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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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Pedro Cabrales reports financial support was provided by US Department of Defense. Pedro Cabrales reports financial support was provided by National Institutes of Health. Andre F. Palmer is an inventor on U.S. patent application PCT/US2022/041743. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Abstract: 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., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Shock Society.)

Lung transplantation is hampered by the lack of suitable donors. Previously, donors that were thought to be marginal or inadequate were discarded. However, new and exciting technology, such as ex vivo lung perfusion (EVLP), offers lung transplant providers extended assessment for marginal donor allografts. This dynamic assessment platform has led to an increase in lung transplantation and has allowed providers to use donors that were previously discarded, thus expanding the donor pool. Current perfusion techniques use cellular or acellular perfusates, and both have distinct advantages and disadvantages. Perfusion composition is critical to maintaining a homeostatic environment, providing adequate metabolic support, decreasing inflammation and cellular death, and ultimately improving organ function. Perfusion solutions must contain sufficient protein concentration to maintain appropriate oncotic pressure. However, current perfusion solutions often lead to fluid extravasation through the pulmonary endothelium, resulting in inadvertent pulmonary edema and damage. Thus, it is necessary to develop novel perfusion solutions that prevent excessive damage while maintaining proper cellular homeostasis. Here, we describe the application of a polymerized human hemoglobin (PolyhHb)-based oxygen carrier as a perfusate and the protocol in which this perfusion solution can be tested in a model of rat EVLP. The goal of this study is to provide the lung transplant community with key information in designing and developing novel perfusion solutions, as well as the proper protocols to test them in clinically relevant translational transplant models.

Alpha-alpha diaspirin-crosslinked human hemoglobin (DCLHb or ααHb) was a promising early generation red blood cell (RBC) substitute. The DCLHb was developed through a collaborative effort between the United States Army and Baxter Healthcare. The core design feature underlying its development was chemical stabilization of the tetrameric structure of hemoglobin (Hb) to prevent Hb intravascular dimerization and extravasation. DCLHb was developed to resuscitate warfighters on the battlefield, who suffered from life-threatening blood loss. However, extensive research revealed toxic side effects associated with the use of DCLHb that contributed to high mortality rates in clinical trials. This study explores whether scavenging Hb and heme via the apohemoglobin-haptoglobin (apoHb-Hp) complex can reduce DCLHb associated toxicity. Awake Golden Syrian hamsters were equipped with a window chamber model to characterize the microcirculation. Each group was first infused with either Lactated Ringer's or apoHb-Hp followed by a hypovolemic infusion of 10% of the animal's blood volume of DCLHb. Our results indicated that animals pretreated with apoHb-Hb exhibited improved microhemodynamics vs the group pretreated with Lactated Ringer's. While systemic acute inflammation was observed regardless of the treatment group, apoHb-Hp pretreatment lessened those effects with a marked reduction in IL-6 levels in the heart and kidneys compared to the control group. Taken together, this study demonstrated that utilizing a Hb and heme scavenger protein complex significantly reduces the microvasculature effects of ααHb, paving the way for improved HBOC formulations. Future apoHb-Hp dose optimization studies may identify a dose that can completely neutralize DCLHb toxicity., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors would also like to thank the National Institutes of Health (NIH) for funding through grants R01HL162120 and R01HL159862 to A.F.P. and P.C. I.S.P., P.C. and A.F.P. are co-inventors on patent applications PCT/US2020/016267 and PCT/US2020/033836., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Normothermic ex vivo lung perfusion (EVLP) can resuscitate marginal lung allografts to increase organs available for transplantation. During normothermic perfusion, cellular metabolism is more active compared with subnormothermic perfusion, creating a need for an oxygen (O 2 ) carrier in the perfusate. As an O 2 carrier, red blood cells (RBCs) are a scarce resource and are susceptible to hemolysis in perfusion circuits, thus releasing cell-free hemoglobin (Hb), which can extravasate into the tissue space, thus promoting scavenging of nitric oxide (NO) and oxidative tissue damage. Fortunately, polymerized human Hb (PolyhHb) represents a synthetic O 2 carrier with a larger molecular diameter compared with Hb, preventing extravasation, and limiting adverse reactions. In this study, a next-generation PolyhHb-based perfusate was compared to both RBC and asanguinous perfusates in a rat EVLP model. During EVLP, the pulmonary arterial pressure and pulmonary vascular resistance were both significantly higher in lungs perfused with RBCs, which is consistent with RBC hemolysis. Lungs perfused with PolyhHb demonstrated greater oxygenation than those perfused with RBCs. Post-EVLP analysis revealed that the PolyhHb perfusate elicited less cellular damage, extravasation, iron tissue deposition, and edema than either RBCs or colloid control. These results show promise for a next-generation PolyhHb to maintain lung function throughout EVLP., Competing Interests: Disclosure: The authors have no conflicts of interest to report., (Copyright © ASAIO 2024.)

The development of haemoglobin-based oxygen carrier (HBOC) is an excellent supplement to pre-hospital emergency blood transfusions. In this study, a new type of HBOC was prepared by using human cord haemoglobin (HCHb) and glutaraldehyde (GDA) and Bis(3,5-dibromosalicyl) fumarate (DBBF) to modify (DBBF-GDA-HCHb), the changes of physicochemical indexes during its preparation were evaluated, while a traditional type of GDA-HCHb was prepared, and the oxygen-carrying capacity of two type of HBOC was evaluated by a rat model of 135.0% exchange transfusion (ET). Eighteen SD male rats were selected, and were randomly divided into control group (5.0% albumin), DBBF-GDA-HCHb group and GDA-HCHb group. The 12 h survival rate of the C group was 16.67%, and the two HBOC groups were both 83.33%. Compared with GDA-HCHb, DBBF-GDA-HCHb can reduce lactic acid content by supplying oxygen to hypoxic tissues in a more timely manner, and can also can improve the reduction of MAP due to ischaemia.

Hemoglobin wrapped covalently with poly(2-ethyl-2-oxazoline)s (POx-Hb) is characterized physicochemically and physiologically as an artificial O 2 carrier for use as a red blood cell (RBC) substitute. The POx-Hb is generated by linkage of porcine Hb surface-lysines to a sulfhydryl terminus of the POx derivative, with the average binding number of the polymers ascertained as 6. The POx-Hb shows moderately higher colloid osmotic activity and O 2 affinity than the naked Hb. Human adult HbA conjugated with POx also possesses equivalent features and O 2 binding properties. The POx-Hb solution exhibits good hemocompatibility, with no influence on the functions of platelets, granulocytes, and monocytes. Its circulation half-life in rats is 14 times longer than that of naked Hb. Hemorrhagic shock in rats is relieved sufficiently by infusion of the POx-Hb solution, as revealed by improvements of circulatory parameters. Serum biochemistry tests and histopathological observations indicate no acute toxicity or abnormality in the related organs. All results indicate that POx-Hb represents an attractive alternative for RBCs and a useful O 2 therapeutic reagent in transfusion medicine.

Due to several limitations associated with blood transfusion, such as the relatively short shelf life of stored blood, low risk of developing acute immune hemolytic reactions and graft- versus -host disease, many strategies have been developed to synthesize hemoglobin-based oxygen carriers (HBOCs) as universal red blood cell (RBC) substitutes. Recently, zeolite imidazole framework-8 (ZIF-8), a metal-organic framework, has attracted considerable attention as a protective scaffold for encapsulation of hemoglobin (Hb). Despite the exceptional thermal and chemical stability of ZIF-8, the major impediments to implementing ZIF-8 for Hb encapsulation are the structural distortions associated with loading large quantities of Hb in the scaffold as the Hb molecule has a larger hydrodynamic diameter than the pore size of ZIF-8. Therefore to reduce the structural distortion caused by Hb encapsulation, we established and optimized a continuous-injection method to synthesize nanoparticle (NP) encapsulated polymerized bovine Hb (PolybHb) using ZIF-8 precursors (ZIF-8P-PolybHb NPs). The synthesis method was further modified by adding EDTA as a chelating agent, which reduced the ZIF-8P-PolybHb NP size to <300 nm. ZIF-8P-PolybHb NPs exhibited lower oxygen affinity (36.4 ± 3.2 mm Hg) compared to unmodified bovine Hb, but was similar in magnitude to unencapsulated PolybHb. The use of the chemical cross-linker glutaraldehyde to polymerize bovine Hb resulted in the low Hill coefficient of PolybHb, indicating loss of Hb's oxygen binding cooperativity, which could be a limitation when using PolybHb as an oxygen carrier for encapsulation inside the ZIF-8 matrix. ZIF-8P-PolybHb NPs exhibited slower oxygen offloading kinetics compared to unencapsulated PolybHb, demonstrating successful encapsulation of PolybHb. ZIF-8P-PolybHb NPs also exhibited favorable antioxidant properties when exposed to H 2 O 2 . Incorporation of PolybHb into the ZIF-8 scaffold resulted in reduced cytotoxicity towards human umbilical vein endothelial cells compared to unloaded ZIF-8 NPs and ZIF-8 NPs loaded with bovine Hb. We envisage that such a monodisperse and biocompatible HBOC with low oxygen affinity and antioxidant properties may broaden its use as an RBC substitute.

