The evolution of artificial red blood cells and recent advances in state-of-the-art oxygen carrier technologies: a narrative review

Abstract

The demand for homologous blood transfusions has reached an unprecedented level, driven by a declining donor population and the ever-increasing need for blood products. While significant advancements have been made in medical equipment and techniques, a critical gap remains in developing an effective alternative to conventional blood transfusion. Medical research to find a proper blood substitute involves many previous experiments. The search for a blood substitute has been ongoing for patients for whom human blood is unavailable, with a few products showing promise in this field. Recent advancements in medical innovation have begun to address this challenge, notably through the development of artificial oxygen carriers (AOCs). These laboratory-synthesized alternatives to traditional blood transfusions offer a means of bypassing the need for human blood, particularly packed red blood cell (pRBC) transfusions. While AOCs fulfill the singular, critical role of in vivo oxygen delivery, the term is frequently used interchangeably with the broader concept of artificial blood. Various AOC products are currently in different stages of clinical development. Most Notable examples include Perftoran, which has been approved in Russia, Kazakhstan, Ukraine, the Kyrgyz Republic, and Mexico and has been administered to over 35,000 patients. Another significant product, Hemopure, has received clinical use approval in South Africa and Russia and has obtained expanded use approval from the United States Food and Drug Administration (USFDA). This article examines the landscape of AOCs, including their preparation methods, available products, regulatory approval status, current applications, limitations, and potential for future use in medical practice. This review article offers an overview of the different types of AOCs currently available, focusing on their clinical development for human use.

1. INTRODUCTION

In the current era of rapid advancements in medicine and medical technology, one of the persistent challenges in the healthcare sector is the availability of blood products for transfusion. Blood transfusion is a critical component in numerous medical procedures, particularly in surgeries, where it is indispensable for patient survival and recovery.¹ Cardiac surgeries, for instance, alone account for approximately 20% of all transfusions in medical settings. Despite the essential role of blood transfusion, several issues impede its practical use.

A significant challenge is the global shortage of blood donors, which directly impacts the availability of blood products. Additionally, allogeneic blood transfusions involving blood from a donor carry inherent risks, including transfusion-related acute lung injury (TRALI) and transfusion-associated circulatory overload (TACO).² There is also the potential for the transmission of blood-borne infections such as HIV and Hepatitis C, further complicating the transfusion process.³ Furthermore, the difficulty of finding compatible blood for patients with rare blood types (such as Bombay blood type, present in less than 1% of the world’s population) adds another layer of complexity to managing transfusions. Certain religious beliefs, such as those held by Jehovah’s Witnesses, present additional challenges, as some individuals may refuse blood transfusions on religious grounds.⁴ Additionally, human blood requires specialized storage conditions, has a short shelf-life, and lacks portability, further limiting its utility in medical procedures.⁵

In response to these challenges, artificial oxygen carriers (AOC) have been developed. They are synthetically engineered solutions aimed at replicating the oxygen transport function of human blood by facilitating efficient gas exchange. These carriers are designed to bind, transport, and deliver oxygen to various tissues and organs. They are commonly known as “artificial blood,” as they fulfill the essential role of oxygen delivery; however, they do not exhibit the broader physiological functions of natural blood, such as immune defence, nutrient transport, thermoregulation, and coagulation.

2. ARTIFICIAL OXYGEN CARRIER

As described by William Amberson in 1937, “The oxygen capacity of the blood substitute is of vital importance, and there is no substitute for haemoglobin.”⁶ Hence, the search for substitutes for the primary function of blood was initiated globally. The introduction of novel artificial erythrocyte substitutes, also known as AOC, offers a promising solution to this challenge. These substitutes fulfill the primary function of blood—oxygen transport and are often called AOCs.⁷ AOCs possess several advantages over traditional blood products, including longer shelf lives,⁵ biocompatibility with all blood groups (eliminating the need for cross-matching),³ and the absence of any risk for disease transmission. They also offer a prolonged intravascular circulation time and a high affinity for gases such as oxygen and carbon dioxide (CO₂), facilitating efficient gas transport and mitigating the risks associated with allogeneic erythrocyte transfusions.⁴ A brief history of AOCs development is summarized in Table 1.

Table 1.

A brief history of artificial oxygen carrier’s development.

This article provides a classification of AOCs into 2 distinct categories, based on the hemoglobin (Hb) and further subdivided by the state and sources of oxygen-carrying mechanism. Figure 1 shows the qualities of perfect AOC. The classification for understanding is as follows.

Figure 1.

Characteristics of an ideal artificial oxygen carrier.

2.1. Hb-based oxygen carriers

Hemoglobin-based oxygen carriers (HBOCS) can be further classified based on the state of Hb and the source of Hb as follows:

• A.Classification based on the state of Hb

  • Acellular HBOCS

  • Cellular HBOCS

  • Stem cell–derived oxygen carriers (SCOCs)

• B.Classification based on the source of Hb

  • Human Hb-derived HBOCs

  • Animal Hb-derived HBOCs

  • Recombinant Hb-based HBOCs

2.2. Non-HBOCs

• Perfluorocarbon-based oxygen carriers (PFCs)

• Synthetically derived porphyrin-based AOCs

• Micro/nanobubble-mediated oxygen transport

3. Hb-BASED OXYGEN CARRIERS

HBOCs are engineered to mimic the oxygen transport function of endogenous Hb by reversibly binding oxygen at their heme sites. They circulate freely within the plasma which allow improved oxygen delivery to regions experiencing compromised blood flow. HBOCs are developed from outdated human or bovine blood or recombinant sources, which serve as the raw material for their production. Since native Hb cannot be used directly as an oxygen-carrying component due to its instability and associated toxicity, it undergoes chemical modification and microencapsulation to function effectively as an alternative oxygen transporter.⁸

