The history of hemoglobin-based oxygen carriers: a narrative review

Introduction

Hemoglobin-based oxygen carriers (HBOCs) have been explored for more than a century as potential alternatives to allogeneic red blood cell (RBC) transfusion, particularly in settings where blood is unavailable, unsafe, or unacceptable. A modeling study published in 2019 predicted an annual 102-million-unit blood shortage in low- and middle-income countries (LMICs) (1). Allogeneic blood requires time for compatibility matching while incurring costs. Furthermore, some patients decline allogeneic blood for personal, cultural, or religious reasons. Every blood transfusion carries the risk of immunological reactions, not the least of which is that all allogeneic blood suppresses infection and cancer immune systems for some period (potentially life long) after infusion (2). For these scenarios, a shelf-stable, universally compatible oxygen-carrying therapy remains an unmet clinical need.

HBOCs are synthetic or semi-synthetic compounds designed to transport oxygen from the lungs to the tissues, potentially in a similar manner to RBCs without requiring the cellular structure or storage constraints of blood products (3). Despite their promise, clinical translation has been challenging. Multiple products have reached human trials, yet none have achieved full U.S. Food and Drug Administration (FDA) approval due to persistent safety and efficacy concerns (4). A central difficulty in evaluating HBOCs is the absence of a standardized safety benchmark for allogeneic blood itself. Modern blood transfusion practices predate contemporary requirements for randomized controlled trials; therefore, no comprehensive safety profile comparable to an FDA review exists for RBCs. This complicates regulatory pathways for HBOCs, which must demonstrate safety relative to a product with incomplete historical data. In the United States, most opportunities for HBOC use arise through expanded-access mechanisms in patients for whom blood is not an option, making systematic data collection more difficult. The collection of necessary FDA quality, safety and efficacy data from patients who receive HBOCs in isolated “one-off” cases presents immense challenges. Overcoming regulatory hurdles will include (I) understanding of the FDA requirements and limitations and/or (II) designing studies to gather high-quality survival data in austere environments where blood is not an option.

This narrative review article provides a concise overview of the historical evolution of HBOCs, summarizes the scientific and clinical barriers that have shaped their development, and outlines the current state of emerging HBOC technologies. By synthesizing past lessons with ongoing innovations, we aim to clarify the opportunities and challenges that remain in the effort to create a safe, effective, and accessible alternative to allogeneic RBC transfusion.

Background

Hemoglobin, the oxygen-regulatory molecule in RBCs, has long been recognized as a critical component for maintaining tissue oxygenation. The concept of developing hemoglobin-based substitutes emerged as an obvious pharmaceutical way to carry oxygen. If one could take the hemoglobin molecule, manipulate it, and get it to deliver oxygen outside of the cell membranes of RBCs that carry immune signals and without the plasma that has viruses, proteins, and other adverse compounds, then perhaps a pharmaceutical alternative to banked blood could be created. The development of a hemoglobin-based blood substitute has evolved within a limited understanding of tissue oxygen delivery; hemoglobin is complex and has many constraints and regulatory functions.

Early attempts to create HBOCs involved the use of free hemoglobin extracted from human or animal sources. However, these early formulations were associated with significant side effects, such as vasoconstriction, oxidative stress, and renal toxicity, largely due to the instability of free hemoglobin in the bloodstream (5). Over the years, advancements in biochemistry and biotechnology have led to the development of more sophisticated HBOCs, including chemically modified hemoglobin, cross-linked hemoglobin, polymerized hemoglobin, and encapsulated hemoglobin (3). These innovations aim to address the limitations of earlier generations by improving stability, reducing toxicity, and enhancing oxygen delivery to tissues.

