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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.

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.

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

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.

Polymerized human hemoglobin (PolyhHb) is being studied as a possible red blood cell (RBC) substitute for use in scenarios where blood is not available. While the oxygen (Osub2/sub) carrying capacity of PolyhHb makes it appealing as an Osub2/subtherapeutic, the commercial PolyhHb PolyHeme® (Northfield Laboratories Inc.) was never approved for clinical use due to the presence of large quantities of low molecular weight (LMW) polymeric hemoglobin (Hb) species (lt;500 kDa), which have been shown to elicit vasoconstriction, systemic hypertension, and oxidative tissue injury in vivo. Previous bench-top scale studies in our lab demonstrated the ability to synthesize and purify PolyhHb using a two-stage tangential flow filtration purification process to remove almost all undesirable Hb species (gt;0.2 µm andlt;500 kDa) in the material, to create a product that should be safer for transfusion. Therefore, to enable future large animal studies and eventual human clinical trials, PolyhHb synthesis and purification processes need to be scaled up to the pilot scale. Hence in this study, we describe the pilot scale synthesis and purification of PolyhHb. Characterization of pilot scale PolyhHb showed that PolyhHb could be successfully produced to yield biophysical properties conducive for its use as an RBC substitute. Size exclusion high performance liquid chromatography showed that pilot scale PolyhHb yielded a high molecular weight Hb polymer containing a small percentage of LMW Hb species (lt;500 kDa). Additionally, the auto-oxidation rate of pilot scale PolyhHb was even lower than that of previous generations of PolyhHb. Taken together, these results demonstrate that PolyhHb has the ability to be seamlessly manufactured at the pilot scale to enable future large animal studies and clinical trials.

Background The aims of the present study were to evaluate and compare the efficacy of blood-feeding in phlebotomines through industrially processed membranes from the small intestine of pigs (used for the production of commercial sausages) and the skin of euthanized chicks. Methods Laboratory-bred Lutzomyia longipalpis and different field-caught phlebotomine species were subjected to the artificial feeding systems under similar conditions. Paired tests were performed using the control (skin from euthanized chicks) and test membranes (pig small intestine). The feeding rates were compared by paired t-test, and Pearson correlation was used to examine the relationship between the thickness of the membranes and feeding rate. Results The feeding rate was greater with the test membrane than with the control membrane for L. longipalpis (t-test, t = −3.3860, P = 0.0054) but not for the most frequent field-caught species, Nyssomyia antunesi (t-test, t = 0.7746, P = 0.4535). The average thicknesses of the control and test membranes were 184 ± 83 µm and 34 ± 12 µm, respectively (Mann–Whitney U-test, U = 0.00, Z = 2.8823, P = 0.0039); however, there was no correlation between feeding rate and membrane thickness. A moderate positive correlation was observed between the number of phlebotomines that fed and the total number of phlebotomines in the cage for each type of membrane and for each species. Conclusions The test membrane is a viable alternative for the artificial blood-feeding of phlebotomines, and is thus a potential substitute for the skin of animals that are euthanized for this purpose. Feeding rate was independent of membrane thickness. Graphical Abstract

A wide variety of hemoglobin-based oxygen carriers (HBOCs) have been designed for use as red blood cell (RBC) substitutes in transfusion medicine, ex vivo organ perfusion, oxygen delivery to hypoxic tissues, and a myriad of other applications. However, hemoglobin (Hb) derived from annelids (erythrocruorins [Ecs]) comprise a natural class of HBOC, since they are larger in size (30 nm in diameter) and contain more heme groups per molecule (144 heme groups) compared to human Hb (hHb; 5 nm in diameter and 4 heme groups). The larger size of Ec compared to hHb reduces tissue extravasation from the vascular space, thus, reducing vasoconstriction, systemic hypertension, and tissue oxidative injury when used as an RBC substitute. In addition, prior research has shown that Ecs possess slower auto-oxidation rates than hHb at physiological temperature, thus, making them attractive candidates for use as RBC substitutes. Unfortunately, it was also observed that Ecs have a much lower circulatory half-life in vivo compared to other HBOCs. Hence, conjugating polyethylene glycol (PEG) to the surface of Ec was proposed as a simple strategy to increase Ec circulatory half-life. Therefore, in order to inform future in vivo studies with PEGylated Ec, we decided to investigate the structural stability and biophysical properties of variable PEG surface coverage on Ec compared to native Ec. We observed an increase in PEG-Ec diameter and molecular weight (MW) and changes to the quaternary structure, secondary structure, and surface hydrophobicity after PEGylation. There was also an increase in oxygen binding affinity, reduction in oxygen offloading rate, and increase in auto-oxidation rate for increasing PEGylation ratios. Weak dissociation of Ec was also observed after dense PEGylation caused by steric repulsion of the conjugated PEG chains. Hence, we determined an optimum Ec PEGylation ratio that resulted in a substantial size and MW increase along with preservation of oxygen binding properties. In future studies, these materials will be tested in animal models to evaluate pharmacodynamics, pharmacokinetics, tissue oxygenation, microcirculatory responses, and overall safety.