HBOCs demonstrate a sigmoidal oxygen dissociation curve and possess lower p50 values than native human Hb. This characteristic facilitates more efficient oxygen unloading at the tissue level. However, the use of cell-free Hb in its unmodified form presents several clinical challenges. It is known to scavenge nitric oxide (NO), leading to vasoconstriction; it exhibits abnormally high oxygen affinity, is rapidly cleared from the circulation, and has been associated with nephrotoxicity.⁹

Various chemical and structural modifications are applied to cell-free Hb derived from both human and bovine sources to overcome these limitations. These modifications are essential to enhance biocompatibility and functional performance, enabling HBOCs to serve as effective red blood cell (RBC) substitutes. The specific strategies employed in developing and optimizing HBOCs are detailed in the preceding paragraph.⁹

4. CLASSIFICATION OF HBOCs BASED ON THE STATE OF Hb

4.1. Acellular HBOCs

Acellular HBOCs are composed of chemically modified or recombinant Hb that circulates freely in plasma without a surrounding cellular membrane. These modifications are intended to enhance stability, extend half-life, and mitigate toxic effects such as vasoconstriction and oxidative stress.

4.1.1. Polymerized Hb

Polymerization is a chemical modification process that increases the molecular size of Hb by covalently linking multiple Hb units—typically 4 to 5 molecules—thereby forming a macromolecular complex. This increases the circulatory half-life and decreases the rate of renal clearance and extravasation.³ Polymerization is commonly achieved using agents such as glutaraldehyde, which stabilizes the Hb molecule and reduces vasoconstriction commonly seen with unmodified acellular HBOCs.

Examples of polymerized HBOCs include:

• Hemopure: Derived from bovine Hb polymerized with glutaraldehyde.⁵

• PolyHeme: Composed of human Hb polymerized using glutaraldehyde.¹⁰

• Hemoximer (PHP): Produced from human Hb and surface-modified with a polyoxyethylene pyridoxylated polymer.¹¹

4.1.2. Crosslinked Hb

A common limitation of acellular Hb is the tendency for the ferrous iron (Fe²⁺) to oxidize into the ferric form (Fe³⁺), producing methemoglobin, which cannot bind oxygen and may contribute to tissue ischemia.¹² Cross-linking addresses this by chemically binding the alpha or beta chains within the Hb tetramer, preventing dissociation into dimers and minimizing renal filtration and toxicity.⁹ Common chemical cross-linkers include diaspirin and raffinose.

An example of this is:

• HemAssist: A diaspirin-cross-linked human HBOCS.¹³

4.1.3. Polyethylene glycol-conjugated Hb

PEGylation involves the covalent attachment of polyethylene glycol (PEG) chains to the Hb molecule. This modification enhances the molecule’s hydrodynamic size, reduces immunogenicity, and prolongs circulatory residence. PEG-Hb exhibits reduced vasoconstriction, improved viscosity, and modulated oxygen affinity due to altered kinetics of oxygen release from its central cavity.¹⁴ Both human and bovine Hb can be PEGylated; however, bovine PEG-Hb has shown superior performance in preclinical hemorrhagic shock models, including better restoration of mean arterial pressure and blood volume.¹⁵

One commercial example of PEG-Hb is:

• Hemospan: A PEG-modified Hb product aimed at improving oxygen delivery while minimizing vasoactive effects.¹⁰

4.2. Cellular Hb-based oxygen carriers

Cellular HBOCs, by contrast, incorporate Hb within a cellular or cell-like structure (eg, liposomes cell-derived RBCs), emulating the biological characteristics of native erythrocytes and thereby enhancing biocompatibility and circulation time.

4.2.1. Liposome-encapsulated Hb

Cellular encapsulation techniques aim to restore the protective and functional roles of the erythrocyte membrane by enclosing Hb within lipid bilayer vesicles (liposomes). First introduced by Dr. Thomas Chang in 1957, this method confers the biochemical and structural advantages of RBCs to free Hb, including reduced toxicity, enhanced stability, and prolonged circulation.¹⁶ The liposomal membrane serves as a barrier to oxidative damage and limits interaction with vascular endothelium.

An example is:

• TRM-645: A liposome-encapsulated Hb developed by Terumo Corporation.¹⁷

4.2.2. Recombinant Hb

Recombinant Hb is produced via genetic engineering, wherein modified human Hb genes are inserted into host organisms like Escherichia coli or yeast for large-scale expression. This approach allows precise control over Hb structure and function. Recombinant variants can be optimized using mutagenesis to improve oxygen affinity, reduce NO scavenging, and slow autoxidation, thereby enhancing overall therapeutic utility.¹⁸

An example is:

• Optro: The first HBOC product that features genetically engineered, recombinantly modified Hb cross-linked with glycine.¹⁹

4.3. Stem cell–derived oxygen carriers

A more recent advancement involves the generation of RBCs from pluripotent stem cells, including induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). These methods offer a potentially unlimited source of functional RBCs that closely resemble native erythrocytes in structure and performance.²⁰

The process of ex vivo erythropoiesis involves the directed differentiation of hematopoietic stem and progenitor cells (hSPCs) into mature RBCs. This occurs in 3 main stages: lineage commitment, cellular expansion, and terminal maturation. Cytokines such as stem cell factor (SCF) and erythropoietin (EPO) are critical in supporting this process.²¹

Multiple sources of hSPCs have been identified:

• Bone marrow (BM): Historically, the primary source of RBCs.