Rationale and knowledge gap

The development of HBOCs has been driven by the growing demand for alternatives to traditional blood transfusions. Furthermore, HBOCs could play a critical role in emergency and military settings, where rapid access to blood products may be restricted, and in populations that refuse blood products for religious or other reasons. However, the clinical translation of HBOCs has been hindered by a range of challenges, including adverse effects such as hypertension, oxidative damage, and inflammatory responses. Understanding the underlying mechanisms of these side effects and developing strategies to mitigate them are essential for advancing HBOC technology (2).

Despite significant progress in HBOC research, knowledge gaps remain. The mechanisms underlying the vasoactive and pro-inflammatory effects of HBOCs are not fully understood, limiting the ability to design safer formulations (6). Additionally, there is a lack of comprehensive clinical data on the long-term effects of HBOC use, particularly in diverse patient populations and across different clinical scenarios. Furthermore, the field lacks consensus on optimal methods for evaluating HBOC performance, including metrics for assessing oxygen delivery, tissue perfusion, and biocompatibility (7). Addressing these gaps is critical for advancing HBOCs from experimental prototypes to clinically viable products. We present this article in accordance with the Narrative Review reporting checklist (available at https://aob.amegroups.com/article/view/10.21037/aob-2025-1-46/rc).

Methods

This narrative review was developed through a structured, author-driven literature identification process rather than a formal database-defined systematic search (Table 1). Each author was assigned one or more thematic sections based on their expertise (e.g., historical development, biochemical mechanisms, clinical trials, regulatory considerations, and emerging technologies). Authors independently identified, reviewed, and selected literature relevant to their assigned sections.

To ensure adequate breadth and prevent omissions of key work, authors used both targeted searches and backward citation tracking from foundational papers, major reviews, and clinical trial reports. Searches focused on peer-reviewed publications, authoritative historical accounts, regulatory documents, and preclinical and clinical studies related to HBOCs.

Cell-free oxygen carrier

The concept of stroma-free hemoglobin (SFH) emerged as an attractive proposition in the early 20th century. Early investigations focused primarily on the clinical applicability of SFH, while later studies, particularly from the 1940s through the 1960s, examined SFH’s contribution to renal injury in hemolytic transfusion reactions and malarial blackwater fever (8-12).

One of the earliest documented applications was reported by von Stark, who administered subcutaneous hemoglobin into anemic patients but failed to obtain a stable formulation (12). Subsequently, Sellards and Minot developed a more refined preparation from lysed, washed red cells and infused volumes of 5–33 mL into 33 subjects. Although all recipients developed hemoglobinuria, 30 reported no overt adverse effects, whereas three experienced pyrexia, chills, headache, nausea, flushing, and insomnia (13). Additional small trials yielded similar outcomes.

A notable case was described by Amberson et al., where SFH was administered to a woman with postpartum hemorrhage when cross-matched blood was unavailable (14). Her hemodynamic status improved initially, but she later developed oliguria and died of renal failure. The investigators observed hypertension and bradycardia following SFH infusion and concluded that SFH was not suitable for clinical use (14). Subsequent studies confirmed that SFH reduced renal plasma flow, creatinine clearance, insulin clearance, and para-aminohippurate (PAH) clearance (15). In a U.S. Navy-sponsored trial, 47 patients received SFH infusions; over one-third of recipients developed hypertension, bradycardia, and reduced urea clearance (9).

Further refinements in SFH preparation were used in small-scale clinical studies (16). In a noteworthy trial, Savitsky et al. infused purified SFH into eight human volunteers. All developed oliguria with reduced creatinine clearance; two subjects reported abdominal pain, and seven developed bradycardia and hypertension. These effects were attributed to vasoconstriction intrinsic to hemoglobin. Consequently, SFH was abandoned as a potential blood substitute (17).

Meanwhile, animal studies offered a contrasting perspective. A study in dogs by Rabiner et al. reported that purified SFH could circumvent renal toxicity and serve as an oxygen-carrying plasma expander in hemorrhagic shock (16). A subsequent study by Rabiner et al. demonstrated that SFH was lifesaving in dogs undergoing exchange transfusion to critically low hematocrits (18). SFH conferred distinct advantages, including: effective oxygen transport and exchange, improved survival during prolonged hypoxia, absence of renal toxicity in the canine model and properties consistent with those of an “ideal” plasma expander. These developments highlighted the model-dependent nature of SFH toxicity and species-dependent differences in tolerance (17).

