Development of novel oxygen carriers by coupling hemoglobin to functionalized multiwall carbon nanotubes.

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.

How to Ward Off Retinal Toxicity of Perfluorooctane and Other Perfluorocarbon Liquids?

Purpose: Reactive and underfluorinated impurities are acknowledged as a source of cytotoxicity of perfluorocarbon liquids (PFCLs) used as blood substitutes. To determine whether this is also a relevant factor in retinal toxicity, we analyzed eight PFO batches associated with adverse ocular events. Methods: (A) The amount of reactive and underflurinated impurities was analyzed by fluoride-selective potentiometry and expressed as H-value. (B) Cytotoxicity of these batches was determined by an ISO 10993-5-compliant extractive test and compared to published data generated with a direct-contact method. (C) A toxic PFO batch (061014) was purified to remove reactive and underfluorinated impurities. (A) and (B) -measurements were repeated after that. (D) The dose dependence of the H-value and cytotoxicity was determined in a dilution experiment. Results: (A) The batches revealed H-values ranging from 1.400 ppm to 4.500 ppm. (B) All batches induced cell growth inhibition; seven must be classified as cytotoxic. Findings from ISO-conform extractive and direct-contact methods showed no difference. (C) After all reactive and underfluorinated impurities in batch 061014 were removed, the H-value dropped to <10 ppm and cytotoxicity disappeared. (D) Cytotoxicity increases gradually as the H-value rises. Conclusions: The clinical relevance of the H-value as a safety parameter for PFO endotamponades could be proven. The H-value is a measure for reactive and underfluorinated impurities that cause toxicity of PFCLs and should be incorporated in each endotamponade specification with a limit of 10 ppm to prove the effectiveness of the ultra-purification required and ensure a safe product. Despite the fact that an (ISO)-standard literally is a "standard" only, which cannot cover all imaginable possibilities, the incorporation of the H-value determination into the relevant ISO standard has been initiated. If a thorough risk assessment results in risks that cannot be detected and/or managed by the effective standard, additional investigations have to be performed.

The Use of an Acellular Oxygen Carrier in a Human Liver Model of Normothermic Machine Perfusion.

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.

Perfluorodecalin-Filled Poly(n-butyl-cyanoacrylate) Nanocapsules as Potential Artificial Oxygen Carriers: Preclinical Safety and Biocompatibility.

With regard to the development of artificial blood substitutes, perfluorodecalin-filled poly(n-butyl-cyanoacrylate) nanocapsules are already discussed for the use as artificial oxygen carriers. The aim of the present study was to thoroughly investigate the preclinical safety and biocompatibility of the perfluorodecalin-filled poly(n-butyl-cyanoacrylate) nanocapsules prepared by interfacial polymerization. Nanocapsules were assessed for physical and microbial stability. Subsequent to intravenous infusion to anesthetized rats, effects on systemic parameters, microcirculation, circulatory in vivo half-life, acid base/metabolic status, organ damage and biodistribution were evaluated using inter alia 19F-NMR spectroscopy and in vivo microscopy. Perfluorodecalin-filled poly(n-butyl-cyanoacrylate) nanocapsules displayed physical and microbial stability over a period of 4 weeks and the circulatory in vivo half-life was t1/2 = 30 min. In general, all animals tolerated intravenous infusion of the prepared nanocapsules, even though several side-effects occurred. As a consequence of nanocapsule infusion, a transient decrease in mean arterial blood pressure, impairment of hepatic microcirculation, organ/tissue damage of liver, spleen and small intestine, as well as an elevation of plasma enzyme activities such as lactate dehydrogenase, creatine kinase and aspartate aminotransferase could be observed. The assessment of the distribution pattern revealed nanocapsule accumulation in spleen, kidney and small intestine. Perfluorodecalin-filled poly(n-butyl-cyanoacrylate) nanocapsules conformed to basic requirements of drugs under preclinical development but further improvement is needed to establish these nanocapsules as novel artificial oxygen carriers.

Interaction of recombinant octameric hemoglobin with endothelial cells.

Hemoglobin-based oxygen carriers (HBOCs) may generate oxidative stress, vasoconstriction and inflammation. To reduce these undesirable vasoactive properties, we increased hemoglobin (Hb) molecular size by genetic engineering with octameric Hb, recombinant (r) HbßG83C. We investigate the potential side effects of rHbßG83C on endothelial cells. The rHbßG83C has no impact on cell viability, and induces a huge repression of endothelial nitric oxide synthase gene transcription, a marker of vasomotion. No induction of Intermolecular-Adhesion Molecule 1 and E-selectin (inflammatory markers) transcription was seen. In the presence of rHbßG83C, the transcription of heme oxygenase-1 (oxidative stress marker) is weakly increased compared to the two other HBOCs (references) or Voluven (control). This genetically engineered octameric Hb, based on a human Hb ßG83C mutant, leads to little impact at the level of endothelial cell inflammatory response and thus appears as an interesting molecule for HBOC development.