• Peripheral blood (PB): Mobilized using granulocyte colony-stimulating factor (G-CSF).²²

• Umbilical cord blood (UCB): Despite a lower cell yield, UCB is a viable and promising alternative source.²³

iPSC-derived RBCs are obtained by reprogramming somatic cells using transcription factors like OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. These iPSCs are then differentiated into hematopoietic and subsequently erythroid lineages.²⁴ The resulting RBCs can be generated in scalable quantities and tailored for specific antigenic profiles.

A novel product in this category is:

• HEMOXCell: Derived from mesenchymal stem cells cultured in media containing human platelet lysate, HEMOXCell serves as a natural oxygen carrier, particularly effective in oxygenating hypoxic tissue environments and maintaining cell viability.²⁵

5. CLINICAL APPLICATION OF SCOCs

Stem cell–derived RBCs have been used clinically for transfusion therapy, particularly in hematologic disorders such as sickle cell anemia and thalassemia, especially when derived from HLA-matched sibling donors.²⁶ Furthermore, iPSC-derived erythrocytes offer the ability to generate rare or patient-specific blood phenotypes. Importantly, these cells have shown no chromosomal abnormalities during reprogramming and expansion, suggesting genomic stability and safety for therapeutic applications.²⁰

Additionally, stem cell–based oxygen carriers are being explored for managing chronic hypoxia-related conditions,²⁷ expanding their utility beyond transfusion medicine into regenerative and tissue engineering domains.

6. CLASSIFICATION OF HBOCs BASED ON THE SOURCE

1. Human Hb-derived HBOCs: HBOCs derived from human sources are typically produced using outdated or surplus human blood obtained from blood banks. These HBOCs offer high biocompatibility and exhibit minimal immunogenic responses when appropriately purified. However, their use is associated with several challenges, including ethical concerns regarding the utilization of human blood and the limitations imposed by a restricted donor supply.^10,13

2. Examples: HemAssist, PolyHeme.

3. Animal Hb-derived HBOCs: Animal-derived HBOCs are primarily obtained from bovine blood, though other sources such as marine invertebrates (eg, Arenicola marina) and various experimental animal species have also been explored. These HBOCs are advantageous due to the abundance and ready availability of animal blood, reduced ethical restrictions, and Hb oxygen-binding characteristics that are comparable to human Hb. Nonetheless, if not sufficiently purified, there is a potential risk of immunogenic reactions.^28,29

4. Examples: Hemopure (bovine-derived), HemO2life (from Arenicola marina).

5. Recombinant Hb-based HBOCs: Recombinant HBOCs are synthesized through genetic engineering techniques, wherein modified human Hb genes are inserted into microbial hosts such as E coli or yeast to facilitate large-scale expression. This biotechnological approach allows precise control over the structure and functional properties of Hb. Recombinant variants can be further optimized through site-directed mutagenesis to enhance oxygen affinity, reduce NO scavenging, and decrease autoxidation, thereby improving their overall therapeutic efficacy.¹⁹

6. Example: Optro (derived from recombinant Hb).

7. PRECLINICAL STUDIES ON HBOCs

The clinical potential of HBOCs has been extensively evaluated in animal models to assess their effects on various physiological and pathological parameters.

Kawaguchi et al³⁰ investigated the therapeutic impact of Sanguinate, a PEGylated HBOC, in a rodent model following myocardial infarction. The study demonstrated that Sanguinate effectively preserved myocardial tissue, cardiac function, and mitral valve competence post-infarction.³⁰ Similarly, Vandegriff et al³¹ examined the pro-apoptotic effects of Hb in the rat brain using the PEGylated HBOC MP4OX. The results indicated that MP4OX induced significantly lower levels of neuronal apoptosis compared to a purified alpha-cross-linked HBOC formulation.

Additionally, a study conducted on Cynomolgus monkeys (Macaca fascicularis) evaluated the effects of liposome-encapsulated Hb with high oxygen affinity. The findings revealed that this formulation reduced histological damage in the cerebral cortex and preserved the cerebral metabolic rate of oxygen, suggesting neuroprotective benefits under conditions of cerebral stress.³² A comprehensive summary of these animal studies is presented in Table 2.

Table 2.

Preclinical studies of artificial oxygen carriers in animal models.