Advances in hemoglobin biochemistry progressed in parallel to translational efforts. Three-dimensional structural studies of hemoglobin by Perutz and Kendrew provided critical insights, for which they won the Nobel Prize in 1962 (19). Subsequently, the elucidation of hemoglobin’s amino acid composition enabled biochemical modification of the molecule. Bunn et al. demonstrated that chemical cross-linking with bis(N-maleimidomethyl) ether (BME) stabilized the hemoglobin tetramer, preventing dimer dissociation and markedly reducing nephrotoxicity (20).

The possibility of improving safety and securing patentable formulations fueled intensive public and private research. Further scientific development marked the shift toward molecular modification strategies aimed at altering the biochemical behavior and safety profile of hemoglobin for therapeutic use.

Generations of HBOCs

HBOCs have undergone several stages of development, each addressing issues identified with earlier designs, such as oxidative stress, vasoconstriction, and short circulatory half-lives (Table 2). Winslow described these as different “generations” of HBOCs, though consensus on generational classification varies (9). Recent reviews, including those by Jansman and Hosta-Rigau, Sen Gupta, and Chen et al., Hess (2024), and Jahr et al. (2025), emphasize that HBOC evolution is best understood through the lens of chemical and structural modification strategies such as cross-linking, polymerization, conjugation, encapsulation, and recombinant engineering approaches (3,4,21-23). Thus, they grouped them based on how the hemoglobin molecules were modified without assigning rigid generational boundaries.

One of the primary goals of early HBOC research was to stabilize hemoglobin outside of RBCs. Free hemoglobin rapidly breaks apart into smaller non-functional dimers, which are quickly cleared from circulation through the kidneys. This leads to short circulatory half-lives and significant renal damage. Moreover, free hemoglobin scavenges nitric oxide (NO), triggering vasoconstriction and hypertension. To solve this, researchers employed chemical cross-linking to stabilize the hemoglobin tetramer (α₂β₂). Cross-linking is the most emphasized method of hemoglobin modification, resulting in reduced oxygen affinity, improved oxygen transportation, and a better half-life compared to unmodified SFH. It is now recognized that many HBOC-associated toxicities arise not solely from NO scavenging but also from structure-dependent oxidative pathways mediated by the heme prosthetic group, including reactive oxygen species (ROS) generation, ferryl species formation, and sterile inflammatory signaling that are independent yet synergistic with NO-related effects (24,25).

Hess (2024 review) defined seven major toxicities of hemoglobin, in modified and α-α crosslinked forms; uncrosslinked hemoglobin dimerizes and damages the proximal tubule via oxidation which leads to high-output renal failure; NO is bound and oxidized by hemoglobin which leads to systemic and pulmonary vasoconstriction; in Choi cultures of fetal mouse brain, hemoglobin, heme, and iron are excitatory neurotoxins and exposure leads to neuronal death; hemoglobin activates human macrophages leading to secretion of tumor necrosis factor alpha (TNFα) and interleukin-8 (IL-8) and thus, human endothelial cells bind neutrophils and allow neutrophil diapedesis; bacterial endotoxin activity is increased by hemoglobin; hemoglobin supplies Escherichia coli and Listeria monocytogenes with a source of iron that promotes growth; hemoglobin draws water into intravascular spaces and dilutes coagulation proteins because is it osmotically active (4,26). Subsequent structure–toxicity analyses have demonstrated that molecular size, degree of cross-linking or polymerization, and surface chemistry critically influence the magnitude of NO scavenging, oxidative stress, and endothelial activation (4,23). Mechanistic work by Alayash and colleagues has demonstrated that oxidative side reactions arising from heme redox transitions (ferrous → ferric → ferryl states) and associated ROS production contribute substantially to endothelial activation, inflammation, and cellular injury in HBOC exposure (24).