Pullulan is an imperative microbial exo-polymer commercially produced by yeast like fungus Aureobasidium pullulans. Its structure contains maltosyl repeating units which comprises two α-(1 → 4) linked glucopyranose rings attached to one glucopyranose ring through α-(1 → 6) glycosidic bond. The co-existence of α-(1 → 6) and α-(1 → 4) glycosidic linkages endows distinctive physico-chemical properties to pullulan. It is highly biocompatible, non-toxic and non-carcinogenic in nature. It is extremely resistant to any mutagenicity or immunogenicity. The unique properties of pullulan make it a potent candidate for biomedical applications viz. drug delivery, gene delivery, tissue engineering, molecular chaperon, plasma expander, vaccination, etc. This review highlights the potential of pullulan in biomedical research and development.

Veno-venous bypass (VVB) is necessary for maintaining hemodynamic and internal environment stabilities in complex liver surgeries. However, the current VVB strategies require systematic anticoagulation and are time-consuming, leading to unexpected complications. This study aims to overcome these limitations by using a novel magnetic artificial blood vessel constructed with heparin-PLCL core-shell nanofibers. Coaxial electrospinning was used to fabricate core-shell nanofibers with heparin encapsulated into the core layer. The microstructure, physical and chemical properties, hemocompatibility, and heparin release behavior were characterized. The regional anticoagulation magnetic artificial vessel was constructed with these nanofibers and used to perform VVB in a rat liver transplantation model for

Military working dogs (MWDs) are force multipliers that are at risk for severe trauma when employed on the battlefield. When in severe hemorrhagic shock, MWDs require both oxygen- carrying capacity and replacement of vascular volume and coagulation factors. The objective of this study was to evaluate the hemostatic capacity of canine freeze-dried plasma (cFDP) with a Food and Drug Administration (FDA)-approved hemoglobin- based oxygen carrier (HBOC) in an in vitro model of resuscitation. Whole blood (WB) was collected from 10 MWDs, and these samples were diluted by 10%, 25%, or 40% with either cFDP (reconstituted with water), HBOC, cFDP (reconstituted with HBOC), or an equal volume of a 1:1 ratio of cFDP (reconstituted with water) and HBOC. Hemostatic parameters were minimally changed based on evaluation of prothrombin time, activated partial thromboplastin time, fibrinogen and thromboelastography at the 10% and 25% dilutions, and parameters consistent with a hypocoagulability were seen at dilutions of 40%. Based on the results of this study, additional research is warranted to determine if cFDP reconstituted with HBOC is a viable resuscitation product in canine trauma.

In the process of photodynamic therapy (PDT) treatment of tumors, reactive oxygen species (ROS) plays a key role in destroying tumor tissues. However, traditional PDT often has limited ROS killing capacity due to hypoxia in the tumor microenvironment (TME) or obstruction by the ROS defense system, resulting in poor efficacy. Therefore, enhancing the killing effect of ROS on tumors is the core of enhancing the anti‐tumor effect of PDT. In recent years, many studies have developed a series of strategies to enhance the ability of ROS to kill tumors in view of the limitations of the TME on PDT. This article summarizes the commonly used or innovative strategies in recent years, including not only frequently used methods for hypoxia in the TME but also innovative strategies to inhibit the ROS defense system., In recent years, many studies have developed a series of strategies to enhance the ability of reactive oxygen species (ROS) to kill tumors in view of the limitations of the tumor microenvironment (TME) on photodynamic therapy. This article summarizes the commonly used or innovative strategies in recent years, including not only the common methods for TME hypoxia but also innovatively covering the relevant strategies for inhibiting the ROS defense system.