Hemoglobin-based oxygen carriers (HBOCs) are being developed to overcome limitations associated with transfusion of donated red blood cells (RBCs) such as potential transmission of blood-borne pathogens and limited ex vivo storage shelf-life. Annelid erythrocruorin (Ec) derived from the worm Lumbricus terrestris (Lt) is an acellular mega-hemoglobin that has shown promise as a potential HBOC due to the large size of its oligomeric structure, thus overcoming limitations of unmodified circulating cell-free hemoglobin (Hb). With a large molecular weight of 3.6 MDa compared to 64.5 kDa for human Hb (hHb) and 144 oxygen-binding globin subunits compared to the 4 globin subunits of hHb, LtEc does not extravasate from the circulation to the same extent as hHb. LtEc is stable in the circulation without RBC membrane encapsulation and has a lower rate of auto-oxidation compared to acellular hHb, which allows the protein to remain functional for longer periods of time in the circulation compared to HBOCs derived from mammalian Hbs. Surface coatings, such as poly(ethylene glycol) (PEG) and oxidized dextran (Odex), have been investigated to potentially reduce the immune response and improve the circulation time of LtEc in vivo . Polydopamine (PDA) is a hydrophilic, biocompatible, bioinspired polymer coating used for biomedical nanoparticle assemblies and coatings and has previously been investigated for the surface coating of hHb. PDA is typically synthesized via the self-polymerization of dopamine (DA) under alkaline (pH > 8.0) conditions. However, at pH > 8.0, the oligomeric structure of LtEc begins to dissociate. Therefore, in this study, we investigated a photocatalytic method of PDA polymerization on the surface of LtEc using 9-mesityl-10-methylacridinium tetrafluoroborate (Acr-Mes) to drive PDA polymerization under physiological conditions (pH 7.4, 25 °C) over 2, 5, and 16 h in order to preserve the size and structure of LtEc. The resulting structural, biophysical, and antioxidant properties of PDA surface-coated LtEc (PDA-LtEc) was characterized using various techniques. PDA-LtEc showed an increase in measured particle size, molecular weight, and surface ζ-potential with increasing reaction time from t = 2 to 16 h compared to unmodified LtEc. PDA-LtEc reacted for 16 h was found to have reduced oxygen-binding cooperativity and slower deoxygenation kinetics compared to PDA-LtEc with lower levels of polymerization ( t = 2 h), but there was no statistically significant difference in oxygen affinity. The thickness of the PDA coating can be controlled and in turn the biophysical properties can be tuned by changing various reaction conditions. PDA-LtEc was shown to demonstrate an increased level of antioxidant capacity (ferric iron reduction and free-radical scavenging) when synthesized at a reaction time of t = 16 h compared to LtEc. These antioxidant properties may prove beneficial for oxidative protection of PDA-LtEc during its time in the circulation. Hence, we believe that PDA-LtEc is a promising oxygen therapeutic for potential use in transfusion medicine applications.

Red blood cell (RBC) substitutes tested in late-phase clinical trials contained low-molecular-weight hemoglobin species (<500 kDa), resulting in vasoconstriction, hypertension, and oxidative tissue injury; therefore, contributing to poor clinical outcomes. This work aims to improve the safety profile of the RBC substitute, polymerized human hemoglobin (PolyhHb), via in vitro and in vivo screening of PolyhHb fractionated into four molecular weight brackets (50-300 kDa [PolyhHb-B1]; 100-500 kDa [PolyhHb-B2]; 500-750 kDa [PolyhHb-B3]; and 750 kDa to 0.2 μm [PolyhHb-B4]) using a two-stage tangential flow filtration purification process. Analysis showed that PolyhHb's oxygen affinity, and haptoglobin binding kinetics decreased with increasing bracket size. A 25% blood-for-PolyhHb exchange transfusion guinea pig model suggests that hypertension and tissue extravasation decreased with increasing bracket size. PolyhHb-B3 demonstrated extended circulatory pharmacokinetics, no renal tissue distribution, no aberrant blood pressure, or cardiac conduction effects, and may therefore be appropriate material for further evaluation.

Introduction: During the preparation of polyHb-SOD-CAT-CA, the lysate was extracted by toluene. However, due to its serious toxicity and potential application in the production of dangerous explosives, the use of toluene would likely be a restriction of the industrial development of polyHb-SOD-CAT-CA. So, selecting other extraction reagents as alternatives to toluene is necessary to promote the industrialization of polyHb-SOD-CAT-CA., Aims: The objective of this study is to investigate the application of several organic solvents extraction during polyHb-SOC-CAT-CA preparation process, which include n-haxane and diethyl ether, and also to compare with the existing toluene., Methods: After extraction with different extractants, the effects of studied organic extractant on the stability of hemoglobin and enzymes include SOD, CAT and CA through monitoring the property indexes include Hb concentration, MetHb content, oxygen affinity of Hb, enzymes activities and so on., Results: The P50 and Hill coefficient of n-hexane group were higher than that in diethyl ether group and toluene group. The MetHb contents, Hb recoveries and enzymes recoveries of n-hexane group and toluene group were much better than that in diethyl ether group. The SOD activity recovery rate in n-hexane experimental group was slightly lower than that in toluene group. However, the CAT and CA recovery rate of n-hexane group was higher than that in toluene group., Conclusion: The results of this study suggested that the effects of n-hexane on the properties stability and productivity of polyHb-SOD-CAT-CA were nearly similar with that of toluene, indicating potential reliability and feasibility of n-hexane in the future research and development of polyHb- SOD-CAT-CA., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The main goal of this study was to investigate the cardioprotective properties in terms of effects on cardiodynamics of perfluorocarbon emulsion (PFE) in ex vivo-induced ischemia-reperfusion injury of an isolated rat heart. The first part of the study aimed to determine the dose of 10% perfluoroemulsion (PFE) that would show the best cardioprotective effect in rats on ex vivo-induced ischemia-reperfusion injury of an isolated rat heart. Depending on whether the animals received saline or PFE, the animals were divided into a control or experimental group. They were also grouped depending on the applied dose (8, 12, 16 ml/kg body weight) of saline or PFE. We observed the huge changes in almost all parameters in the PFE groups in comparison with IR group without any pre-treatment. Calculated in percent, dp/dt max was the most changed parameter in group treated with 8 mg/kg, while the dp/dt min, SLVP, DLVP, HR, and CF were the most changed in group treated with 16 mg/kg 10 h before ischemia. The effects of 10% PFE are more pronounced if there is a longer period of time from application to ischemia, i.e., immediate application of PFE before ischemia (1 h) gave the weakest effects on the change of cardiodynamics of isolated rat heart. Therefore, the future of PFE use is in new indications and application methods, and PFE can also be referred to as antihypoxic and antiischemic blood substitute with mild membranotropic effects., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A bovine hemoglobin (HbBv) or human adult hemoglobin (HbA) wrapped covalently by human serum albumins (HSAs), hemoglobin-albumin clusters (HbBv-HSA 3 and HbA-HSA 3 ), are artificial O 2 carriers used as a red blood cell substitute. This article describes the physicochemical properties of the HbBv-HSA 3 and HbA-HSA 3 solutions, and their abilities to restore the systemic condition after resuscitation from hemorrhagic shock in anesthetized rats. The HbBv-HSA 3 and HbA-HSA 3 , which have high colloid osmotic activity, showed equivalent solution characteristics and O 2 binding parameters. Shock was induced by 50% blood withdrawal. Rats exhibited hypotension and significant metabolic acidosis. After 15 min, the rats were administered shed autologous blood (SAB), HbBv-HSA 3 , HbA-HSA 3 , or Ringer's lactate (RL) solution. Survival rates, circulation parameters, hematological parameters, and blood gas parameters were monitored during the hemorrhagic shock and for 6 h after administration. All rats in the SAB, HbBv-HSA 3 , and HbA-HSA 3 groups survived for 6 h. The HbBv-HSA 3 and HbA-HSA 3 groups restored mean arterial pressure after the resuscitation. No remarkable difference was observed in the time courses of blood gas parameters in any resuscitated group except for the RL group. Serum biochemical tests showed increases in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the HbBv-HSA 3 and HbA-HSA 3 groups compared to the SAB group. Therefore, we observed other rats awakened after resuscitation with HbA-HSA 3 for 7 days. The blood cell count, AST, and ALT recovered to the baseline values by 7 days. All the results implied that HbBv-HSA 3 and HbA-HSA 3 clusters provide restoration from hemorrhagic shock as an alternative material for SAB transfusion., (© 2022 Wiley Periodicals LLC.)