Intramolecular cross-linked hemoglobin

Intramolecular cross-linked hemoglobin refers to molecules where covalent bonds are created within the hemoglobin tetramer, usually between the two α-subunits or two β-subunits using a site-specific crosslinker, to prevent dissociation. These cross-links can be formed using reagents such as diaspirin or raffinose. Early studies in the 1980s and 1990s demonstrated that intramolecular cross-linking, for example, with pyridoxal-based compounds, could approximately double the circulation half-life and maintain good oxygen-carrying function in animal studies (27).

A notable example is Diaspirin Cross-Linked Hemoglobin (DCLHb), which involves α-α cross-linking. DCLHb was first developed by Walder and colleagues at the University of Iowa, subsequently licensed to Baxter Healthcare, and developed initially through a collaborative effort between Baxter and the United States Army, then continued by Baxter alone (28). DCLHb was produced and marketed by Baxter Healthcare (Deerfield, IL, USA) under the trade name HemAssist. DCLHb used a cross-linker called bis(3,5-dibromo salicyl) fumarate (DBBF) to connect Lys99 residues on both α-subunits in the deoxygenated state (29). Other reagents explored for cross-linking included 2,5-double isothiocyanate benzene sulfonate (DIBS), 1,3-butadiene bicyclic oxidate (BUDE), glutaraldehyde (GDA), and pyridoxal 5'-phosphate (PLP).

DCLHb was one of the first cross-linked HBOCs to reach large clinical trials in the 1990s. The JAMA trauma study showed that it circulated longer than free hemoglobin but also caused side effects such as hypertension and myocardial ischemia (Table 3) (30). These effects were later traced to NO scavenging and oxidative reactions, problems now recognized as major causes of vasoconstriction and toxicity in early HBOCs. Baxter’s HemAssist was the first HBOC that advanced to some of the phase II and phase III trials; however, they ultimately stopped all trials and discontinued HemAssist (38-40).

Although intramolecular cross-linking solved the issue of tetramer dissociation, it did not fully eliminate these side effects. Modern research is now moving toward more advanced designs, such as engineered hemoglobin variants, PEGylated conjugates, encapsulated formulations, and recombinant molecules, which aim to maintain hemoglobin stability while reducing NO scavenging and oxidative stress. Recent clinical and preclinical analyses have emphasized that remaining toxicity risks are linked to heme-driven redox mechanisms, including ROS and ferryl intermediates that can trigger sterile inflammation and mitochondrial dysfunction beyond NO interactions (25).

Advances in nanomaterial-related HBOCs, including liposome-encapsulated hemoglobin and polymer nanocapsules reviewed by Zhu et al. (2024), demonstrate that encapsulation strategies can simultaneously address vasoactivity, oxidative toxicity, and circulatory half-life limitations while enabling applications beyond hemorrhagic shock, including ischemic stroke, cancer therapy, and wound healing (41). Nanostructured carriers such as liposomal vesicles, polymeric nanoparticles, and metal-organic-framework encapsulates (e.g., Hb@ZIF-8) have shown improved colloidal stability, controlled oxygen release, and reduced immune clearance, suggesting broader utility in acute and chronic hypoxia contexts.

Intramolecularly cross-linked hemoglobin remains an active area of investigation, with recent structure-guided modification strategies described by Kim et al. (2024) demonstrating that advances in site-specific cross-linking chemistries, such as azido acyl methyl phosphate reagents and bio-orthogonal CuAAC “click” coupling, generating larger, more stable hemoglobin conjugates that retain oxygen affinity while reducing vasoactivity (42). These larger conjugates are less likely to extravasate and scavenge NO, reducing vasoactive side effects, and may hold promise for ex vivo organ perfusion applications (42,43). However, no intramolecularly cross-linked hemoglobin product has received regulatory approval for routine clinical use, and clinical translation is limited to investigational and compassionate use settings.