Down selection of polymerized bovine hemoglobins for use as oxygen releasing therapeutics in a guinea pig model.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) are being developed as resuscitative fluids for use in multiple medical applications and in lieu of blood transfusion. However, cardiovascular, central nervous system, and renal adverse events have largely impeded progress. This has prompted a need to evaluate novel down selection approaches for HBOCs prior to in-depth preclinical and clinical safety testing. In the present study, polymerized bovine Hbs (PolybHbs) were prepared with increasing ratios of glutaraldehyde to bovine Hb (10:1, 20:1, 30:1, and 40:1). The optimal PolybHb candidate selection was based on a priori determined in vivo response to include a long circulating PolybHb with no measurable renal exposure, minimal cardiovascular response, limited oxidation to metHb in vitro, or in circulation and absence of acute end organ toxicity. Guinea pigs were dosed via a 50% blood for PolybHb exchange transfusion. Data suggested that the 30:1 preparation exhibited maximum circulatory exposure (AUC(0)(-∞)) with the lowest level of oxidation (plasma metHb formation) and minimal (< 10%) blood pressure elevation. Additionally, the 30:1 preparation was absent renal iron deposition as well as abnormal glomerular/tubular histopathology or serum creatinine elevation. Clearance pathways predominantly followed those consistent with endogenous Hb clearance based pathways. Therefore, data confirmed the ability to select a single PolybHb from a small library of HBOCs based on a priori determined characteristics. Moreover, the approach to down selection described could be applied to enhance the early predictability of human safety for this class of biological therapeutics to optimize for specific indications.

Basic science offers a challenge for developing hemoglobin based oxygen carriers into therapeutic agents.

Experimental data, obtained during the course of several years, are connected into a coherent picture, which may help research for the development of HBOCs as therapeutic agents. Oxygen affinity, scavenging of nitric oxide, and yield of production of hemoglobin based oxygen carriers were the areas under consideration.

A new polyethyleneglycol-derivatized hemoglobin derivative with decreased oxygen affinity and limited toxicity.

A new protocol is described for derivatization of hemoglobin with polyethyleneglycol (PEG) via reaction of the unmodified native hemoglobin with an activated amine-reacting polyethylene glycol derivative which, unlike protocols previously described, leads to formation of a peptide bond between hemoglobin and PEG. Dioxygen binding and peroxide reactivities of the derivatized hemoglobin are examined, and found to be within reasonable limits, with the particular observation that, unlike with a few other derivatization protocols, the dioxygen affinity is slightly lower than that of native Hb. In cell culture tests (human umbilical vein epithelial cells, HUVEC), the derivatization protocol induces no toxic effect. These results show promise towards applicability for production of hemoglobin-based blood substitutes.

Tissue oxidative metabolism after extreme hemodilution with PEG-conjugated hemoglobin.

NADH-localized fluorometry was used as a noninvasive technique to monitor changes in the energy state of intact tissue (muscle and connective tissue), without anesthesia, as a function of blood plasma O(2)-carrying capacity in the hamster window chamber model. Acute moderate isovolemic hemodilution was induced by two isovolemic hemodilution steps: in the first step, 6% 70-kDa dextran (Dex70) was used to induce an acute anemic state (18% Hct); in the second step, exchange transfusion of polyethylene glycol (PEG) maleimide-conjugated Hb (4 g/dl, PEG-Hb) or Dex70 (6 g/dl) was used to reduce erythrocytes to 75% of baseline (11% Hct). PEG-Hb had six copies of PEG (5 kDa) conjugated to each human Hb (0.48 g PEG/g Hb) through extension arm-facilitated chemistry. Systemic parameters, microvascular perfusion, functional capillary density, intravascular and interstitial Po(2), and intracellular NADH fluorescence were monitored. Mean arterial blood pressure after extreme hemodilution was statistically significantly reduced for Dex70 compared with PEG-Hb. The presence of PEG-Hb in the circulation maintained positive acid-base balance. While microvascular blood flows were not different, functional capillary density was significantly higher for PEG-Hb than Dex70. Arteriolar Po(2) was higher in the presence of PEG-Hb than Dex70, but tissue and venular Po(2) were not different. Cellular energy metabolism (intracellular O(2)) in the tissues was improved with PEG-Hb. Moderate hemodilution to 18% Hct (6.4 g Hb/dl) brings tissue O(2) delivery to the verge of inadequacy. Extreme hemodilution to 11% Hct (3.7 g Hb/dl) produces tissue anoxia, and high-O(2)-affinity PEG-Hb (Po(2) at which blood is 50% saturated with O(2) = 4 Torr, 1.1 g Hb/dl) only partially decreases anaerobic metabolism without increasing tissue Po(2).