Carbon monoxide (CO) is expected to attenuate the progression of obliterative bronchiolitis (OB), which is a serious complication after lung transplantation. However, issues in terms of feasible exogenous CO supply, such as continuousness and safety, remain unsolved. Here, we applied nano red blood cells, namely hemoglobin vesicles (Hb-V), as a CO cargo based on the biomimetic concept and investigated the therapeutic potential of CO-loaded Hb-V on OB in orthotopic tracheal transplant model mice. The CO-loaded Hb-V was comprised of negatively charged liposomes encapsulating carbonylhemoglobin with a size of ca. 220 nm. The results of histological evaluation showed that allograft luminal occlusion and fibrosis were significantly ameliorated by treatment with CO-loaded Hb-V compared to treatment with saline, cyclosporine, and Hb-V. The therapeutic effects of CO-loaded Hb-V on OB were due to the suppression of M1 macrophage activation in tracheal allografts, resulting from decreased IL-17A production. Furthermore, the expression of TNF-α and TGF-β in tracheal allografts was decreased by CO-loaded Hb-V treatment but not saline and Hb-V treatment, indicating that CO liberated from CO-loaded Hb-V inhibits epithelial-mesenchymal transition. These findings suggest that CO-loaded Hb-V exerts strong therapeutic efficacy against OB via the regulation of macrophage activation by IL-17A and TGF-β-driven epithelial-mesenchymal transition., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Blood sources used for insect colonies and their effects on fecundity and fertility have been studied in multiple mosquito species, but the effect of anticoagulants that prevent clotting of blood has received minimal attention. Here, we identify the effect two anticoagulants have on the mortality, fecundity, and fertility of Culex quinquefasciatus (Sebring and BCS strains) and Aedes aegypti Liverpool. Each mosquito species was provided with one of three treatments: direct feeding on live chicken (LC), blood from freshly exsanguinated chicken treated with heparin (EXS) or commercially purchased chicken blood treated with Alsever's solution (ART). No significant effect of treatment on mortality was observed. Both Cx. quinquefasciatus Sebring and BCS strains demonstrated a significant effect of treatment type on fecundity with the number of eggs laid for LC being 1.40-fold higher than EXS and 2.14-fold higher than ART for Sebring. For BCS strain mosquitoes, LC was 1.55-fold higher than ART, and EXS was 1.57-fold higher than ART, but there was no significant difference between LC and EXS. For Ae. aegypti mosquitoes, only a significant difference in mean egg counts was observed between LC and ART treatments, with LC laying 1.46-fold more eggs . No significant effect on fertility was observed among any mosquitoes for any treatment. These results demonstrate the negative effect of anticoagulants on the fecundity for multiple mosquito taxa. This may affect the ability of labs to produce large numbers of mosquitoes or colonize wild mosquito populations and should be taken into account when considering colony maintenance or vector biology research.

We aimed to investigate the resuscitative efficacy of hemoglobin vesicles (HbVs) as a red blood cell (RBC) substitute for the initial treatment of severe postpartum hemorrhage (PPH). Twenty-five pregnant rabbits underwent cesarean section; uncontrolled hemorrhage was induced by transecting the right uterine artery to establish a severe PPH model. During the first 30 min, all rabbits were administered 6% hydroxyethyl starch (HES) of an equivalent volume to the hemorrhage every 5 min. Thereafter, they received any of the following three isovolemic fluids for resuscitation every 5 min: RBCs with platelet-poor plasma (RBC/PPP) (n = 8), 6% HES (n = 7), or HbVs with 25% human serum albumin (n = 10). After surgical hemostasis at 60 min, survival was monitored until 12 h. No rabbits receiving only HES infusion survived beyond 6 h, whereas all rabbits receiving RBC/PPP transfusion survived. The rabbits receiving HbV infusion showed significantly higher mean arterial pressure and hemoglobin levels than the HES-receiving rabbits, and 8 of 10 rabbits survived for 6 h. The HbV group showed significantly higher survival than the HES group but worse survival than the RBC/PPP group. In conclusion, HbV infusion for severe PPH effectively prevents lethal hemorrhagic shock in a pregnant rabbit model., (© 2021. The Author(s).)

Liposome-encapsulated hemoglobin vesicles (HbV) can serve as a blood substitute with oxygen-carrying capacity comparable to that of human blood and lethal hemorrhage is associated with lethal arrhythmias. To investigate the resuscitation effect of HbV on lethal hemorrhage and anti-arrhythmogenesis, we performed optical mapping analysis (OMP) and electrophysiological study (EPS) in graded blood exchange (85% blood loss) in the rat model. We also measured cardiac autonomic activity, as assessed by heart rate variability (HRV), and changes in plasma norepinephrine and left ventricle ejection fraction (LVEF) by echocardiography. Pathological study on Connexin43 was performed. A 5% albumin (ALB group), washed rat erythrocytes (wRBC group), and HbV (HbV group) were used as a resuscitation fluid. The survival effects over 24 hours were examined. All rats died in the ALB group, whereas almost all survived for 24-hours period in wRBC and HbV groups. OMP showed impaired action potential duration dispersion (APDd) in the ALB group, whereas normal APDs in HbV and wRBC groups. Lethal arrhythmias were induced by EPS in the ALB group, but not in wRBC and HbV groups. HRV indices, LVEF, Connexin43 were preserved in HbV and wRBC groups. Lethal hemorrhage causes lethal arrhythmias in the presence of impaired APDd. HbV acutely rescues lethal hemorrhage by preventing lethal arrhythmias and preserving arrhythmogenic factors., (© 2021 International Center for Artificial Organs and Transplantation and Wiley Periodicals LLC.)

Background: The interest in vascular substitutes has recently increased. We evaluated the feasibility of using a homologous parietal peritoneum as a vascular substitute for venous reconstruction during abdominal surgery., Methods: The inferior vena cava was replaced with a homologous parietal peritoneum after cross-linking with glutaraldehyde in 36 rabbits. At 7, 14, and 28 days, the patency rate, outer and inner graft diameters, histology, and immunohistochemistry were evaluated., Results: Both the 7- and 14-day groups maintained vascular patency. Vascular patency was maintained in 3 rabbits in the 28-day group. The inner diameters of the anastomotic sites were 6.23 ± 0.18, 5.64 ± 0.16, and 2.34 ± 0.21 mm in the 7-day, 14-day, and 28-day groups, respectively. The midpoint inner diameters of the homologous parietal peritoneum grafts were 624 ± 0.46, 5.74 ± 0.26, and 2.14 ± 0.28 mm in each group, respectively. Endothelial cell proliferation on the homologous parietal peritoneum graft surfaces in all groups was based on the histological findings from the first group. Multiple neovascularizations of the homologous parietal peritoneum graft were found in the 14- and 28-day groups, indicating neo-media formation. Acute inflammation appeared to progress to the entire layer of the homologous parietal peritoneum graft without an intraluminal thrombus, but the graft was patent in the 14-day group. In the 28-day group, 6 rabbits showed near-total occlusion and a thrombus formed in the homologous parietal peritoneum graft at the anastomosis site with severe stricture; however, the rabbits were alive and had collateral vessel formation., Conclusion: Using homologous parietal peritoneum is feasible for venous reconstruction in abdominal surgery., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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 (HbFe 2+ ) 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 H 2 O 2 , Tyr, and HbFe 2+ . HbFe 2+ and pPolyHb reduced cell cycle arrest, apoptosis, LDH release, and MDA formation. These results showed that reducing oxidative damage induced by H 2 O 2 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., (© 2021 International Center for Artificial Organs and Transplantation and Wiley Periodicals LLC.)