Sr. no.	AOC product	Category of AOC	Year	Animal model	Objective	Results	References
1.	Oxycyte	PFC	2008	Rats (adult male Sprague-Dawley)	To know the effect of oxycyte after an LFPI on cognitive recovery and mitochondrial oxygen consumption treatment	It showed improvement of cognitive recovery and abated the loss of CA3 neuronal cells	33
2.	PFC nanoemulsion	PFC	2012	Mouse insulinoma beta cells (MIN-6, passages 30–40)	To optimize the nanoscale perfluoroemulsion through evaluation of different critical factors like materials, emulsification time, and particle size with stability	It demonstrates particle size affecting transportation of oxygen and enhanced micelle size decrease diffusion of oxygen	34
3.	Polymerized bovine Hbs	HBOC	2012	Male Hartley guinea pigs (350–450 g)	To investigate the selection of polymerized bovine Hbs from different ratios as oxygen therapeutics	30:1 preparation of polybHbs showed better circulatory response with low oxidation. Also, less elevation of blood pressure, less iron is deposition in the liver was observed	35
4.	Oxycyte	PFC	2013	Yorkshire swine	To evaluate the seizure latency (oxygen toxicity of CNS) and duration after PFC administration with 6 ATA of oxygen in swine	The result exhibited safety during the use of PFC to treat DCS	36
5.	Oxycyte	PFC	2013	Yorkshire swine	To know the effect of decreasing the dose of oxycyte (3 cc/ kg) in swine model of DCS	It improves injury of the spinal cord, but it did not significantly increase the survival benefit	37
6.	HbPs: 700 nm	HBOC	2013	Male C57BL/6 adult mice	New Hb particles content assembled to 80% of local Hb content of RBC	No oversupply of oxygen, nitric oxide scavenging limited, and non-vasoconstrictor behavior	38
7.	NVX-108 (perfluorocarbon dodecafluoropentane)	PFC	2014	Male Sprague-Dawley rats	To know the effects of NVX 108 on cerebral microvasculature in rat	It provides more oxygen and increases oxygen transport in brain tissue except for vasoactivity (systemic or cerebral)	39
8.	HbPs: 700 nm	HBOC	2014	Wistar rats (3-mo old)	To solve the vasoconstriction problem caused by nitric oxide scavenging and checking oversupply of oxygen	No oversupply of oxygen, nitric oxide scavenging limited	40
9.	MP4OX (PEGylated HBOC)	HBOC	2014	Sprague-Dawley rats (14 wk old)	To evaluate the properties of Hb that accord apoptosis in rat brain and if like this signs aid cryoprotection or damage	MP4OX showed low levels of apoptosis than control group	31
10.	OxyVita C	HBOC	2014	Rats	To evaluate effects (vasoactive) of OxyVita C on cerebral pial arteriole diameters and systemic blood pressure	In small and medium-sized pial arterioles, no vasoconstriction was observed. Moreover, no cerebral vasoconstriction was observed	41
11.	LEH	HBOC	2014	Male Sprague Dawleys rats (250–300 g)	To assess the effects of HDASPEG2K-LEH on the immunity of mice	The modified LEH is immune neutral	42
12.	PLGA-PEG/PFC emulsion	PFC	2015	HCT 116 cells (in vitro); Rat (in vivo)	To assess the PLGA-PEG/PFOB emulsion effect on HCT 116 cell viability, intracellular ROS production, and for detection of the hypoxic condition, reoxygenation by expression of HIF-1α To assess oxygen transport through new administration way “pulmonary delivery” in rats	Cell viability and intracellular ROS exposed hypoxia-reoxygenation injury in HCT 116 cells which were sub-lethal and HIF-1α contributed to cell viability; PLGA-PEG/PFC emulsion increased oxygen transport which improved lung ventilation in rats	43
13.	LEH	HBOC	2015	Male Sprague Dawleys rats (250–300 g)	To evaluate the effect of LEH on resuscitation with 45% hypovolemic shock	Correct oxygen deficits recuperate the cerebral metabolism and build a pro-survival phenotype	44
14.	Perftoran	PFC	2016	Rat (Sprague-Dawley, male, 300–450 g)	To evaluate the properties (potential vasoactive) of perftoran by measurement of pial arteriolar diameters in a healthy rat brain	It does not increase vasoconstriction in the pial arterioles of the brain. Additionally, it does not elevate systemic blood pressure compared with the control group	45
15.	Oxycyte	PFC	2016	Yorkshire swine	As PFCs showed 50 times more oxygenation than human plasma, so they wanted to evaluate the intravenous dose of the PFC emulsion whether it ameliorate the tissue oxygenation and alleviate the OALI effects	After treatment of OALI, it ameliorates oxygen transport in the blood and histology of lung	46
16.	LEH	HBOC	2016	Male Sprague Dawleys rats (250–300 g)	To investigate the capability of LEH to prevent hemorrhagic shock and to supply more oxygen	Reduce hemorrhagic shock-related pro-inflammatory cytokines and injury markers to the critical organs. Decline the plasma levels of corticosterone (stress hormone)	47
17.	A-AOC	PFC	2017	Female Wistar rats (Rattus norvegicus, 225–275 g, age- 4 mo)	To prove the function of albumin-derived perfluorocarbon as novel AOCs in a rat Langendorff-heart perfusion model	It preserved the rat heart function because of good oxygen transportation	48
18.	A-AOC	PFC	2017	Male Wistar rats (Rattus norvegicus)	To re-test the improvement of in vivo evaluation of biocompatibility of new nanocapsule (A-AOCs)	This new nanocapsule showed better biocompatibility and longer half-life circulation	49
19.	A-AOC	PFC	2017	Male Wistar rats (Rattus norvegicus, 400–450 g)	To evaluate the physicochemical characteristics and pharmacological performance of albumin-derived perfluorocarbon-based AOCs	In healthy rats, intravenous administration is well tolerated. Except for the spleen (some minor tissue damage, maybe due to effects of dose), any objectionable effects were not observed	50
20.	LEH with high oxygen affinity	HBOC	2017	Male dB/dB mice	To investigate the effect of LEH during skin wound healing in diabetic mice	LEH quickly healed the wound in dB/dB mice. In addition, decreased hypoxia, inflammation, and raised surface perfusion, in situ cell proliferation was observed	51
21.	LEH	HBOC	2017	Cynomolgus monkeys	To assess the high O2 affinity of LEH is better than the low O2 affinity of LEH using PET during MCAO and reperfusion	High O2 affinity of LEH reduces histological damage in the cerebral cortex and protects the cerebral metabolic rate of O2	32
22	DDFPe	PFC	2018	Male Yorkshire swine (78 ± 5 kg)	To evaluate the dodecafluoropentane efficacy compared with FWB after resuscitation	DDFPe administration with FFP does not enhance survival rate or ameliorate oxygen transport	52
23.	SanguinateTM (PEGylated carboxyhemoglobin)	HBOC	2018	Male spontaneous hypertensive rats	To understand the effect of SanguinateTM (SG) in collateral and reperfusion CBF and brain injury during MCAO	Preventing declination of reperfusion CBF, increased collateral flow and sustaining it for 1.5 h of ischemia	53
24.	HbMP-700	HBOC	2018	Mouse (Mus musculus)	To investigate the influence of newly introduced HbMP-700 on vasoconstriction along with genetic toxicity	No toxicity and clinical signs were observed during the experiment. It impeded premature oversupply of oxygen and vasoconstriction and also gives high oxygen affinity	54
25.	SanguinateTM (PEGylated carboxyhemoglobin)	HBOC	2018	Lewis rats (5 wk old)	To investigate the effect of sanguinate after MI	Preserve the myocardium, heart function, and mitral competence after MI	30
26.	HbMP-700	HBOC	2018	Mouse lymphoma model	To investigate oxidative pressure, vasoconstriction effect, and genetic toxicity	The high affinity of oxygen obstructs the oversupply of premature oxygen and avoids vasoconstriction of small blood vessel	54
27.	A-AOC	PFC	2020	Male Wistar rats (318–430 g; 11 wk old)	To prevent DCS	A-AOC decreased DCS lesions and mortality significantly	55
28.	A-AOC	PFC	2020	Male Wistar rats (Rattus norvegicus, 430–460 g)	To evaluate the albumin-derived capsule function in a normovolemic hemodilution rat model	After being treated with this capsule, animals showed high arterial blood pressure, stable body temperature and pH, higher partial pressure of oxygen and as well as lower partial pressure of CO2. Finally, this capsule impeded hypoxic tissue damage	56
29.	PNPH (aka SanFlow)	HBOC	2020	Male Hartley guinea pigs (650 ± 110 g)	To determine the effect of small amount transfusion of PNPH in TBI plus hemorrhagic shock model guinea pigs	It gives neuroprotection to the guinea pig brain from secondary neurodegeneration	57
30.	Oxygent (w/v 60% PFC) or Perftoran (w/v 20% PFC)	PFC	2021	Sheep (juvenile female Dorser (Dorper); 3–4 mo old, 18–32 kg)	To know the extent effects of platelet-like mechanism, inflammation after PFC treatment	After PFC infusion, there were no changes in inflammatory cell lines. After oxygent infusion, decreased no of platelet found on day 4 which was corrected on day 7. In the case of perftoran infusion, no platelet effect was found	58
31.	Oxycyte	PFC	2021	Juvenile male sheep (weight 24.4 ± 2.10 kg)	To assess the effect of PFC oxycyte™ in severe DCS in ovine model	After the onset of DCS, oxycyte™ reduce injury of the spinal cord although did not decrease the mortality rate	59