Hemoglobin-based oxygen carriers: From mechanisms of toxicity and clearance to rational drug design.

Hemoglobin-based oxygen carriers (HBOCs) have been developed to support blood oxygen transport capacity during hemorrhagic shock, hemolysis and ischemic insult. Existing product candidates have demonstrated considerable efficacy in experimental animal models and in clinical trial subjects; however, severe adverse safety signals that appeared in recent phase II and phase III clinical trials involving certain HBOCs have in part hindered further development and licensing. Emerging insights into hemoglobin (Hb) toxicity as well as physiologic Hb scavengers such as haptoglobin and CD163 that are capable of detoxifying extracellular Hb in vivo suggest that alternative product candidates could be designed. Together with novel animal models and biomarkers tailored to monitor the effects of extracellular Hb, a new generation of HBOCs can be envisioned.

Non-toxicity of IV injected perfluorocarbon oxygen carrier in an animal model of liver regeneration following surgical injury.

Lethal dose experiments in animals have demonstrated that second-generation perfluorocarbon oxygen carriers are remarkably non-toxic. However, this non-toxicity has not previously been demonstrated in a liver failure scenario. A surgical liver damage and regeneration model in rats was selected using a well-controlled cross tabulated study design. A large number of physiological, biochemical, and hematological parameters were measured. No indications were found that intravenously injected perfluorooctyl bromide emulsion was toxic at the concentrations employed, in either healthy or severe liver injury scenarios. Neither was there any significant impact on the rate of liver regeneration following the injuries. Bearing in mind prior human clinical studies, it is therefore safe to assume that perfluorocarbon emulsions are also non-toxic in bioartificial liver treatments.

Intrinsic toxicity of hemoglobin: how to counteract it.

The development of safe and effective blood substitutes is of great importance in both civilian and military medicine. The currently tested hemoglobin (Hb)-based oxygen carriers, however, have toxicity and efficacy problems. A number of unwanted effects have been observed in human trials, creating doubts about their clinical usefulness. In some subjects, vasoconstriction and decreased blood flow to the vital organs, heart attack, stroke, systemic inflammation, organ damage, and even death, have been attributed to the transfusion of these experimental products. Hb is a well-known pressor agent and strong oxidant, although the full understanding of its intrinsic toxicity is yet to be uncovered. In particular, the complete mechanism of Hb-induced vasoconstriction needs full elucidation. Knowledge of the biological events that trigger the induction of genes upon treatment with redox-active Hb, as well as its catabolism, is still incomplete. It seems that our limited knowledge of free Hb effects in vivo is the main reason for not yet having a viable substitute of human blood. The future for universal red cell substitutes is in the new-generation products that address all of Hb's intrinsic toxicity issues.

Radical producing and consuming reactions of hemoglobin: how can we limit toxicity?

Hemoglobin has a range of enzymatic activities that can affect its putative pharmacological role as an extracellular oxygen carrier. In the presence of peroxides, deoxyhemoglobin and methemoglobin can produce free radicals and induce lipid peroxidation. Oxyhemoglobin can oxidize the free radical nitric oxide to nitrate, yet deoxyhemoglobin can produce nitric oxide from nitrite. These enzymatic reactions can induce or diminish toxic side reactions when hemoglobin is added in vivo. For example the removal of the free radical vasodilator nitric oxide, or the addition of the lipid-derived vasoconstrictor F2-isoprostane, will both alter blood flow and blood pressure. In order to determine the dominant effects it is necessary to design molecules with differing radical reactivities. Molecules have been designed with these modifications and this article will review their role in determining mechanism as well as their possible functionality as blood substitutes.

Nitrite reductase activity of hemoglobin as a systemic nitric oxide generator mechanism to detoxify plasma hemoglobin produced during hemolysis.