Background: Hemoglobin (Hb)-based oxygen (O 2 ) carriers (HBOCs) are being developed as alternatives to red blood cells and blood when these products are unavailable. Clinical trials of previous HBOC generations revealed side effects, including hypertension and vasoconstriction, that were not observed in preclinical studies. Large molecular weight (MW) polymerized bovine Hb (PolybHb) represents a new class of HBOC with promising results. We evaluated the safety profile of PolybHb after an exchange transfusion (ET) in guinea pigs (GPs). This study compares changes in indices of cardiac, inflammatory, and organ function after ET with high (R-state) and low (T-state) O 2 affinity PolybHb with high MW., Study Design and Methods: Guinea pigs underwent a 20% ET with PolybHb. To assess the implication of PolybHb ET on the microcirculation, hamsters instrumented with a dorsal window chamber were subjected to a similar volume ET., Results: T and R-state PolybHb did not induce significant alterations in cardiac function. T-state PolybHb induced mild vasoconstriction shortly after transfusion, while R-state did not have acute effects on microvascular tone., Conclusion: Large MW PolybHbs were found to be safe and efficacious in increasing O 2 carrying capacity and the O 2 affinity of the PolybHb did not affect O 2 delivery or extraction by tissues in relevant preclinical models. In conclusion, these results suggest that both T-state and R-state PolybHb are safe and do not impair O 2 delivery. The results are encouraging and support further evaluation of high MW PolybHbs and their future feasibility compared to allogenic blood in a trauma model., (© 2020 AABB.)

Medical support for traumatic haemorrhage is lacking for far-forward combat units. VIR-HBOC (haemoglobin-based oxygen carrier) is a novel biological therapeutic under development as a field-stable resuscitation fluid. HBOCs have a long history of complications, chief among them is vasoconstrictive hypertension, which must be resolved before efficacy testing. As such, VIR-HBOC was compared against Lactated Ringers (LRS; vehicle) and a cross-linked haemoglobin (ααHb; a known vasoactive HBOC) in a rat topload model. Twenty-three male, Sprague Dawley rats were randomly assigned to receive a 10% infusion (estimated total blood volume) of one test article while normotensive and under anaesthesia. Cardiovascular, blood chemistry and oximetry, microvascular arteriolar diameters, and interstitial tissue oxygenation parameters were measured. Circulatory half-life was calculated by plasma total haemoglobin. Treatment with ααHb caused immediate increases in mean arterial pressure compared to LRS and VIR-HBOC groups, and corresponding arteriolar vasoconstriction ( p  < .05), which did not occur for LRS or VIR-HBOC. Circulatory half-lives for VIR-HBOC and ααHb were calculated as 340 and 157 min, respectively. This first report of VIR-HBOC showed no evidence of a hypertensive or vasoactive effect. It was well-tolerated over the eight-hour time course of this topload model, which warrants further investigation in studies of haemorrhagic shock.

Volume resuscitation is an important early treatment for haemorrhagic shock. Haemoglobin-based oxygen carrier (HBOC) can expand the volume and provide oxygen for tissues. Vascular leakage is common complication in the process of haemorrhagic shock and resuscitation. The aim of this study was to observe the effects of HBOC (a bovine-derived, cross-linked tetramer haemoglobin oxygen-carrying solution, 0.5 g/L) on vascular leakage in rats after haemorrhagic shock. A haemorrhagic shock rat model and hypoxic vascular endothelial cells (VECs) were used. The role of intercellular junctions and endothelial glycocalyx in the protective effects of HBOC and the relationship with mitochondrial function were analysed. After haemorrhagic shock, the pulmonary vascular permeability to FITC-BSA, Evans Blue was increased, endothelial glycocalyx was destroyed and the expression of intercellular junction proteins was decreased. After haemorrhagic shock, a small volume of HBOC solution (6 ml/kg) protected pulmonary vascular permeability, increased structural thickness of endothelial glycocalyx, the levels of its components and increased expression levels of the intercellular junction proteins ZO-1, VE-cadherin and occludin. Moreover, HBOC significantly increased oxygen delivery and consumption in rats, improved VEC mitochondrial function and structure. In conclusion, HBOC mitigates endothelial leakage by protecting endothelial glycocalyx and intercellular junctions through improving mitochondrial function and tissue oxygen delivery.

The reliance on blood is a limiting factor for mass rearing of mosquitoes for Sterile-Insect-Technique (SIT) and other mosquito-based control strategies. To solve this problem, we have developed SkitoSnack, a formulated diet for Aedes aegypti (L) mosquitoes, as an alternative for vertebrate blood. Here we addressed the question if long-term yellow fever mosquito culture with SkitoSnack resulted in changed life history traits and fitness of the offspring compared to blood-raised mosquitoes. We also explored if SkitoSnack is suitable to raise Asian tiger mosquitos, Aedes albopictus (L.), and the human bed bug, Cimex lectularius (L). We measured life history traits for 30th generation SkitoSnack-raised Ae. aegypti and 11th generation SkitoSnack-raised Ae. albopictus, and compared them with control mosquitoes raised on blood only. We compared meal preference, flight performance, and reproductive fitness in Ae. aegypti raised on SkitoSnack or blood. We also offered SkitoSnack to bed bug nymphs. We found that long-term culture with SkitoSnack resulted in mosquitoes with similar life history traits compared to bovine blood-raised mosquitoes in both species we studied. Also, Ae. aegypti mosquitoes raised on SkitoSnack had similar flight performance compared to blood raised mosquitoes, were still strongly attracted by human smell and had equal mating success. Minimal feeding occurred in bed bugs. Our results suggest that long-term culture with the blood-meal replacement SkitoSnack results in healthy, fit mosquitoes. Therefore, artificial diets like SkitoSnack can be considered as a viable alternative for vertebrate blood in laboratory mosquito culture as well as for mosquito mass production for Sterile-Insect-Technique mosquito control interventions. SkitoSnack was not suitable to induce engorgement of bed bugs., Competing Interests: The authors have declared that no competing interests exist.

Backgrounds: Intravenous transfusion sometimes encounters difficulty under prehospital conditions when peripheral vessels are collapsed and inaccessible. We investigated whether the cellular type hemoglobin-based oxygen carriers (Hemoglobin Vesicles: HbVs) allow intraosseous administration into blood circulation for the resuscitation of rabbits with severe hemorrhagic shock., Study Design and Methods: New Zealand white rabbits (2.5 kg average) were set in severe hemorrhagic shock [mean arterial pressure (MAP): 21 ± 2 mm Hg, Hb 5.1 ± 0.8 g/dL]. Immediately thereafter, 12 mL/kg of HbVs, 5% human serum albumin (HSA), autologous whole blood (WB), stored red blood cells (RBCs) or 36 mL/kg of Lactated Ringer's (LR) were intraosseously transfused, followed by an additional intraosseous transfusion with 8 mL/kg of HSA (following HbV, HSA or stored RBC transfusion), or WB or 24 mL/kg of LR (following LR transfusion), respectively., Results: Intraosseous transfusion of HbVs increased MAP (48 ± 9 mm Hg) and improved hypohemoglobinemia (7.1 ± 0.6 g/dL) as well as WB or RBC transfusion. In contrast, neither HSA nor LR improved hemodynamics or Hb levels. Seven out of 10 rabbits receiving HbVs survived for 24 hours, while only one out of 10 rabbits receiving LR survived (WB and RBC; 100% survivals, HSA; 30% survival)., Conclusions: Intraosseous infusion of HbVs might be an effective initial treatment to maintain hemodynamics during acute hemorrhagic shock. This approach could be used in emergency situations in which access to peripheral vessels is difficult., (© 2020 AABB.)