A-AOC = albumin-derived perfluorocarbon-based artificial oxygen carrier, ATA = atmosphere absolute, CBF = cerebral blood flow, CNS = central nervous system, DCS = decompression sickness, DDFPe = dodecafluoropentane, FFP = fresh frozen plasma, FWB = fresh whole blood, Hb = hemoglobin, HbMP = hemoglobin microparticles, HBOC = hemoglobin-based oxygen carriers, HbPs = hemoglobin particles, HIF-1α = hypoxia-inducible factor 1-alpha, LEH = liposome-encapsulated hemoglobin, LFPI = lateral fluid percussion injury, MCAO = middle cerebral artery occlusion, MI = myocardial infarction, OALI = oleic acid lung injury, PEG = polyethylene glycol, PET = positron emission tomography, PFC = perfluorocarbon-based oxygen carriers, PFOB = Per-fluoro octyl bromide, PLGA = poly (lactic-co-glycolic acid), PNPH = polynitroxylated PEGylated hemoglobin, RBC = red blood cell, TBI = traumatic brain injury.

8. HBOCs: CURRENT PRODUCTS AND CLINICAL STATUS

1. HemAssist: HemAssist was the first HBOC to receive FDA approval for clinical trials. It consisted of 2,3-diaspirin cross-linked Hb derived from expired human blood. The product showed favorable tolerance during Phase I and II trials. However, in Phase III trials, patients demonstrated an increased risk of complications, including pancreatitis and myocardial infarction. Additionally, trauma patients enrolled in the US Phase III trials exhibited significantly higher mortality rates compared to the control group, although the European Phase III trials showed more balanced results. Due to these concerns, HemAssist was discontinued in 1999.¹³

2. PolyHeme: PolyHeme is a product derived from expired human blood. The product underwent Phase I, II, and III trials and showed positive outcomes, including elevated Hb levels in patients compared to those receiving allogenic blood products. Despite these favorable results, PolyHeme failed to meet the non-inferiority 30-day mortality endpoint, leading to the FDA’s denial of approval in May 2009.⁶⁰

3. HemoLink: HemoLink is a cross-linked and polymerized Hb solution stabilized with o-raffinose. The product successfully passed Phase I and II clinical trials and showed promising results in Phase III trials, demonstrating the ability to save patients’ lives.⁶¹ However, despite its success in trials, HemoLink did not receive marketing approval. Clinical trials for the product were halted in 2003, and the company declared bankruptcy in 2005, bringing an end to its development.⁶²

4. Hemopure (Hb-201): Hemopure is one of the most promising HBOC products. It consists of cross-linked bovine Hb.⁶³ Clinical trials in humans, particularly in cardiac settings, showed that Hemopure improved coronary oxygenation and perfusion. Its small molecular size facilitates efficient oxygen uptake and release, allowing for effective gaseous exchange. Studies have also demonstrated that Hemopure can restore cerebral circulation and function in animal models for up to 4 hours postmortem. Furthermore, it showed potential for perfusing other vital organs, such as the liver and kidneys, suggesting its ability to replace allogeneic erythrocyte transfusions. The Hemopure has expanded use approval in the US after FDA approval.⁶⁴