Polymerized hemoglobin

After the DCLHb experience, from the 1990s to 2000s, researchers shifted toward hybrid or newer strategies, combining intramolecular stabilization with PEGylation, surface shielding, encapsulation (in liposomes), and recombinant engineering (mutations to reduce NO affinity or oxidation), all aimed at maintaining stability while reducing NO scavenging and oxidative stress. To further increase molecular size and reduce extravasation, intermolecular polymerization was performed. By linking multiple hemoglobin tetramers together using agents such as GDA, scientists produced high-molecular-weight polymers that stayed intravascular longer and were less nephrotoxic. This approach led to the development of several notable products, including PolyHeme (Northfield Laboratories, Inc.), Hemopure (HbO₂ Therapeutics), and Oxyglobin (HbO₂ Therapeutics). Polymerized HBOCs displayed improved plasma retention and reduced renal toxicity. However, vasoactivity persisted due to continued NO scavenging, and oxidative tissue injury remained problematic. Work by Palmer and colleagues has shown that controlled fractionation and PEGylation of polymerized hemoglobins can further modulate biophysical properties and reduce toxicity while preserving oxygen delivery capacity (44,45). Among these, Oxyglobin gained approval for veterinary use, while others, such as PolyHeme and Hemopure, failed to achieve widespread clinical acceptance after late-stage trials (44,46).

Polyethylene glycol (PEG) conjugation and surface shielding

PEG is biologically inert, biocompatible, and known to reduce immunogenicity (a concept pioneered in the 1970s for interferon and enzymes). PEG conjugation was introduced to the hemoglobin structure in the late 1990s, attaching long, flexible PEG chains to the hemoglobin surface. PEGylation increased molecular size further and forms a hydration shell around the hemoglobin molecule, which improves circulatory half-life and shields the protein from NO and oxidative interactions compared to unmodified or polymerized hemoglobin. PEGylated HBOCs [e.g., MP4 (Sangart, Inc.), Sanguinate (Prolong Pharmaceuticals), and Euro-PEG-Hb (EuroBloodSubstitutes Project)] demonstrated longer circulatory half-lives and improved safety profiles, though high oxygen affinity remained a limitation for clinical use (43). Although PEGylation reduces direct endothelial interaction and NO scavenging, it does not fully suppress heme-mediated oxidative pathways, which remain a mechanistically important contributor to observed adverse effects (25).

Importantly, analyses by Natanson et al. (2008) and Estep (2025) demonstrate that MP4OX (also known as MP4; Sangart, Inc.) exhibited a higher incidence of myocardial infarction (MI) compared to cross-linked and polymerized HBOCs at comparable doses (6,47). Estep’s analysis suggests this increased risk correlates with higher rates of autoxidation and heme loss from the PEGylated formulation, leading to enhanced oxidative stress and potentially increased thrombotic risk rather than coronary vasoconstriction. Notably, assessment of in vivo blood flows across multiple species and HBOC formulations did not detect reductions in coronary blood flow even when systemic vasoconstriction occurred, arguing against coronary vasoconstriction as the primary mechanism of MI (47). Whether this represents a general limitation of PEGylation or is specific to the MP4 formulation remains unclear, but these findings indicate that PEGylation is not a universal solution for reducing HBOC toxicity.

Products such as MP4OX and PEG-Hb prototypes demonstrated markedly improved tolerability compared with earlier cross-linked HBOCs, though clinical efficacy remained limited. Recent biophysical analyses further indicate that PEGylation reduces endothelial interaction and NO scavenging primarily through steric shielding rather than elimination of underlying redox activity (23,45). Some advances have been made in PEG-conjugated HBOCs, demonstrating potentially improved safety profiles compared to earlier generations of modified HBOCs (45,48).