Background: Haemoglobin vesicles (HbVs) are red blood cell (RBC) substitutes with a phospholipid bilayer membrane and a polyethylene modified surface (diameter=250 nm; P 50 =28 Torr). They can be preserved for years and can be used in patients of all blood types without the risk of infection. Their oxygen affinity can be modified by changing the allosteric effectors., Methods: Left pneumonectomy was performed under mechanical ventilation on rats, followed by rapid exsanguination of ~30% of the total circulating blood volume. Rat RBCs shed in 5% human serum albumin (HSA) solution (rat RBC), HbV with high oxygen affinity in 5% albumin solution (low-P 50 HbV, P 50 =9 Torr), normal HbV suspended in 5% albumin (HbV, P 50 =28 Torr) or 5% HSA was infused for resuscitation. Haemodynamics and oxygenation were evaluated., Results: Systemic arterial blood pressure significantly decreased after exsanguination and increased after each infusion. In the HbV, low-P 50 HbV and rat RBC groups, all rats were liberated from mechanical ventilation and blood pressure was stabilised, whereas 50% of the rats in the HSA group died within 1 hour after weaning from mechanical ventilation. The PaO 2 in arterial blood for 1 hour after liberation from mechanical ventilation in the rat RBC, HbV and low-P 50 HbV groups was 59.4±12.5, 58.3±10.1 and 70.5±14.5 mm Hg, respectively. The PaO 2 in the low-P 50 HbV group was significantly higher than those in the rat RBC and HbV groups (p=0.05 for both). Serum lactate elevations due to hypoxic damage were minimised by HbV, low-P 50 HbV as well as rat RBCs., Conclusions: The oxygen-carrying ability of HbV was comparable to that of rat RBCs, even under impaired lung function after pneumonectomy. HbVs with high oxygen affinity may have more beneficial effects on oxygenation in pulmonary resection., Competing Interests: Competing interests: HS is an inventor on patents related to the production and utilisation of haemoglobin vesicles., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Despite all the attempts to create advanced hemoglobin (Hb)-based oxygen carriers (HBOCs) employing an encapsulation platform, major challenges including attaining a high Hb loading and long circulation times still need to be overcome. Herein, the fabrication, for the first time, of nanoparticles fully made of Hb (Hb-NPs) employing the electrospray technique is reported. The Hb-NPs are then coated by antioxidant and self-polymerized poly(dopamine) (PDA) to minimize the conversion of Hb into nonfunctional methemoglobin (metHb). The PDA shell is further functionalized with poly(ethylene glycol) (PEG) to achieve stealth properties. The results demonstrate that the as-prepared Hb-NPs are hemo- and biocompatible while offering antioxidant protection and decreasing the formation of metHb. Additionally, decoration with PEG results in decreased protein adsorption onto the Hb-NPs surface, suggesting a prolonged retention time within the body. Finally, the Hb-NPs also preserve the reversible oxygen-binding and releasing properties of Hb. All in all, within this study, a novel HBOCs with high Hb content is fabricated and its potential as an artificial blood substitute is evaluated., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The effects of liposome-encapsulated hemoglobin with high O 2 affinity (h-LEH), an artificial O 2 carrier in skeletal muscle, were studied by in situ fatigue resistance test in fast-type plantaris (PLT) and slow-type soleus (SOL) muscles with or without ischemia in the rat. The distal tendons of PLT and SOL muscles were isolated in situ and individually attached to the force transducers to record the developed tension in response to stimuli (80 Hz tetanus train, 1.5 minutes) to the ipsilateral sciatic nerve. The fatigue resistance test (five sets separated by 2-minute rests) was evaluated in terms of tension attenuation (fatigue) from the initial to the last tension (A) during each set, attenuation of the initial (B) or last tension (C) in each set, as compared to the first set in the presence or absence of ischemia or h-LEH (10 mL/kg). While ischemia significantly enhanced fatigue only in PLT, h-LEH showed no effect regardless of the perfusion pattern (normal/ischemia) or muscle-type (PLT/SOL) during each set (A). In parameter (B), set-by-set fatigue development was observed in PLT, whereas h-LEH-SOL showed a trend of advanced fatigue resistance. Such trends became clear in the parameter C (last tension), because h-LEH-SOL exerted, rather than decreased, the tension enhancement regardless of the presence or absence of ischemia, whereas there were no h-LEH effects in PLT. In addition, faster recovery of the nicotinamide adenine dinucleotide content in the muscle after 10 minutes of all fatigue tests was observed in h-LEH-SOL, while saline-SOL still showed a significantly higher value than that of control. These results suggested that additional O 2 supply by h-LEH may accelerate the tricarboxylic acid cycle/electron transport chain in slow-type aerobic SOL muscle containing abundant mitochondria and contribute to the faster removal of muscle fatigue substances such as lactate., (© 2019 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Background: Cardiac surgery using cardiopulmonary bypass carries a high risk of bleeding and need for blood transfusion. Blood administration is associated with increased rates of morbidity and mortality. Perioperatively, strategies are often employed to reduce blood transfusions in high-risk patients or in situations where blood transfusion is contraindicated. Normovolemic hemodilution is a blood conservation technique used during cardiac surgery that involves replacement of blood with fluids. SANGUINATE ® (PEGylated carboxyhemoglobin bovine) is a novel hemoglobin-based oxygen carrier that can deliver oxygen effectively to tissues in the presence of severe hypoxia. The use of a hemoglobin-based oxygen carrier during hemodilution may augment tissue oxygen delivery and reduce blood transfusion., Methods: Six standardized cardiopulmonary bypass runs simulating normovolemic hemodilution using varying proportions of bovine whole blood and SANGUINATE were performed. Pump speed, flow rate, line pressures, hemoglobin concentration, oxygenation, and degree of anticoagulation were assessed at regular intervals. Membrane oxygenators and arterial line filters were inspected for evidence of clotting following each run., Results: Increases in the pressure drop across the membrane oxygenator were detected during runs 5 and 6. Median activated clotting time values were able to be maintained at goal during the runs, and SANGUINATE did not appear to be thrombogenic. Hemoglobin concentration decreased following the addition of SANGUINATE. Oxygenation was maintained during all runs that included SANGUINATE., Conclusion: SANGUINATE does not impact the performance of the cardiopulmonary bypass circuit in a bovine whole blood model. The results support further evaluation of SANGUINATE in the setting of normovolemic hemodilution and cardiopulmonary bypass.

Gradual rewarming from hypothermic to normothermic is a novel perfusion modality with superior outcome to sudden rewarming to normothermic. However, the identification of an oxygen carrier that could function at a temperature range from 4 to 7°C or whether it is necessary to use oxygen carrier during kidney rewarming, remains unresolved. This study was designed to test the use of a hemoglobin-based oxygen carrier (HBOC) during gradual kidney rewarming as an alternative to simple dissolved oxygen. In this study, 10 rat kidneys were randomly divided into the control and the HBOC group. In the control group, no oxygen carrier was used during rewarming perfusion and the perfusion solution was oxygenated only by applying diffused carbogen flow. The protocol mimicked a donor after circulatory death (DCD) kidney transplantation, where after 30 minutes warm ischemia and 120 minutes cold storage in University of Wisconsin solution, the DCD kidneys underwent gradual rewarming from 10 to 37°C during 90 minutes with or without HBOC. This was followed by 30 minutes of warm ischemia in room temperature to mimic the anastomosis time and 120 minutes of reperfusion at 37°C to mimic the early post-transplant state of the graft. The HBOC group demonstrated superior kidney function which was highlighted by higher ultrafiltrate production, better glomerular filtration rate and improved sodium reabsorption. There was no significant difference between the 2 groups regarding the hemodynamics, tissue injury, and adenosine triphosphate levels. In conclusion, this study suggests better renal function recovery in DCD kidneys after rewarming with HBOC compared to rewarming without an oxygen carrier., (© 2019 The Authors. Artificial Organs published by Wiley Periodicals, Inc., on behalf of International Center for Artificial Organ and Transplantation (ICAOT).)

Compromised microcirculation and endothelial dysfunction are hallmarks of sickle cell disease (SCD). EAF PEG Haemoglobin (Hb) and EAF PEG Albumin (Alb) represent a novel class of semisynthetic colloidal supra plasma expanders that improve microcirculation. The therapeutic activity of supra plasma expanders to attenuate vaso-occlusion and restore the haemodynamic functions in patients with SCD has been investigated using NY1DD, a transgenic mouse model of mild SCD without anaemia. Vaso-occlusion and perturbation of haemodynamics are amplified in NY1DD by hypoxia-reoxygenation protocol. EAF P5K6 Alb and Alb T12 (Alb conjugated with 12 copies of antioxidant tempo) attenuate vaso-occlusion when infused at the start of reoxygenation. However, only EAF PEG Alb restores haemodynamics close to levels in control C57BL. EAF P5K6 Alb-T12, active plasma expander conjugated with antioxidant, completely clears vaso-occlusion and restores normal haemodynamics. EAF PEG Hb also completely clears vaso-occlusion and restores normal haemodynamics. Pretreating NY1DD with EAF PEG Hb protects it from hypoxia reoxygenation-induced damages. EAF P5K6 Alb T12 attenuates the endothelial dysfunction in S + S Antilles mice as reflected by the vasodilatory response of its arteries and arterioles to vaso-dilators. Active plasma expanders are novel therapeutics to restore normal haemodynamics in SCD patients to improve tissue oxygenation during episodes of painful vaso-occlusive crisis. Abbreviations: 2-IT: 2-immothiolane; Mal-T: 4-Maleimido tempo; Alb: human serum albumin (HSA); Alb-T12: human albumin conjugated with 12 copies of tempo; EAF: extension arm facilitated; EAF PEG Hb: extension arm facilitated PEGylated haemoglobin; EAF PEG Alb: extension arm facilitated PEGylated albumin; EAF P3K6 Hb: extension arm facilitated PEGylated haemoglobin conjugated with 6 copies of PEG3K; EAF P5K6 Alb T12: extension arm facilitated PEGylated albumin conjugated with 6 copies of PEG5K and 12 copies of tempo; Hb: haemoglobin; HAS: human serum albumin (Alb); PEG: polyethylene glycol; MP4: MalPEG Hb, is formulated at 4.2 g/dL in lactated Ringer's solution, a product of Sangart; SCD: sickle cell disease; NO: nitric oxide; SEC: size exclusive chromatography; Vrbc: red cell velocity; Q: volumetric flow rates, Q; SNP: sodium nitroprusside.