5. HemO2life: HemO2life is a novel HBOC derived from the natural Hb of Arenicola marina, a polychaete species that has existed for over 450 million years. This species is found along the intertidal zones of the east-Atlantic shoreline of France, extending from the North Sea to Biarritz. The molecular weight of HemO2life is approximately 50 times that of human Hb, enabling it to bind up to 156 oxygen molecules, compared to the 4 molecules human Hb can carry. It is a naturally occurring polymerized Hb exhibiting antioxidant properties similar to superoxide dismutase.⁶⁵ HemO2life has shown promising results in treating COVID-19 patients, improving survival rates, reducing intubation times, and decreasing oxygen support needs during ventilator shortages. It is currently approved for donor organ preservation in the European Union.⁶⁶

6. Optro: Optro, developed by Somatogen Inc. and Eli Lilly, is the first HBOC product that features genetically engineered, recombinantly modified Hb cross-linked with glycine. Modifying the human Hb gene minimizes the dissociation of Hb molecules into dimers, maintaining their oxygen affinity. The recombinant DNA technology used to produce Optro involves inserting the human Hb gene into E coli or yeast, which then produces the Hb. Despite its promising design, Optro did not receive FDA approval due to excessive vasoconstriction caused by significant NO scavenging and was discontinued in 1999.¹⁹

7. Hemospan: Hemospan is a PEG-conjugated HBOC that exhibits improved oxygen affinity, reduced vasoconstriction, and minimized NO scavenging effects due to its PEG formulation. This modification also enhances the product’s size and water hydration levels. In Phase I and II clinical trials, Hemospan demonstrated no serious complications. However, Phase III trials conducted in the USA and Europe, which compared Hemospan to Voluven as a volume expander during surgery, yielded disappointing results, leading to its termination in 2015. Side effects like hypertension, myocardial infarction, high mortality, acute renal failure, transient ischemic attack were encountered in the study group. Criticism has been raised regarding the study’s design, suggesting that Hemospan should have been tested as a blood substitute rather than a volume expander.⁶⁷

8. Sanguinate: Sanguinate is a PEGylated, carbon monoxide (CO)-hybridized form of bovine Hb. Its structure is modified to improve its ability to bypass microcirculatory obstructions and efficiently deliver oxygen to ischemic tissues.⁶⁸ Sanguinate has been used successfully in over 100 emergency patients with severe anemia, particularly in situations where transfusion is contraindicated. Its anti-inflammatory, anti-vasoconstriction, and plasma-expanding properties have made it a promising option for treating conditions like stroke, inflammatory diseases, and sepsis. It completed Phase II clinical trials in 2017.⁶⁹

9. HemoxiMer (PHP): HemoxiMer is a chemically modified human Hb conjugated with pyridoxal and polyoxyethylene. Initially developed as an HBOC, it was later repurposed as a NO scavenger for conditions involving excessive NO. While Phase I and II trials were completed, Phase III trials in both the USA and Europe were unsuccessful, leading to the cessation of its development as an HBOC. However, HemoxiMer still holds potential as a therapeutic for conditions requiring NO modulation.¹³

10. OxyVita: OxyVita is a next-generation HBOC derived from bovine blood. It is produced using zero-link polymerization technology, a method that creates a super polymer of Hb tetramers without the use of linking agents. This technology results in Hb molecules with a molecular weight of approximately 17 Mega Daltons. OxyVita is designed for use when RBC transfusion is unavailable or undesirable for severe hemorrhage and hemorrhagic shock.⁴¹ Its development is in progress. Table 3 provides an overview of the various HBOC products and their current status.

Table 3.

Hemoglobin-based oxygen carriers: current products and clinical status.