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.

Previously, our lab developed high molecular weight (MW) tense (T) quaternary state glutaraldehyde polymerized bovine hemoglobins (PolybHbs) that exhibited reduced vasoactivity in several small animal models. In this work, we prepared PolybHb in the T- and relaxed (R) quaternary state with ultrahigh MW (> 500 kDa) with varying cross-link densities and investigated the effect of MW on key biophysical properties (i.e., O(2) affinity, cooperativity (Hill) coefficient, hydrodynamic diameter, polydispersity, polymer composition, viscosity, gaseous ligand-binding kinetics, auto-oxidation, and haptoglobin-binding kinetics). To further optimize current PolybHb synthesis and purification protocols, we performed a comprehensive meta-data analysis to evaluate correlations between procedural parameters (i.e. cross-linker:bovine Hb (bHb) molar ratio, gas/liquid exchange time, temperature during sodium dithionite addition, and number of diafiltration cycles) and the biophysical properties of both T-state and R-state PolybHbs. Our results showed that, the duration of the fast-step auto-oxidation phase of R-state PolybHb increased with decreasing glutaraldehyde:bHb molar ratio. Additionally, T-state PolybHbs exhibited significantly higher bimolecular rate constants for binding to haptoglobin and unimoleular O(2) offloading rate constants compared to R-state PolybHbs. The methemoglobin (metHb) level in the final product was insensitive to the molar ratio of glutaraldehyde to bHb for all PolybHbs. During tangential flow filtration processing of the final product, 14 diafiltration cycles was found to yield the lowest metHb level.

Laser-induced interstitial thermotherapy (LITT) is applied to ex-vivo porcine livers. An artificial blood vessel is used to study the cooling effect of larger blood vessels in proximity to the ablation zone. The same setting is simulated using a model based on partial differential equations (PDEs) for temperature, radiation, and tissue damage. The simulated temperature distributions are compared to temperature data obtained from MR thermometry. The study shows that the quality and resolution of the thermometry data is sufficient to validate and improve modeling approaches. Furthermore, the data can be used to identify missing model parameters as well as the exact placement of the laser applicator in relation to the imaging plane., arXiv admin note: text overlap with arXiv:1910.12515

A bovine hemoglobin (HbBv) or human adult hemoglobin (HbA) wrapped covalently by human serum albumins (HSAs), hemoglobin-albumin clusters (HbBv-HSA

For the past thirty years, hemoglobin-based oxygen carriers (HBOCs) have been under development as a red blood cell substitute. Side-effects such as vasoconstriction, oxidative injury, and cardiac toxicity have prevented clinical approval of HBOCs. Recently, high molecular weight (MW) polymerized human hemoglobin (PolyhHb) has shown positive results in rats. Studies have demonstrated that high MW PolyhHb increased O

Increasingly growing problems in vascular access for long-term hemodialysis lead to a considerable demand for synthetic small diameter vascular prostheses, which usually suffer from some drawbacks and are associated to high failure rates. Incorporating the concept of in situ tissue engineering (TE) into synthetic small diameter blood vessels, for example, thermoplastic poly(ether urethane) (TPU) ones, could provide an alternative approach for vascular access that profits from the advantages of excellent mechanical properties of synthetic polymer materials (early cannulation) and unique biointegration regeneration of autologous neovascular tissues (long-term fistulae). In this study, a kind of heparinized small diameter (d = 2.5 mm) TPU/poly(ε-caprolactone) (TPU/PCL-Hep) bi-layered blood vessels was electrospun with an inner layer of PCL and an outer layer of TPU. Afterward, the inner surface heparinization was conducted by coupling H

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.

Bioengineering an effective, small diameter (6 mm) artificial vascular graft for use in bypass surgery when autologous grafts are unavailable remains a persistent challenge. Commercially available grafts are typically made from plastics, which have high strength but lack elasticity and present a foreign surface that triggers undesirable biological responses. Tissue engineered grafts, leveraging decellularized animal vessels or derived de novo from long-term cell culture, have dominated recent research, but failed to meet clinical expectations. More effective constructs that are readily translatable are urgently needed. Recent advances in natural materials have made the production of robust acellular conduits feasible and their use increasingly attractive. Here, we identify a subset of natural materials with potential to generate durable, small diameter vascular grafts.