Pathological hypoxia-induced organ dysfunction contributes to the high mortality of sepsis. Because of the microcirculation dysfunction following severe sepsis, it is difficult for erythrocytes to transport oxygen to hypoxic tissues. Haemoglobin-based oxygen carriers (HBOCs) are capable of delivering oxygen to hypoxic tissues. The aim of this study is to observe the potential benefits of a novel bovine-derived, non-polymerized, cell-free HBOC solution, YQ23, on sepsis in rats. Cecum ligation and puncture was performed to induce sepsis in Sprague-Dawley rats. Effects of Lactate Ringer's solution (LR), YQ23, and whole blood on oxygen delivery and consumption, mitochondrial function, organ protection and animal survival were observed. LR failed to restore oxygen delivery and the therapeutic effects were limited, whereas low dosage of YQ23 and whole blood significantly increased the tissue oxygen delivery and consumption, improved the mitochondrial function of heart, liver, kidney and intestine, prevented the vital organs injuries and improved the animal survival. The effects of 0.15 g·kg -1 YQ23 resembled that of the whole blood. In addition, YQ23 did not induce renal toxicity, severe oxidative effect and acute vasoconstriction. Thus, YQ23 is a safe and effective resuscitation fluid for sepsis.

Hypoxia drives sickle cell disease (SCD) by inducing sickle cell haemoglobin to polymerize and deform red blood cells (RBC) into the sickle shape. A novel carboxyhaemoglobin-based oxygen carrier (PEG-COHb; PP-007) promotes unsickling in vitro by relieving RBC hypoxia. An in vivo rat model of vaso-occlusive crisis (VOC) capable of accommodating a suite of physiological and microcirculatory measurements was used to compare treatment with PEG-COHb to a non-oxygen carrying control solution (lactated ringer's [LRS]). Male Sprague-Dawley rats were anesthetized and surgically prepared to monitor microvascular interstitial oxygenation (P ISF O 2 ), cardiovascular parameters and blood chemistry. Human homozygous SCD RBCs were isolated and exchange transfused into the rats until the distal microcirculation of the exteriorized spinotrapezius muscle was hypoxic and RBC aggregates were visualized. VOC was left untreated (Sham) or treated 15 min later with PEG-COHb or LRS and observed for up to 4 h. Treatment with PEG-COHb showed better improvement of P ISF O 2 , end-point lactate, mean arterial pressure and survival duration compared to Sham and LRS. Restoring P ISF O 2 was associated with relieving the RBC aggregates driving VOC, which then affected other study metrics. Compared to LRS, PEG-COHb's oxygen-carrying properties were key to improved outcomes.

Hemoglobin-vesicles (HbVs) are artificial oxygen carriers encapsulating purified and concentrated hemoglobin solution in phospholipid vesicles (liposomes) and their safety and efficacy as a transfusion alternative have been evaluated. Because of the absence of enzymatic methemoglobin reduction system in HbV, the level of ferric methemoglobin (metHb) increases gradually after intravenous administration. Our previous studies clarified that the glycolytic electron energies, charged as NAD(P)H in red blood cells (RBC), are donated to reduce metHb compartmentalized in HbV via a water-soluble electron mediator such as methylene blue [MB; 3,7-bis(dimethylamino)phenothiazinium chloride], which freely shuttle across both RBC biomembrane and HbV lipid membrane. Herein, we tried to test repeated injections of MB after the massive HbV administration (28 mL/kg) to hemorrhagic shocked Wistar rats ( n  = 3). MB was injected (3.1 mg/kg) at 7, 24 and 48 h after HbV administration. Every MB injection showed rapid reduction of metHb and gradual reversal increase. As a result, the functional life span of HbV was significantly extended over 60 h. It is expected that further optimization of injection scheduling will decrease the total amount of MB and prolong the functional life span of HbV.

As an oxygen-transporting protein, free hemoglobin (Hb) often suffers from the disadvantage of undesirable stability and short blood circulation, which severely impairs the potential clinical applications as the blood substitute. In this work, Hb was facilely encapsulated into a kind of metal-organic frameworks (MOFs) (ZIF-8) inspired by the natural biomineralization process. The obtained ZIF-8 encapsulating Hb (ZIF-8@Hb) showed the small hydrodynamic size of 180.8 nm and neutral zeta potential of -2.1 mV by adjusting the ratio of Hb in ZIF-8 frameworks. Intriguingly, Hb encapsulated by ZIF-8 exhibited significantly enhanced stability in alkaline, oxidation, high temperature, or enzymatic environment compared with free Hb because of the excellent protective MOF coatings. More importantly, the negative charge of Hb neutralized the original positive charge of ZIF-8, which led to the better biocompatibility, lower protein adsorption, and macrophage uptake of ZIF-8@Hb than bare ZIF-8 nanoparticles. Furthermore, ZIF-8@Hb displayed extended blood circulation with the elimination half-life of 13.9 h as well as reduced nonspecific distribution in normal organs compared with free Hb or ZIF-8 nanoparticles. With the above advantages, ZIF-8@Hb showed significantly extended survival time of mice in a disease model of hemorrhagic shock compared with free Hb or bare ZIF-8 nanoparticles. Overall, this work offers a high-stable and long-circulating oxygen carrier platform, which may find wide applications as a blood substitute to treat various oxygen-relevant diseases.

Background: We previously developed substitutes for red blood cells (RBCs) and platelets (PLTs) for transfusion. These substitutes included hemoglobin vesicles (HbVs) and fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV, H12)-coated, adenosine diphosphate (ADP)-encapsulated liposomes [H12-(ADP)-liposomes]. Here, we examined the efficacy of combination therapy using these substitutes instead of RBC and PLT transfusion in a rabbit model with trauma-induced massive hemorrhage with coagulopathy., Study Design and Methods: Thrombocytopenia (PLT count approximately 40,000/μL) was induced in rabbits by repeated blood withdrawal and isovolemic transfusion with autologous RBCs. Thereafter, lethal hemorrhage was induced in rabbits by noncompressible penetrating liver injury. Subsequently, H12-(ADP)-liposomes with platelet-poor plasma (PPP), platelet-rich plasma (PRP), or PPP alone were administered to stop bleeding. Once achieving hemostasis, HbVs, allogenic RBCs, or 5% albumin were transfused into rabbits to rescue them from fatal anemia following massive hemorrhage., Results: Administration of H12-(ADP)-liposomes/PPP as well as PRP (but not PPP) effectively stopped liver bleeding (100% hemostasis). The subsequent administration with HbVs as well as RBCs after hemostasis markedly rescued rabbits from fatal anemia (75% and 70% survivals for 24 hr, respectively). In contrast, 5% albumin administration rescued none of the rabbits., Conclusion: Combination therapy with H12-(ADP)-liposomes and HbVs may be effective for damage control resuscitation of trauma-induced massive hemorrhage., (© 2019 AABB.)