Sr. no.	Product name	Manufacturer	Source	Hb modification	Clinical indication	Side effects	Current status	References
1.	HemAssist	Baxter Corporation in collaboration with the U.S. military	Human	α-α Crosslinked with diaspirin	Hemorrhagic shock	Cerebrovascular emergency (stroke), hypertension, MI, high mortality	First HBOC to get FDA approval for clinical trials. However, in phase III trials, it increased mortality in cardiac surgery, trauma, and stroke patients. Terminated in 1999	4, 13, 70
2.	PolyHeme	Northfield Laboratories, Evanston, Illinois	Human	Polymerized Hb	Bleeding disorder, trauma, surgery	Acute renal failure, transient ischemic attack, hypertension, MI, cerebrovascular emergency (stroke), high mortality	Phase III trials were completed in 2007; however, in 2009, it failed BLA due to adverse effects. FDA denied its approval in 2009, subsequently company went bankrupt, leading to the discontinuation of PolyHeme’s clinical use	10, 62
3.	HemoLink	Hemosol Inc., Mississauga, Canada	Human	Crosslinked with O-raffinose	Cardiac surgery, acute normovolemic hemodilution	MI, transient ischemic attack, severe cardiotoxicity, cerebrovascular emergency (stroke), hypertension, high mortality	HemoLink did not receive marketing approval. Clinical trials for the product were halted in 2003, and the company declared bankruptcy in 2005, bringing an end to its development	62, 71
4.	Hemopure	Biopure Corporation, Cambridge, Massachusetts	Bovine	Polymerization with glutaraldehyde	Hemorrhagic shock, acute coronary syndrome, MI, coronary occlusion, severe anemia, perioperative transfusion	Methemoglobinemia, increased liver enzyme, oliguria, hypertension	Under the trade name Oxyglobin, Biopure developed a similar product which received veterinary use approval in the U.S. in 1997 and in Europe in 1998. Hemopure was approved for clinical use in South Africa in 2001 and in Russia in 2006. FDA granted Expanded (compassionate use) Access Program (EAP) approval for life-threatening anemic patients, for whom blood is not an option	10, 28
5.	HemO2life	Hemarina, Morlaix, France	Arenicola marina (AmEc) Lumbricus terrestris (LtEc)	Natural extracellular biopolymer Hb-erythrocruorin	Hemorrhagic shock, sickle cell anemia, organ preservation, and in COVID-19 patients	-	Phase I clinical trials successfully completed in 2018. Phase II and III trials are in progress. Approved for clinical use by the European Union for donor organ preservation. It could improve COVID-19 patients’ survival	29, 65, 66
6.	Optro	Somatogen, Boulder, Colorado	Genetically engineered Hb	Recombinant DNA technology	Cardiothoracic surgery	Significant nitric oxide scavenging	Phase II trials were unsuccessful due to excessive vasoconstriction associated with its use. Terminated in 1999	19, 62
7.	Hemospan	Sangart Inc., San Diego, California	Human	Maleimide-PEGylated modified Hb	Hemorrhagic shock, hypotension, ischemia	Hypertension, MI, high mortality, acute renal failure, transient ischemic attack	Reached Phase III trials. Terminated in 2015	67, 72
8.	Sanguinate	Prolong Inc., South Plainfield, New Jersey	Bovine	PEGylated carboxy-Hb	Sickle cell anemia, vaso-occlusive crisis, renal insufficiency, cerebral ischemia	Musculoskeletal detrimental events, vertigo, lethargy	Phase II clinical trials completed in 2017. Further development in progress	13, 73
9.	HemoxiMer or PHP (Pyridoxylated Hb)	Apex Bioscience, Chapel Hill, North Carolina	Human	Conjugated PHP and crosslinked human Hb	SIRS with shock	Hypertension, high mortality, MI, cerebrovascular emergency (stroke)	Phase II completed; Phase III terminated in 2011 as AOC, but acts as a nitric oxide scavenger	28, 74
10.	OxyVita	University of Maryland in 1999 and further advanced by OXYVITA Inc.	Bovine	Polymerized zero-linked bovine Hb	Severe hemorrhage and hemorrhagic shock	-	Preclinical trials are ongoing. Further development in progress	75, 76
11.	Hemotech	Hemobiotech Inc., West Dallas, Texas	Bovine	Cross-linked with ATP (intramolecularly) and adenosine, glutathione (intermolecularly)	Acute hemorrhage	-	Phase I trials completed. Further development in progress	10, 77, 78

AOC = artificial oxygen carrier, BLA = biologic license application, FDA = Food and Drug Administration, Hb = haemoglobin, HBOC = hemoglobin-based oxygen carriers, MI = myocardial infarction, PHP = pyridoxylated Hb, SIRS = systemic inflammatory response syndrome.

9. LIMITATIONS OF HBOCs

Despite their potential as blood substitutes, HBOCs face several significant challenges that have hindered their widespread clinical adoption. A primary concern associated with acellular HBOCs is their propensity to scavenge NO from the vasculature. This depletion of NO disrupts endothelial function, resulting in vasoconstriction, oxidative stress, and, in some cases, hypertensive cardiovascular responses.³⁵ To mitigate these adverse effects, numerous structural and chemical modifications have been explored to optimize the pharmacological performance and safety profile of HBOC formulations.³⁵

Another major limitation involves the immunogenicity of modified Hb molecules. Repeated administration of acellular HBOCs has been associated with immune system activation, characterized by increased macrophage accumulation and alterations in T-cell and lymphocyte populations. Both genetically engineered and chemically modified Hbs have demonstrated the potential to elicit immune-mediated clearance, thereby reducing their circulatory half-life and therapeutic efficacy.¹⁷

Physicochemical stability is also a critical issue for certain acellular HBOCs, as these formulations may degrade or lose functional capacity over time, compromising their shelf-life and marketability. Furthermore, oxygen-binding dynamics remain suboptimal in several HBOC candidates, leading to premature oxygen release in arterioles. This aberrant release can induce reflex vasoconstriction and hypertension, thus counteracting the intended therapeutic benefits. Specifically, PEG-conjugated HBOCs, while designed to improve plasma retention and reduce immunogenicity, pose additional risks such as vascular extravasation and endocytosis, which may contribute to transient hypertensive episodes.⁷⁹

In parallel, SCOCs are emerging as a promising alternative, yet they too face considerable translational barriers. Achieving complete functional maturation, immune compatibility, and large-scale, cost-effective production remains a persistent challenge. A particularly critical limitation is the difficulty in guiding stem cells to differentiate into fully enucleated, adult-type erythrocytes with physiologically relevant Hb concentrations, a prerequisite for efficient oxygen delivery.⁸⁰ Additionally, the residual presence of undifferentiated stem cells poses concerns related to immune rejection and tumorigenic potential, significantly impacting safety considerations.

Large-scale manufacturing of SCOCs is further complicated by high production costs and issues with storage stability, both of which are essential for clinical feasibility. Moreover, regulatory complexities and ethical considerations present additional hurdles that must be addressed before SCOCs can transition from bench to bedside.⁸¹

These cumulative challenges highlight the critical need for ongoing research and development efforts focused on improving the safety, stability, and clinical efficacy of both HBOCs to advance their viability as transfusion alternatives.

10. NON-HBOCs

These oxygen carriers do not rely on Hb for oxygen transport; instead, they employ chemical modifications to perform this function. Within this category, perfluorocarbon-based AOCs are the most extensively researched and represent a predominant class. Other non–Hb-based AOC alternatives, such as SCOCs, synthetically derived porphyrin-based AOCs, and micro/nanobubble-mediated oxygen transport systems, are still in the developmental stages and have not yet been extensively studied.