Background The most common materials of artificial blood vessels are polyethylene terephthalate and polytetrafluoroethylene. But polycarbonate polyurethane (PCU) is an ideal material for vascular prostheses because of their excellent characteristics. As far as we know, our artificial blood vessel is the first type of hybrid PCU/polyester three-layered large-diameter artificial blood vessel in the world. Objective The purpose of this preclinical animal experiment is to evaluate the hemocompatibility, histocompatibility, effectiveness, and safety of the three-layered large-diameter artificial blood vessel in sheep. Methods The artificial blood vessels took place of the initial segments of the sheep’s thoracic aorta by end-to-end anastomosis. Results All of the 14 sheep are male, their average body weight (BW) was 30.57 ± 3.95 kg. All 14 artificial blood vessels successfully replaced the thoracic aortas. 5 sheep did not survive to the end of the experiment, while the remaining 9 sheep did. After the surgery, the blood biochemical and blood routine indicators fluctuate slightly within the normal range. The angiography showed that the implanted artificial blood vessels were unobstructed without obvious stenosis or expansion. 24 weeks after surgery, the lumen surfaces of the artificial blood vessels were covered by endothelia in different degrees, and the average endothelialization rate was 69.44% (range: 20% to 100%). Conclusions This artificial blood vessel is the first to use PCU in large-diameter artificial vascular grafts. It has excellent blood compatibility, wonderful biocompatibility, high endothelialization rate, and 100% patency.

We aimed to establish an animal model of abdominal aortic vascular replacement in mongrel dogs to investigate the effect of extracorporeal radiotherapy on the intima. Twenty healthy mongrel dogs were randomly divided into four groups: 5-week control group, 5-week radiotherapy group, 10-week control group and 10-week radiotherapy group. We first performed an artificial vascular replacement of the abdominal aortic segment. The radiotherapy group received external radiotherapy with a dose of 7 Gy for 4 days. The thickness of neointimal hyperplasia, immunoreactivity and expression of proliferation-related factors were detected by hematoxylin and eosin (HE) staining, immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR )and western blotting at 5 and 10 weeks after the reconstruction. The results showed that the intimal thickness of the artificial blood vessel in the 5- and 10-week radiotherapy groups was thinner than that in the control groups by HE staining. The immunoreactivity and expression levels of Skp2, c-Myc and CyclinE1 were significantly decreased in the radiotherapy groups than those in control groups by immunohistochemistry, qRT-PCR and western blotting. On the contrary, immunoreactivity and expression levels of P27kip1 were increased. In conclusion, we discovered that postoperative external radiotherapy significantly decreases the intimal hyperplasia of artificial blood vessels by regulating c-Myc-Skp2-P27-CyclinE1 network.

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.

The effects of liposome-encapsulated hemoglobin with high O2 affinity (h-LEH), an artificial O2 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 O2 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.

In blood, the primary role of red blood cells (RBCs) is to transport oxygen via highly regulated mechanisms involving hemoglobin (Hb). Hb is a tetrameric porphyrin protein comprising of two α- and two β-polypeptide chains, each containing an iron-containing heme group capable of binding one oxygen molecule. In military as well as civilian traumatic exsanguinating hemorrhage, rapid loss of RBCs can lead to suboptimal tissue oxygenation and subsequent morbidity and mortality. In such cases, transfusion of whole blood or RBCs can significantly improve survival. However, blood products including RBCs present issues of limited availability and portability, need for type matching, pathogenic contamination risks, and short shelf-life, causing substantial logistical barriers to their prehospital use in austere battlefield and remote civilian conditions. While robust research is being directed to resolve these issues, parallel research efforts have emerged toward bioengineering of semisynthetic and synthetic surrogates of RBCs, using various cross-linked, polymeric, and encapsulated forms of Hb. These Hb-based oxygen carriers (HBOCs) can potentially provide therapeutic oxygenation when blood or RBCs are not available. Several of these HBOCs have undergone rigorous preclinical and clinical evaluation, but have not yet received clinical approval in the USA for human use. While these designs are being optimized for clinical translations, several new HBOC designs and molecules have been reported in recent years, with unique properties. The current article will provide a comprehensive review of such HBOC designs, including current state-of-the-art and novel molecules in development, along with a critical discussion of successes and challenges in this field.