Normothermic machine perfusion presents a novel platform for pretransplant assessment and reconditioning of kidney grafts. Maintaining the metabolic activity of a preserved graft at physiologic levels requires an adequate oxygen supply, typically delivered by crystalloid solutions supplemented with red blood cells. In this study, we explored the feasibility of using a synthetic hemoglobin-based oxygen carrier (HBOC) in human kidney normothermic perfusion. Fourteen discarded human kidneys were perfused for 6 hours at a mean temperature of 37°C using a pressure-controlled system. Kidneys were perfused with a perfusion solution supplemented with either HBOC (n = 7) or packed red blood cells (PRBC) (n = 7) to increase oxygen-carrying capacity. Renal artery resistance, oxygen extraction, metabolic activity, energy stores, and histological features were evaluated. Throughout perfusion, kidneys from both groups exhibited comparable behavior regarding vascular flow (P = .66), oxygen consumption (P = .88), and reconstitution of tissue adenosine triphosphate (P = .057). Lactic acid levels were significantly higher in kidneys perfused with PRBC (P = .007). Histological findings were comparable between groups, and there was no evidence of histological damage caused by the HBOC. This feasibility experiment demonstrates that a HBOC solution can offer a logistically more convenient off-the-shelf alternative to PRBC in normothermic machine perfusion of human kidneys., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Preparation of stable and effective artificial oxygen carriers (AOCs) is a promising strategy to temporarily replace transfused blood and solve tissue hypoxia. Developing hemoglobin (Hb) loaded particles is one of the main ways to prepare suitable AOCs. Particles with a hierarchical micro/nanostructure can be loaded with plenty of proteins and have attracted great attention. Therefore, multiwall carbon nanotubes (MWCNTs) were chosen to fabricate AOCs. To improve the Hb-loading capacity of MWCNTs, functionalized MWCNTs, including carboxyl-functionalized MWCNTs (MWCNT-COOH), amino-functionalized MWCNTs (MWCNT-NH2), and heparin-conjugated MWCNTs (MWCNT-Hep), were prepared. Then, in this study, Hb was coupled to the functionalized MWCNTs to fabricate the AOCs. The functionalized MWCNTs and the AOCs were characterized by FTIR, SEM, TEM, and zeta potential analysis. The oxygen/Hb-loading capacity of the AOCs was also measured. The adverse effects of the AOCs on human umbilical vein endothelial cells (HUVECs) and human red blood cells (RBCs) were evaluated. The results showed that (1) the functional groups were grafted on the surface of the MWCNTs, and Hb was bound to the functionalized MWCNTs, thus the AOCs were successfully prepared; (2) MWCNT-Hep-Hb had the most stable dispersibility (i.e., the most negative zeta potential) in 0.9 wt% NaCl solution (MWCNT-Hep-Hb < MWCNT-COOH-Hb < MWCNT-Hb < MWCNT-NH2-Hb < 0); (3) MWCNT-Hep had the best Hb-loading capability, which was three times that of purified MWCNTs; (4) with concentrations increased up to 400 μg mL-1, MWCNT-Hep-Hb still had the highest cell viability (97.63% > 80%, ISO 10993-5:2009) and excellent blood biocompatibility. Therefore, MWCNT-Hep-Hb might be a satisfactory candidate as a blood substitute.

Background: Hemoglobin-based oxygen carriers (HBOCs) have proven useful for supplementing oxygen delivery when red cells are unavailable; however, HBOCs do not promote hemostasis. The need for prehospital bridges to blood transfusion informed this study which sought to determine the impact of HBOCs on coagulation, with or without cotransfusion of freeze-dried plasma (FDP)., Methods: Treatment was simulated in vitro by replacing whole blood volume (or whole blood prediluted with 25% plasmalyte A as a hemodilution model) with HBOC-201, FDP, or both at ratios of 10% to 50% of original volume. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, complete blood count, viscosity, thromboelastography (TEG), and platelet adhesion to collagen under flow were evaluated. Subsequently, tissue plasminogen activator was added to model hemorrhagic shock effects on fibrinolysis., Results: Substituting blood with HBOC resulted in dose-dependent decreases in fibrinogen and cells, which lengthened PT (+61% at highest dose) and aPTT (+40% at highest dose) and produced TEG parameters consistent with dilutional coagulopathy. While substituting blood with FDP decreased cell counts accordingly, fibrinogen, PT, aPTT, and TEG parameters were not statistically changed. When HBOC and FDP were combined 1:1 for volume replacement, observed HBOC-only detriments were mitigated: PT and aPTT were increased by 17% and 11%, respectively, at the highest doses. In prediluted samples, similar trends were seen with exacerbated differences. Platelet adhesion to collagen was directly affected by hematocrit. Samples containing both HBOC and tissue plasminogen activator were highly susceptible to fibrinolysis., Conclusion: A dose equivalent to 1 unit to 2 units each of HBOC-201 and FDP had a modest impact on functional coagulation measures and is reasonable to consider for clinical study as a part of early transfusion intervention. Higher doses may impart hemodilution risks similar to resuscitation with crystalloid or other colloids in coagulation-compromised patients. Further study of HBOC effects on fibrinolysis is also indicated., Study Type: In vitro laboratory study.

Artificial blood is an innovative concept of transfusion medicine where specifically designed compounds perform the task of transport and delivery of oxygen in the body to replace this function of allogenic human blood transfusion. Several molecules have been developed in the past few decades to achieve this objective and continous refinements are being continuously made in the quest of the ideal blood substitute. Currently, available technology manufactures artificial blood from haemoglobin obtained from outdated human/bovine blood (Haemoglobin Based Oxygen Carriers) or utilizing Perfluorocarbons. These synthetic blood substitutes are advantageous in that they do not require compatibility testing, are free from blood borne infections, have prolonged shelf life and do not require refrigeration. Artificial blood is projected to have a significant impact on the development of medical care in the future. It can complement the current blood products for transfusion and create a stable supply of safe and effective products. It is likely to reduce the requirements of blood transfusions drastically especially in settings of trauma and surgery thereby reducing the reliance on banked donated blood., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

We describe the results of adding a new biological agent HEMO 2 life ® to a standard preservation solution for hypothermic static lung preservation aiming to improve early functional parameters after lung transplantation. HEMO 2 life ® is a natural oxygen carrier extracted from Arenicola marina with high oxygen affinity developed as an additive to standard organ preservation solutions. Standard preservation solution (Perfadex ® ) was compared with Perfadex ® associated with HEMO 2 life ® and with sham animals after 24 h of hypothermic preservation followed by lung transplantation. During five hours of lung reperfusion, functional parameters and biomarkers expression in serum and in bronchoalveolar lavage fluid (BALF) were measured. After five hours of reperfusion, HEMO 2 life ® group led to significant improvement in functional parameters: reduction of graft vascular resistance (p < .05) and increase in graft oxygenation ratio (p < .05). Several ischemia-reperfusion related biomarkers showed positive trends in the HEMO 2 life ® group: expression of HMG B1 in serum tended to be lower in comparison (2.1 ± 0.8 vs. 4.6 ± 1.5) with Perfadex ® group, TNF-α and IL-8 in BALF were significantly higher in the two experimental groups compared to control (p < .05). During cold ischemia, expression of HIF1α and histology remained unchanged and similar to control. Supplementation of the Perfadex ® solution by an innovative oxygen carrier HEMO 2 life ® during hypothermic static preservation improves early graft function after prolonged cold ischemia in lung transplantation.

Background: Clinical resuscitative treatment of traumatic hemorrhage involves transfusion of RBC, platelets and plasma in controlled ratios. However, use of such blood components, especially platelets, present many challenges including availability, portability, contamination risks, and short shelf-life, which limit the use of platelet transfusions outside of large trauma centers such as remote civilian hospitals and austere prehospital settings. This has prompted significant research in platelet substitutes that may resolve the above issues while providing platelet-mimetic hemostatic action. In this framework, we have developed a synthetic platelet surrogate, SynthoPlate, by integrative decoration of platelet function mimetic peptides on a biocompatible lipid nanovesicle platform. We have previously demonstrated hemostatic capability of SynthoPlate in correcting tail-bleeding time in thrombocytopenic mice. Building on this, we hypothesized that SynthoPlate transfusion would decrease bleeding in a murine model of acute hemorrhagic shock., Methods: A validated model of uncontrolled intraperitoneal hemorrhage, via liver laceration was used to induce hemorrhagic shock in mice. SynthoPlate, control (unmodified) particles, and normal saline were administered as pretreatment and recue infusions to mice undergoing liver laceration and evaluated for hemostatic benefit by determining differences in blood loss and monitoring real-time hemodynamic data., Results: Pretreatment SynthoPlate transfusion resulted in significant reduction of blood loss following hemorrhage, compared with control particles or normal saline treatment (0.86 ± 0.16 g control particles [CP] vs. 0.84 ± 0.13 g normal saline [NS] vs. 0.68 ± 0.09 g SynthoPlate, p < 0.005). SynthoPlate transfused mice demonstrated improved hemodynamics taking significantly longer to develop post-injury hypotension (168.3 ± 106.6 seconds CP vs. 137 ± 58 seconds NS vs. 546.7 ± 329.8 seconds SynthoPlate, p < 0.05). SynthoPlate infusion following liver laceration, that is, rescue transfusion, also resulted in a significant decrease in blood loss (0.89 ± 0.17 g CP vs. 0.92 ± 0.19 g NS vs. 0.69 ± 0.18 g SynthoPlate, p < 0.05)., Conclusion: Transfusion of SynthoPlate particles reduces blood loss in a murine model of liver injury, and SynthoPlates may represent a viable transfusion product for the mitigation of blood loss in acute, severe hemorrhagic shock.

