactive hydroxyl radical (OH•) generated by the reaction between Hb's iron (Fe2+) and hydrogen peroxide (H2O2). However, recent research on both cellular and acellular Hbs revealed that the protein engages in enzymatic-like activity when challenged with H2O2, resulting in the formation of a highly reactive ferryl heme (Fe4+) that can target other biological molecules before it self-destructs. Accumulating evidence from several in vitro and in vivo studies are summarized in this review to show that Hb's pseudoperoxidase activity is physiologically more dominant than the Fenton reaction and it plays a pivotal role in the pathophysiology of several blood disorders, storage lesions associated with old blood, and in the toxicity associated with the infusion of Hb-derived oxygen therapeutics.

Facing Trauma and Surgical Emergency in Space: Hemorrhagic Shock.

Although the risk of trauma in space is low, unpredictable events can occur that may require surgical treatment. Hemorrhage can be a life-threatening condition while traveling to another planet and after landing on it. These exploration missions call for a different approach than rapid return to Earth, which is the policy currently adopted on the International Space Station (ISS) in low Earth orbit (LEO). Consequences are difficult to predict, given the still scarce knowledge of human physiology in such environments. Blood loss in space can deplete the affected astronaut's physiological reserves and all stored crew supplies. In this review, we will describe different aspects of hemorrhage in space, and by comparison with terrestrial conditions, the possible solutions to be adopted, and the current state of the art.

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.

Research of storable and ready-to-use artificial red blood cells (hemoglobin vesicles) for emergency medicine and other clinical applications.

Hemoglobin (Hb) is the most abundant protein in blood, with concentration of about 12-15 g/dl. The highly concentrated Hb solution (35 g/dl) is compartmentalized in red blood cells (RBCs). Once Hb is released from RBCs by hemolysis during blood circulation, it induces renal and cardiovascular toxicities. To date, hemoglobin-based oxygen carriers of various types have been developed as blood substitutes to mitigate the Hb toxicities. One method is Hb encapsulation in phospholipid vesicles (liposomes). Although the Hb toxicity can be shielded, it is equally important to ensure the biocompatibility of the liposomal membrane. We have developed Hb-vesicles (HbV). A new encapsulation method using a rotation-revolution mixer which enabled efficient production of HbV with a high yield has considerably facilitated R&D of HbV. Along with our academic consortium, we have studied the preclinical safety and efficacy of HbV extensively as a transfusion alternative, and finally conducted a phase I clinical trial. Moreover, carbonyl-HbV and met-HbV are developed respectively for an anti-inflammatory and anti-oxidative agent and an antidote for poisons. This review paper specifically presents past trials of liposome encapsulated Hb, biocompatible lipid bilayer membranes, and efficient HbV preparation methods, in addition to potential clinical applications of HbV based on results of our in vivo studies.

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.

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.

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.

Preparation of Artificial Red Blood Cells (Hemoglobin Vesicles) Using the Rotation-Revolution Mixer for High Encapsulation Efficiency.

Hemoglobin vesicles (Hb-V) are artificial red blood cells encapsulating highly concentrated hemoglobin (Hb) in liposomes comprising phospholipids, cholesterol, negatively charged lipids, and polyethylene glycol (PEG)-conjugated phospholipids. Safety and efficacy of Hb-V as a transfusion alternative have been extensively studied. For this study, we prepared Hb-V using the kneading method with a rotation-revolution mixer as an alternative to the conventional extrusion method. We optimized the kneading operation parameters to obtain Hb-V with a high yield. Results show that the Hb encapsulation efficiency was increased dramatically up to 74.2%, which is higher than that of the extrusion method (20%) because the kneading method enabled mixing of a highly concentrated carbonylhemoglobin (HbCO) solution (40 g/dL) and a considerably large amount of powdered lipids in only 10 min. The high viscosity of the Hb-lipid mixture paste (ca. 103-105 cP) favorably induces frictional heat by kneading and increases the paste temperature (ca. 60 °C), which facilitates lipid dispersion and liposome formation. During the kneading operation using a thermostable HbCO solution, Hb denaturation was prevented. Hb-V prepared using this method showed no marked changes in particle sizes, Hb denaturation, or Hb leakage from liposomes during two years of long-term storage-stability tests. Collectively, these results demonstrate that the kneading method using a rotation-revolution mixer shows good potential as a new method to produce Hb-V.

Development of a Lyophilization Process for Long-Term Storage of Albumin-Based Perfluorodecalin-Filled Artificial Oxygen Carriers.

Every day, thousands of patients receive erythrocyte concentrates (ECs). They are indispensable for modern medicine, despite their limited resource. Artificial oxygen carriers (AOCs) represent a promising approach to reduce the need for ECs. One form of AOCs is perfluorodecalin-filled albumin-based nanocapsules. However, these AOCs are not storable and need to be applied directly after production. In this condition, they are not suitable as a medicinal product for practical use yet. Lyophilization (freeze drying) could provide the possibility of durable and applicable nanocapsules. In the present study, a suitable lyophilization process for perfluorodecalin-filled nanocapsules was developed. The nanocapsules were physicochemically characterized regarding capsule size, polydispersity, and oxygen capacity. Even though the perfluorodecalin-filled albumin-based nanocapsules showed a loss in oxygen capacity directly after lyophilization, they still provided a remarkable residual capacity. This capacity did not decline further for over two months of storage. Furthermore, the nanocapsule size remained unaltered for over one year. Therefore, the AOCs were still applicable and functional after long-term storage due to the successful lyophilization.

19F Magnetic Resonance Imaging and Spectroscopy in Neuroscience.

1H magnetic resonance imaging (MRI) has established itself as a key diagnostic technique, affording the visualization of brain anatomy, blood flow, activity and connectivity. The detection of other atoms (e.g. 19F, 23Na, 31P), so called hetero-nuclear MRI and spectroscopy (MRS), provides investigative avenues that complement and extend the richness of information that can be gained from 1H MRI. Especially 19F MRI is increasingly emerging as a multi-nuclear (1H/19F) technique that can be exploited to visualize cell migration and trafficking. The lack of a 19F background signal in the brain affords an unequivocal detection suitable for quantification. Fluorine-based contrast material can be engineered as nanoemulsions, nanocapsules, or nanoparticles to label cells in vitro or in vivo. Fluorinated blood substitutes, typically nanoemulsions, can also carry oxygen and serve as a theranostic in poorly perfused brain regions. Brain tissue concentrations of fluorinated pharmaceuticals, including inhalation anesthetics (e.g. isoflurane) and anti-depressants (e.g. fluoxetine), can also be measured using MRS. However, the low signal from these compounds provides a challenge for imaging. Further methodological advances that accelerate signal acquisition (e.g. compressed sensing, cryogenic coils) are required to expand the applications of 19F MR imaging to, for instance, determine the regional pharmacokinetics of novel fluorine-based drugs. Improvements in 19F signal detection and localization, combined with the development of novel sensitive probes, will increase the utility of these multi-nuclear studies. These advances will provide new insights into cellular and molecular processes involved in neurodegenerative disease, as well as the mode of action of pharmaceutical compounds.