MP4OX (Sangart, Inc., San Diego, CA, USA) reached late-phase trials but was discontinued between 2013–2014 due to funding and regulatory challenges (49). Despite these setbacks, PEGylation remains a pivotal innovation. It was the first modification to substantially reduce vasoactivity and extend plasma half-life (10–30 hours), providing a foundation for modern HBOC design. But it still did not fully mimic RBCs’ autoregulation, oxygen delivery remained more “fixed” (low P₅₀), and production was expensive. Recent data suggest that combining PEGylation with polymerization may mitigate heme loss; Khan et al. (2025) demonstrated that PEGylated polymerized hemoglobins retained similar heme release rates compared to their precursors, unlike PEGylated monomeric hemoglobin, which showed elevated heme release (45). Current research integrates PEGylation chemistry with encapsulation and recombinant engineering in hybrid platforms designed to further decouple oxygen delivery from vasoactive and oxidative side effects (4,23,45). Understanding and limiting redox-driven mechanisms, specifically ROS generation and ferryl hemoglobin formation associated with free heme moieties and heme dissociation, is now a central focus of next-generation HBOC design strategies (50).

Recombinant engineering and redox-active mutations

Next-generation HBOC design increasingly relies on recombinant engineering to address heme retention, redox instability, and oxidative toxicity at the molecular level, complementing chemical modification strategies employed in earlier generations. Loss of the heme prosthetic group and subsequent formation of ferryl hemoglobin (Fe⁴⁺=O) species are now recognized as central drivers of oxidative tissue injury, lipid peroxidation, and sterile inflammation, representing a critical unresolved mechanism in HBOC toxicity (24,50).

Khan et al. (2025) demonstrated that the β-F41K mutation significantly reduces heme dissociation rates while preserving physiologically relevant oxygen affinity and decreasing auto-oxidation, thereby directly targeting heme-mediated oxidative pathways rather than NO scavenging alone (51). By stabilizing heme-globin interactions, this mutation limits the formation of downstream ferryl species and secondary ROS. This strategy addresses heme-driven redox cycling rather than relying on molecular size or steric shielding to mitigate toxicity.

Complementary strategies include engineering redox-active tyrosine residues (e.g., βT84Y, γL96Y) that facilitate ferryl hemoglobin reduction by endogenous antioxidants such as ascorbate, thereby decreasing lipid peroxidation and oxidative stress without compromising oxygen delivery (52,53). Among these, γL96Y was identified as presenting the best profile of oxidative protection without loss of protein stability or function (53). These mutations enhance intrinsic redox cycling capacity, mimicking protective antioxidant pathways normally present in RBCs.

Cooper et al. (2024) further demonstrated that combining tyrosine substitutions with phenylalanine insertions within the heme pocket (αL29F, γV67F) can simultaneously reduce NO scavenging and oxidative lipid damage (53). PEGylated recombinant variants incorporating these mutations showed no increase in blood pressure following infusion and significantly improved survival in rat hemorrhagic shock models. These findings underscore how structure-guided recombinant design can decouple oxygen delivery from vasoactive and oxidative side effects, representing a rational pathway toward clinically translatable HBOC platforms.

Expanded applications for HBOC use

Alternative uses for HBOCs in limited patient populations and clinical settings other than trauma and cardiac surgery are being evaluated. HBOCs have been investigated for use in perfusing transplantable organs in animal models and in humans. In rat models, HBOC-201 was used for oxygenated warming perfusion of rat livers from cold temperatures; rat livers rewarmed with HBOC-201 showed lower lactate levels compared to cold-stored grafts (54). In humans, normothermic machine perfusion (NMP) has been used in donor livers to assist with hepatobiliary viability assessments prior to transplant, but NMP requires an oxygen-carrying perfusion solution, usually RBCs (55). Using a protocol of sequential dual hypothermic oxygenated machine perfusion (DHOPE), then controlled oxygenated rewarming (COR), then NMP with HBOC-201 with added perfusion fluids, seven initially declined deceased donor livers were placed on this protocol. Five of the seven livers were eventually felt to be transplantable, and the 3-month graft survival of the five transplanted livers was 100%.