Several adverse events have been associated with the infusion of hemoglobin-based oxygen carriers (HBOCs), including transient hypertension, gastrointestinal, pancreatic/liver enzyme elevation, and cardiac/renal injury in humans. Although several mechanisms have been suggested, the basis of HBOC toxicity is still poorly understood. Scavenging of vascular endothelial nitric oxide (NO) and heme-mediated oxidative side reactions are thought to be the major causes of toxicity. However, based on more recent preclinical studies, oxidative pathways (driven by the heme prosthetic group) seem to play a more prominent role in the overall toxicity of free Hb or HBOCs. HBOCs display a diversity of physicochemical properties, including molecular size/cross-linking characteristics leading to differences in oxygen affinity, allosteric, redox properties, and even oxidative inactivation by protein/heme clearing mechanisms. These diverse characteristics can therefore be manipulated independently, leaving open the possibility of engineering a safe and effective HBOC. To date, several antioxidative strategies have been proposed to counteract the redox side reactions of current generation HBOCs.

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.

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.

Hemoglobin-based oxygen carriers (HBOCs) are modified stroma-free hemoglobin molecules used in developing a blood substitute for therapeutic usage. In order to prevent hemoglobin dissociation, glutaraldehyde (GTA) was used to generate high-molecular weight heterogeneous crocodile hemoglobin (Poly-cHb). This work, Poly-cHb was created using various GTA concentrations, ranging from 0.025-0.150% (v/v). Physicochemical properties were investigated that were comparable GTA polymerized human hemoglobin (Poly-hHb). This study has revealed that GTA polymerization increases the molecular size of Native-cHbs from 14.10 nm over a range from 16.31 to 54.27 nm. Moreover, this polymerization alters the secondary structure and heme environment by decreasing the helicity ratio from 1.00 to 0.95 at the highest condition and exhibits hypochromic shift of the Soret band to be 0.88 times lower than the native. However, all Poly-cHbs still possessed higher oxygen affinity than that of Poly-hHbs with average P50 values of 13 and 21 mmHg, respectively. Although, polymerization affected the overall Poly-cHb structure slightly, but compensated by decreasing the denaturation level to lower than 10%. Thus, it is interesting to note that Poly-cHb may advantageously provide effective oxygen carriage and ability for pasteurization, which may benefit the search for new alternative hemoglobin sources for HBOC development.

Background Crystalloids are first line in fluid resuscitation therapy, however there is a lack of evidence-based recommendations on the volume to be administered. Therefore, we aimed at comparing the systemic hemodynamic and respiratory effects of volume replacement therapy with a 1:1 ratio to the historical 1:3 ratio. Methods Anesthetized, ventilated rats randomly included in 3 groups: blood withdrawal and replacement with crystalloid in 1:1 ratio (Group 1, n = 11), traditional 1:3 ratio (Group 3, n = 12) and a control group with no interventions (Group C, n = 9). Arterial blood of 5% of the total blood volume was withdrawn 7 times, and replaced stepwise with different volume rations of Ringer’s acetate, according to group assignments. Airway resistance (Raw), respiratory tissue damping (G) and tissue elastance (H), mean arterial pressure (MAP) and heart rate (HR) were assessed following each step of fluid replacement with a crystalloid (CR1-CR6). Lung edema index was measured from histological samples. Results Raw decreased in Groups 1 and 3 following CR3 (p

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.

Hemoglobin (Hb)-based oxygen carriers (HBOC) are modified extracellular proteins, designed to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects, in part linked to the intrinsic oxidative toxicity of Hb. Previously a redox-active tyrosine residue was engineered into the Hb β subunit (βF41Y) to facilitate electron transfer between endogenous antioxidants such as ascorbate and the oxidative ferryl heme species, converting the highly oxidizing ferryl species into the less reactive ferric (met) form. We inserted different single tyrosine mutations into the α and β subunits of Hb to determine if this effect of βF41Y was unique. Every mutation that was inserted within electron transfer range of the protein surface and the heme increased the rate of ferryl reduction. However, surprisingly, three of the mutations (βT84Y, αL91Y and βF85Y) also increased the rate of ascorbate reduction of ferric(met) Hb to ferrous(oxy) Hb. The rate enhancement was most evident at ascorbate concentrations equivalent to that found in plasma (< 100 μM), suggesting that it might be of benefit in decreasing oxidative stress in vivo. The most promising mutant (βT84Y) was stable with no increase in autoxidation or heme loss. A decrease in membrane damage following Hb addition to HEK cells correlated with the ability of βT84Y to maintain the protein in its oxygenated form. When PEGylated and injected into mice, βT84Y was shown to have an increased vascular half time compared to wild type PEGylated Hb. βT84Y represents a new class of mutations with the ability to enhance reduction of both ferryl and ferric Hb, and thus has potential to decrease adverse side effects as one component of a final HBOC product., Graphical abstract fx1, Highlights • Cell free Hemoglobin Based Oxygen Carriers can deliver oxygen to tissue. • Oxidation of ferrous hemoglobin to ferric and ferryl leads to oxidative toxicity. • Insertion of tyrosine residues enhances reduction of ferryl to ferric hemoglobin. • Some mutants (e.g. βT84Y) also enhance ferric to ferrous reduction by ascorbate. • βT84Y decreases cellular oxidative stress and enhances vascular retention in vivo.