The development of hemoglobin (Hb)-based oxygen carriers (HBOCs) has been hampered because of safety concerns in humans. Chemical and/or genetic modifications of the Hb introduce varied structural and conformational constraint on the molecule that resulted in proteins with diverse allosteric responses, nitrosative and oxidative side reactions. Here, we present for the first time a comprehensive biochemical and biophysical comparison of human, bovine, and genetically engineered HBOCs that have been tested in humans. We evaluate oxygen equilibrium and ligand binding kinetics under different experimental conditions as well as their autoxidation kinetics, redox reactions, and heme release. We determined the effects of HBOCs on cellular redox states and mitochondrial respiration. Taken together, these experiments provide a better understanding of the relationship between the structure-function and oxidative reactivity of these proteins. One can therefore select independently among these diverse properties to engineer a safe and effective HBOC with improved biochemical/biophysical characteristics.

Red blood cells (RBCs) possess intact cyto-architectures while haemoglobin (Hb) is a cell-free, homogeneous solution. Both RBCs and Hb are generalized oxygen carriers. In this paper, kinetic studies on oxygen-releasing of high concentration of Hb and RBCs under various conditions were carried out regarding Hb and RBCs as fluids. Among them, Hb under specific conditions was seen as the simplest Hb-based oxygen carrier (HBOC), Also, factors affecting the oxygen releasing of Hb and RBCs, including osmotic pressure, viscosity and allosteric agent, have been well studied. Analysis of the results from the measurement above showed that kinetics of oxygen releasing of either pure Hb or the simplest HBOCs was obviously different from that of RBCs. The oxygen-releasing time of Hb was shorter and the oxygen-releasing rates of Hb were quicker than those of RBCs under various conditions. Therefore, as fluids, only by changing the milieus it exists in, Hb could not achieve the expected oxygen-releasing effect on the microcirculation so well as RBCs do in the same system, irrespective of the interaction between the fluids and blood vessels. Furthermore, kinetic properties of HBOCs must be considered and matched with those of RBCs in the study of HBOCs.

The development of hemoglobin-based oxygen carriers (HBOCs) is a persistent and urgent need in biomedicine. As a potential HBOCs, Dextran-hemoglobin (Dex-bHb) has been developed over the past years. The novel Dex-bHb, whose thiol group of Cys-93(β) was reversibly protected, was produced and the characteristics were evaluated in our previous study. Herein, blood compatibility was characterized in terms of the red blood cell aggregation and hemolysis rate in vitro, and Dex-bHb showed no obvious effects. After intravenous administration of Dex-bHb to golden Syrian hamsters with hemorrhages shock, it showed mean arterial pressure recovery, blood flow increase and the organ protection from serious hemorrhage injury. Consequently, Dex-bHb is hopeful to be a safe and available blood substitute.

Background: Delayed cerebral ischemia (DCI) after subarachnoid hemorrhage (SAH) has been linked to focal reductions in cerebral blood flow (CBF) and microvascular impairments in oxygen delivery. Effective therapies that restore flow and oxygen transport to vulnerable brain regions are currently lacking. SANGUINATE is a dual-action carbon monoxide-releasing and hemoglobin-based oxygen transfer agent with efficacy in animal models of focal brain ischemia and tolerability in patients with sickle cell disease., Methods: We performed a safety and proof-of-principle study in 12 SAH patients at risk of DCI across three escalating doses (160, 240, and 320 mg/kg). We used 15 O-PET (performed at baseline, after SANGUINATE and at 24 h) to evaluate efficacy for improving CBF and restoring flow-metabolism balance (assessed by oxygen extraction fraction [OEF]) to vulnerable regions (defined as baseline OEF ≥ 0.50)., Results: SANGUINATE resulted in a transient rise in mean arterial pressure (116 ± 15-127 ± 13 mm Hg, p = 0.001) that normalized by 24 h and allowed three patients with DCI to be weaned off vasopressors. No adverse events were noted during infusion. Global CBF did not rise (43 ± 8-46 ± 9 ml/100 g/min) although a trend was seen at the highest dose (45 ± 7-51 ± 9, p = 0.044). However, a significant 16% rise in regional CBF associated with reduction in OEF was seen in vulnerable regions, but did not persist at 24 h., Conclusions: We demonstrated that this novel agent can improve regional CBF and may improve oxygen supply-demand balance. Clinical studies (likely with repeat dosing) are required to evaluate whether this effect can prevent DCI or cerebral infarction.

Blood is a fluid connective tissue where living cells are suspended in noncellular liquid matrix. The cellular components of blood render gas exchange (RBCs), immune surveillance (WBCs) and hemostatic responses (platelets), and the noncellular components (salts, proteins, etc.) provide nutrition to various tissues in the body. Dysfunction and deficiencies in these blood components can lead to significant tissue morbidity and mortality. Consequently, transfusion of whole blood or its components is a clinical mainstay in the management of trauma, surgery, myelosuppression, and congenital blood disorders. However, donor-derived blood products suffer from issues of shortage in supply, need for type matching, high risks of pathogenic contamination, limited portability and shelf-life, and a variety of side-effects. While robust research is being directed to resolve these issues, a parallel clinical interest has developed toward bioengineering of synthetic blood substitutes that can provide blood's functions while circumventing the above problems. Nanotechnology has provided exciting approaches to achieve this, using materials engineering strategies to create synthetic and semi-synthetic RBC substitutes for enabling oxygen transport, platelet substitutes for enabling hemostasis, and WBC substitutes for enabling cell-specific immune response. Some of these approaches have further extended the application of blood cell-inspired synthetic and semi-synthetic constructs for targeted drug delivery and nanomedicine. The current study provides a comprehensive review of the various nanotechnology approaches to design synthetic blood cells, along with a critical discussion of successes and challenges of the current state-of-art in this field. WIREs Nanomed Nanobiotechnol 2017, 9:e1464. doi: 10.1002/wnan.1464 For further resources related to this article, please visit the WIREs website., (© 2017 Wiley Periodicals, Inc.)

Background: Normothermic machine perfusion of the liver (NMP-L) is a novel technique that preserves liver grafts under near-physiological conditions while maintaining their normal metabolic activity. This process requires an adequate oxygen supply, typically delivered by packed red blood cells (RBC). We present the first experience using an acellular hemoglobin-based oxygen carrier (HBOC) Hemopure in a human model of NMP-L., Methods: Five discarded high-risk human livers were perfused with HBOC-based perfusion fluid and matched to 5 RBC-perfused livers. Perfusion parameters, oxygen extraction, metabolic activity, and histological features were compared during 6 hours of NMP-L. The cytotoxicity of Hemopure was also tested on human hepatic primary cell line cultures using an in vitro model of ischemia reperfusion injury., Results: The vascular flow parameters and the perfusate lactate clearance were similar in both groups. The HBOC-perfused livers extracted more oxygen than those perfused with RBCs (O2 extraction ratio 13.75 vs 9.43 % ×10 per gram of tissue, P = 0.001). In vitro exposure to Hemopure did not alter intracellular levels of reactive oxygen species, and there was no increase in apoptosis or necrosis observed in any of the tested cell lines. Histological findings were comparable between groups. There was no evidence of histological damage caused by Hemopure., Conclusions: Hemopure can be used as an alternative oxygen carrier to packed red cells in NMP-L perfusion fluid.

The use of albumin for resuscitation has not proven as beneficial in human trials as expected from numerous animal studies. One explanation could be the practice of adding fatty acid (FA) during manufacture of pharmaceutical albumin. During ischemia, unbound free FAs (FFA) in the circulation could potentially induce cellular damage. We hypothesized that albumins with higher available binding capacities (ABC) for FFAs may prevent that damage. Therefore, we developed a technique to measure ABC, determined if pharmaceutical human serum albumin (HSA) has decreased ABC compared with FA-free bovine serum albumin (BSA), and if binding capacity would affect hemolysis when blood is mixed with exogenous FFA at levels similar to those observed in shock. The new assay used exogenous oleic acid (OA), glass fiber filtration, and a FFA assay kit. RBC hemolysis was determined by mixing 0 to 5 mM OA with PBS, HSA, FA-free BSA, or FA-saturated BSA and measuring plasma hemoglobin after incubation with human blood. 5% HSA contained 4.7±0.2 mM FFA, leaving an ABC of 5.0 ± 0.6 mM, compared with FA-free BSAs ABC of 7.0 ± 1.3 mM (P < 0.024). Hemolysis after OA was reduced with FA-free BSA but increased with FA-saturated BSA. HSA provided intermediate results. 25% solutions of FA-free BSA and HSA were more protective, while 25% FA-saturated BSA was more damaging than 5% solutions. These findings suggest that increased FA saturation may reverse albumin's potential benefit to lessen cellular damage and may explain, at least in part, its failure in human trauma studies.