Hemoglobin (Hb) potently inactivates the nitric oxide (NO) radical via a dioxygenation reaction forming nitrate (NO(3)(-)). This inactivation produces endothelial dysfunction during hemolytic conditions and may contribute to the vascular complications of Hb-based blood substitutes. Hb also functions as a nitrite (NO(2)(-)) reductase, converting nitrite into NO as it deoxygenates. We hypothesized that during intravascular hemolysis, nitrite infusions would limit the vasoconstrictive properties of plasma Hb. In a canine model of low- and high-intensity hypotonic intravascular hemolysis, we characterized hemodynamic responses to nitrite infusions. Hemolysis increased systemic and pulmonary arterial pressures and systemic vascular resistance. Hemolysis also inhibited NO-dependent pulmonary and systemic vasodilation by the NO donor sodium nitroprusside. Compared with nitroprusside, nitrite demonstrated unique effects by not only inhibiting hemolysis-associated vasoconstriction but also by potentiating vasodilation at plasma Hb concentrations of <25 muM. We also observed an interaction between plasma Hb levels and nitrite to augment nitroprusside-induced vasodilation of the pulmonary and systemic circulation. This nitrite reductase activity of Hb in vivo was recapitulated in vitro using a mitochondrial NO sensor system. Nitrite infusions may promote NO generation from Hb while maintaining oxygen delivery; this effect could be harnessed to treat hemolytic conditions and to detoxify Hb-based blood substitutes.

Peroxidase activity of hemoglobin towards ascorbate and urate: a synergistic protective strategy against toxicity of Hemoglobin-Based Oxygen Carriers (HBOC).

Acellular hemoglobins developed as oxygen bridging agents with volume expanding properties ("blood substitutes") are prone to autoxidation and oxidant-mediated structural changes in circulation. In the presence of hydrogen peroxide and either ascorbate or urate we show that ferric hemoglobin functions as a true enzymatic peroxidase. The activity saturates with both substrates and is linearly dependent on protein concentration. The activity is enhanced at low pH with a pKa of 4.7, consistent with protonation of the ferryl species (Fe(IV)-OH) as the active intermediate. To test whether these redox reactions define its behaviour in vivo we exchanged transfused guinea pigs with 50% polymerized bovine Hb (PolyHbBv) and monitored plasma levels of endogenous ascorbate and urate. Immediately after transfusion, met PolyHbBv levels increased up to 30% of total Hb and remained at this level during the first 24 h post transfusion. Plasma ascorbate decreased by 50% whereas urate levels remained unchanged after transfusion. A simple kinetic model, assuming that ascorbate was a more active ferric heme reductase and peroxidase substrate than urate, was consistent with the in vivo data. The present finding confirms the primary and secondary roles of ascorbate and urate respectively in maintaining the oxidative stability of infused Hb.

Inhaled nitric oxide enables artificial blood transfusion without hypertension.

BACKGROUND: One of the major obstacles hindering the clinical development of a cell-free, hemoglobin-based oxygen carrier (HBOC) is systemic vasoconstriction. METHODS AND RESULTS: Experiments were performed in healthy mice and lambs by infusion of either murine tetrameric hemoglobin (0.48 g/kg) or glutaraldehyde-polymerized bovine hemoglobin (HBOC-201, 1.44 g/kg). We observed that intravenous infusion of either murine tetrameric hemoglobin or HBOC-201 induced prolonged systemic vasoconstriction in wild-type mice but not in mice congenitally deficient in endothelial nitric oxide (NO) synthase (NOS3). Treatment of wild-type mice by breathing NO at 80 ppm in air for 15 or 60 minutes or with 200 ppm NO for 7 minutes prevented the systemic hypertension induced by subsequent intravenous administration of murine tetrameric hemoglobin or HBOC-201 and did not result in conversion of plasma hemoglobin to methemoglobin. Intravenous administration of sodium nitrite (48 nmol) 5 minutes before infusion of murine tetrameric hemoglobin also prevented the development of systemic hypertension. In awake lambs, breathing NO at 80 ppm for 1 hour prevented the systemic hypertension caused by subsequent infusion of HBOC-201. CONCLUSIONS: These findings demonstrate that HBOC can cause systemic vasoconstriction by scavenging NO produced by NOS3. Moreover, in 2 species, inhaled NO administered before the intravenous infusion of HBOC can prevent systemic vasoconstriction without causing methemoglobinemia.

How to evaluate blood substitutes for endothelial cell toxicity.