Acute stroke causes complex, pathological, and systemic responses that have not been treatable by any single medication. In this study, using a murine transient middle cerebral artery occlusion stroke model, a novel therapeutic strategy is proposed, where blood replacement (BR) robustly reduces infarctions and improves neurological deficits in mice. Our analyses of immune cell subsets suggest that BR therapy substantially decreases neutrophils in blood following a stroke. Electrochemiluminescence detection demonstrates that BR therapy reduces cytokine storm in plasma and ELISA demonstrates reduced levels of matrix metalloproteinase-9 (MMP-9) in the plasma and brains at different time points post-stroke. Further, we have demonstrated that the addition of MMP-9 to the blood diminishes the protective effect of the BR therapy. Our study is the first to show that BR therapy leads to profoundly improved stroke outcomes in mice and that the improved outcomes are mediated via MMP-9. These results offer new insights into the mechanisms of stroke damage.

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

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

Suspensions of hemoglobin microparticles (HbMPs) are promising tools as oxygen therapeutics. For the approval of clinical studies extensive characterization of these HbMPs with a size of about 750 nm is required regarding physical properties, function, pharmaco-kinetics and toxicology. The standard absorbance measurements in blood gas analyzers require dissolution of red blood cells which does not work for HbMP. Therefore, we have developed a robust and rapid optical method for the quality and functionality control of HbMPs. It allows simultaneous determination of the portion of the two states of hemoglobin oxygenated hemoglobin (oxyHb) and deoxygenated hemoglobin (deoxyHb) as well as the content of methemoglobin (metHb). Based on the measurement of collimated transmission spectra between 300 nm and 800 nm, the average extinction cross section of HbMPs is derived. A numerical method is applied to determine the composition of the HbMPs based on their wavelength-dependent refractive index (RI), which is a superposition of the three different states of Hb. Thus, light-scattering properties, including extinction cross sections can be simulated for different compositions and sizes. By comparison to measured spectra, the relative concentrations of oxyHb, deoxyHb, metHb are accessible. For validation of the optically determined composition of the HbMPs, we used X-ray fluorescence spectrometry for the ratio of Fe(II) (oxyHb/deoxyHb) and Fe(III) (metHb). High accuracy density measurements served to access heme-free proteins, size was determined by dynamic light scattering and analytical centrifugation and the shape of the HbMPs was visualized by electron and atomic force microscopy.

Developing biocompatible, synthetic oxygen carriers is a consistently challenging task that researchers have been pursuing for decades. Perfluorocarbons (PFC) are fascinating compounds with a huge capacity to dissolve gases, where the respiratory gases are of special interest for current investigations. Although largely chemically and biologically inert, pure PFCs are not suitable for injection into the vascular system. Extensive research created stable PFC nano-emulsions that avoid (i) fast clearance from the blood and (ii) long organ retention time, which leads to undesired transient side effects. PFC-based oxygen carriers (PFOCs) show a variety of application fields, which are worthwhile to investigate. To understand the difficulties that challenge researchers in creating formulations for clinical applications, this review provides the physical background of PFCs’ properties and then illuminates the reasons for instabilities of PFC emulsions. By linking the unique properties of PFCs and PFOCs to physiology, it elaborates on the response, processing and dysregulation, which the body experiences through intravascular PFOCs. Thereby the reader will receive a scientific and easily comprehensible overview why PFOCs are precious tools for so many diverse application areas from cancer therapeutics to blood substitutes up to organ preservation and diving disease.