Along with perfusing liver grafts, HBOC-201 is being investigated for the perfusion of donor kidneys prior to transplantation. In rat models, HBOC-201 was used to gradually rewarm five hypothermic kidneys compared to using no oxygen carrier to rewarm five hypothermic kidneys. The kidneys rewarmed with HBOC-201 were found to have higher ultrafiltrate production, improved glomerular filtration rate, and better sodium reabsorption (56). Another study used a porcine model to test NMP of kidneys with no oxygen carrier, HBOC-201, or RBCs for 360 min at 37 ℃ (57). The kidneys perfused with HBOC-201 and RBCs showed lower lactate and aspartate aminotransferase levels compared to kidneys perfused with no oxygen-carrier, indicating that an oxygen carrying perfusate was desirable for NMP of kidneys. The kidneys perfused with RBCs showed higher urine production and creatinine clearance; methemoglobin levels increased 45% in the HBOC-201 perfused group, suggesting that HBOC-201 may be used as an RBC alternative but not for extended periods of time. As well as perfusing transplantable organs, HBOC-201 is under investigation for ex-vivo normothermic limb perfusion (EVNLP) in a porcine model with muscle contractility preserved for approximately 11 hours (58).

Case reports and case series have described HBOCs being used selectively to treat life-threatening anemia in specific patient populations. In a notable case of a patient with an alloantibody to Rh17 for whom no compatible RBC units were available, Hemopure (HBOC-201) was used for management of critical anemia secondary to acute lymphoblastic leukemia (59). In a case series of three patients with sickle cell crisis for whom RBC transfusion was not an option, HBOC-201 was used to successfully support the patients to discharge; two patients were managed with greater than 20 units of the HBOC (60). Another case series described three patients with sickle cell disease who were supported with Expanded Access use of HBOC-201 (61).

Discussion

The narrative review describes the growth of the HBOC field, from inception to contemporary times. The central drive of HBOC development has been to create an oxygen transport system that mimics the oxygen-carrying function of RBCs. Invention and manufacture of HBOCs aim to address several issues related to RBC transfusions, such as shortages of donor RBCs, the risks associated with blood transfusions, providing an alternative for patients with reservations about receiving blood, offering an off-the-shelf product that does not require blood typing, and ensuring readily available blood substitutes in remote or battlefield settings.

The earliest form of HBOCs consisted of SFH solutions, which were purified soluble hemoglobin prepared by lysing RBCs and removing the stroma and cell membranes. Although these HBOCs had conceptual advantages over donor RBCs, such as room temperature stability and not requiring crossmatches before transfusion, they also presented significant pharmacokinetic and physiological limitations, including rapid clearance, low oncotic pressure, and inadequate oxygen unloading. Clinical studies revealed severe adverse reactions, including renal toxicity, vasoconstriction, oxidative stress, methemoglobinemia, and systemic inflammatory effects.

HBOCs developed via polymerized hemoglobin, designed to overcome the limitations of stroma-free HBOCs, were shown to have rapid clearance with oxidative and vasoconstrictive side effects. In this case, hemoglobin was chemically crosslinked using agents like GDA or o-raffinose, which created larger molecules that slowed renal clearance and prolonged circulation while reducing NO scavenging effects. However, despite significant advancements, polymerized hemoglobin HBOCs still exhibited limitations and adverse effects during clinical trials, such as reduced but persistent NO scavenging, systemic and pulmonary hypertension, increased rates of MI, oxidative stress, methemoglobinemia, systemic inflammatory response, and hepatotoxicity.