The most common and widely transplanted tissue worldwide is blood. But concerns about safety and adequacy of blood transfusion have fostered 20 years of research into blood substitutes such as oxygen carriers based on modified hemoglobin (Hb). Chemically modified or genetically engineered Hb developed as oxygen therapeutics are designed to restore blood volume and to correct oxygen deficit due to ischemia in a variety of clinical settings. Uncontrolled oxidative reactions mediated by large amounts of cell-free Hb and their reactions with various oxidant/antioxidant and cell signalling systems emerge as an important pathway of toxicity. Hemoglobin can react with oxygen and NO, leading to the production of reactive oxygen or nitrogen species. Inside the bloodstream, oxidized Hb and ROS/RNS are in direct contact with endothelial cells (EC). Thus, chain reactions may trigger molecular and cellular biology, causing oxidative stress-related pathologies. This editorial presents an overview of interactions between Hb (modified or not) and EC. We also propose a wide range of techniques and methods to assess oxidative stress and inflammation responses of EC after exposure to Hb. This editorial can serve as a guide to evaluate in vitro toxicity of new Hb molecules.

All hemoglobin-based oxygen carriers are not created equally.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) also known as "blood substitutes" have been under active clinical development over the last two decades. Cell-free Hb outside its natural protective red blood cell environment, as is the case with all HBOCs, has been shown to be vasoactive in part due to the scavenging of vascular endothelial nitric oxide (NO) and may in some instances induce heme-mediated oxidative stress. Chemical modification intended to stabilize HBOCs in the tetrameric or polymeric forms introduces conformational constraints that result in proteins with diverse allosteric responses as well as oxidative and nitrosative redox side reactions. Intra and inter-molecular cross-linking may in some instances also determine the interactions between HBOCs and normal oxidative inactivation and clearance mechanisms. Oxygen and oxidative reactions of normal and several cross-linked Hbs as well as their interactions with endogenous plasma protein (haptoglobin) and cellular receptor pathways (macrophage CD163) differ significantly. Therefore, safety and efficacy may be addressed by designing HBOCs with modifications that limit hypertension, minimize heme destabilization and take into account endogenous Hb removal mechanisms to optimize exposure times for a given indication.

Effects of endogenous ascorbate on oxidation, oxygenation, and toxicokinetics of cell-free modified hemoglobin after exchange transfusion in rat and guinea pig.

Chemically modified hemoglobin (Hb) solutions are promising oxygen therapeutics; however, these agents are prone to intravascular oxidation. Using a 50% exchange transfusion (ET) model with bovine polymerized hemoglobin (PolyHbBv), we examined heme oxidation, oxygenation markers, and toxicokinetics in rats, an ascorbic acid (AA)-producing species, and in guinea pigs, a non-AA-producing species. Plasma AA decreased by 50% in guinea pigs after ET, but it was unchanged in rats for the first 20 h post-ET. Both species cleared PolyHbBv from the circulation at similar rates. However, exposure to ferric PolyHbBv over time was 5-fold greater in the guinea pig. Mass spectrometry analysis of plasma revealed oxidative modifications within the tetrameric fraction of PolyHbBv in guinea pig. Oxygen equilibrium curves of PolyHbBv measured in plasma after ET were more left-shifted in guinea pigs compared with rats, consistent with increased ferric PolyHbBv formation. Renal hypoxia-inducible factor (HIF)-1alpha, whose activity strictly depends on the partial pressure of oxygen increased over time, and it correlated inversely with circulating ferrous PolyHbBv in both species. Interestingly, HIF-1alpha activity was greater in guinea pigs compared with rats at 72 h post-ET. Mean arterial pressure increases were also greater in guinea pigs; however, minimal differences in cardiac and renal pathology were observed in either species. The present findings suggest the importance of plasma AA in maintaining the stability of acellular Hb susceptible to oxidation, and they may be relevant to humans, which display a similar plasma/tissue antioxidant status to guinea pig.

First-generation blood substitutes: what have we learned? Biochemical and physiological perspectives.

Chemically modified or recombinant hemoglobin (Hb)-based oxygen carriers (HBOCs) have been developed as oxygen therapeutics or 'blood substitutes' for use in a variety of clinical settings. Oxidative and nitrosative reactions of acellular Hb can limit the effectiveness and compromise the safety of HBOCs. The reactions between Hb and biologically relevant redox active molecules may also perturb redox sensitive signaling pathways. In recent years, systematic in vitro and in vivo structural and functional evaluation of several HBOCs has been carried out and, in some cases, delineated the 'structural' origin of their toxicity. This enables potential protective strategies against Hb-mediated side reactions to be rationally suggested. Here the authors provide an overview of their research experiences, novel insights into the molecular basis of toxicities of these products and some lessons learned.