Intramolecularly cross-linked HBOCs were developed based on the idea of stabilizing hemoglobin as a single macromolecule. Crosslinking stabilizes hemoglobin tetramers, eliminating the possibility of forming nephrotoxic dimers. Additionally, it increased their circulatory half-life to 6–12 hours, compared to just 1–2 hours for uncrosslinked free hemoglobin. However, like polymerized hemoglobin, intramolecularly cross-linked HBOCs had adverse effects that limited their clinical success. The key issues included NO scavenging, leading to vasoconstriction and hypertension, auto-oxidation to methemoglobin, generation of ROS, and altered oxygen affinity.

PEGylated HBOCs improved molecular stability, plasma retention, and reduced vasoactive and oxidative effects, which were significant limitations of previous HBOC generations. While PEG-conjugated HBOCs are considered safer than earlier versions, they are not without side effects. Concerns include endothelial oxidative stress due to ROS formation, mild NO scavenging effects, volume overload, and immunogenicity.

Encapsulated HBOCs were designed to closely mimic the physiological properties of RBCs. These HBOCs consist of purified hemoglobin enclosed in a biocompatible membrane, providing a physiological barrier between hemoglobin and the vascular endothelium, which helps to reduce toxic effects such as renal toxicity and NO scavenging, as well as mitigating the rapid clearance issues associated with other HBOC formulations. However, they still face potential limitations, such as membrane instability and premature clearance due to uptake by the reticuloendothelial system. Currently, these HBOCs primarily remain in preclinical and early translational stages. Of all the developed HBOC agents, Hemopure has received approval for clinical use in South Africa and Russia; however, in the USA, it is classified as an investigational new drug available for compassionate use on a case-by-case basis (61).

The literature describing HBOCs spans nearly one hundred years, with over 1,100 citations on PubMed. The goals of this narrative review were to concisely describe the history and development of HBOCs, summarize the scientific and clinical barriers that have shaped their advancement, and lay out the current state of emerging HBOC technologies. Detailed discussions of the biochemistry of various HBOC formulations, perfluorocarbon-based oxygen carriers (ABL-101), as well as the future directions of HBOCs were out of scope of this review. Additionally, out of scope was discussion of HEMO2life (Hemarina), a cell-free oxygen carrier derived from the marine lugworm (Arenicola marina) (62). Limitations of this review include the references selected by the authors, rather than the authors performing a database-defined systematic literature search. Author-directed literature reviews focused on peer-reviewed publications, authoritative historical accounts, regulatory documents, and preclinical and clinical studies related to HBOCs. References selected were in English. Key words may have been inadvertently excluded from the literature review.

Conclusions

The early quest for HBOC invention was to develop a universal RBC substitute. Future directions may evolve toward RBC substitutes for niche populations, such as patients with limited availability of rare antigen-negative RBC units, combat settings, and individuals who cannot receive blood products for various reasons, as well as for ex vivo applications such as organ preservation prior to transplant.

Multiple generations of HBOCs have been evaluated, but clinical success so far has been limited by significant challenges, particularly oxidative stress-related toxicities. Newer approaches, including encapsulated HBOCs, are currently being investigated and aim to more closely replicate the structure and function of native RBCs. Continued innovation holds promise for future clinical applications in this field. However, these products remain largely in the preclinical stage. Further randomized, outcome-focused clinical trials are required to establish their safety and potential role in clinical practice.

Acknowledgments

Many thanks to Dr. John R. Hess for sharing his valuable perspective and experience with HBOCs.

Footnote

Provenance and Peer Review: This article was commissioned by the Guest Editor (Mark T. Friedman) for the series “Artificial Blood” published in Annals of Blood. The article has undergone external peer review.

Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at https://aob.amegroups.com/article/view/10.21037/aob-2025-1-46/rc

Peer Review File: Available at https://aob.amegroups.com/article/view/10.21037/aob-2025-1-46/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://aob.amegroups.com/article/view/10.21037/aob-2025-1-46/coif). The series “Artificial Blood” was commissioned by the editorial office without any funding or sponsorship. G.K.G. reports a NIH/NCI Cancer Center Support Grant (P30CA008748), unrelated to this submitted work. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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