10.1. Perfluorocarbon-based oxygen carriers

The potential of perfluorocarbons (PFCs) as AOCs was first recognized in 1966, when Clark and Gollan⁸² demonstrated that mice could survive immersion in oxygenated PFC liquids, revealing their remarkable capacity to dissolve and deliver respiratory gases. This pivotal discovery has since catalyzed extensive research into PFCs for biomedical applications, particularly as substitutes to Hb-based oxygen transport systems.

Chemically, PFCs are fully fluorinated synthetic hydrocarbons, typically composed of carbon chains ranging from 8 to 10 atoms. In these molecules, all hydrogen atoms are replaced by fluorine, resulting in the formation of strong and highly polar carbon–fluorine (C–F) bonds.⁹ These halogenated compounds can be linear, cyclic, or polycyclic in structure and exhibit unique physicochemical properties, including chemical inertness, thermal stability, high density, and immiscibility with water and biological fluids.⁹

The exceptional chemical stability of PFCs arises from fluorine’s high electronegativity, low polarizability, and high ionization energy. These attributes yield strong intramolecular C–F bonds while minimizing intermolecular forces, such as van der Waals interactions.⁸³ As a result, PFCs exhibit low surface energy and high resistance to chemical reactions, contributing to their stability, hydrophobicity, and gas-like behavior in liquid form.

Due to these characteristics, PFCs can dissolve significant quantities of respiratory gases—up to 50 times more oxygen than plasma—by occupying intermolecular spaces within their molecular matrix.⁸⁴ Gas solubility in PFCs adheres to Henry’s law, where solubility increases linearly with partial pressure. This contrasts with Hb’s sigmoidal oxygen dissociation curve, making PFCs uniquely suited for applications requiring predictable and passive gas exchange.⁵⁶

At room temperature, high molecular weight PFCs exist as liquids and must be emulsified with biocompatible surfactants for in vivo use due to their hydrophobicity and immiscibility with plasma. In contrast, low molecular weight PFCs may be gaseous and potentially inhalable.⁸⁵ Recent advancements in synthetic chemistry and emulsification techniques have enabled the creation of stable PFC nanoemulsions suitable for clinical applications.⁸⁴

Structurally, PFC molecules adopt a helical conformation with C–F dipoles aligned along the helix axis, differing from the planar configurations typical of hydrocarbons.⁸³ This molecular geometry, combined with weak intermolecular cohesion, enables PFCs to behave like fluids with high gas solubility and low viscosity. Their oxygen and CO₂ transport capacity remains largely unaffected by temperature changes, further distinguishing them from Hb-based systems.⁴

Moreover, PFCs are nanoscale in size—approximately 100 times smaller than RBCs—which allows them to access microvascular and ischemic regions that erythrocytes cannot reach.⁸⁶ This feature underscores their promise in treating conditions such as localized hypoxia and impaired microcirculation.

10.1.1. Preclinical evaluation of PFCs in animal models

Numerous studies have been conducted using animal models to assess the clinical potential of various perfluorocarbon (PFC) formulations. Wrobeln et al⁵⁰ investigated an albumin-derived PFC product (A-AOC) in Wistar rats and reported that intravenous administration of A-AOC was well tolerated in healthy animals, depending on the administered dose, with no observable adverse effects. Similarly, Moon-Massat et al demonstrated that NVX-108 enhanced oxygen delivery and increased oxygen transport to brain tissue in a rodent model.²⁸ In a swine model, Mahon et al³⁷ found that the administration of Oxycyte, a PFC-based product, contributed to improved recovery following spinal cord injury, although it did not significantly improve overall survival rates.

Further evidence from studies conducted on sheep models indicated that PFC administration does not lead to a significant reduction in platelet count, nor is there any observed correlation between PFC concentration and alterations in platelet activation or inhibition. Consequently, the risk of thrombosis or bleeding following intravenous infusion of PFCs is considered low.⁵⁸

A comprehensive summary of these animal studies is presented in Table 2. These findings highlight the distinctive properties of PFCs and support their potential utility in medical applications, particularly in enhancing oxygen delivery and serving as blood substitutes.

10.1.2. Current developments in PFCs

A variety of perfluorocarbon (PFC) formulations are currently available, each at different stages of clinical development.

Fluosol-DA was the first PFC-based product to receive approval from the USFDA in 1989. It was initially employed to facilitate oxygen delivery during percutaneous transluminal coronary angioplasty (PTCA) procedures and was also recommended for conditions such as hemorrhage, emergency transfusion, cerebral ischemia, and CO poisoning.^75,87 However, following several years of clinical use, concerns emerged regarding adverse effects, including thrombocytopenia, leukocytosis, and respiratory complications. As a result, FDA approval was rescinded, and Fluosol-DA was withdrawn from the market in 1994.^84,88

Perftoran is another prominent PFC-based oxygen carrier, having been administered to more than 35,000 patients for the treatment of hemorrhagic anemia and ischemia resulting from trauma or surgical interventions. It is one of the most extensively used AOCs. Perftoran received regulatory approval for clinical use in both civilian and military populations in Russia in 1996 and was subsequently approved in Kazakhstan (1995), Ukraine (2005), and the Kyrgyz Republic (2006). Between 2005 and 2010, it was also marketed under the name Perftec in Mexico.^89,90

Oxypherol was investigated primarily for its potential use in organ perfusion; however, its clinical adoption was limited due to its exceptionally long biological half-life of approximately 500 days, making it a less favorable candidate.⁸⁴

Oxyfluor, another PFC-based formulation, was evaluated in a porcine model of cardiopulmonary bypass, where it demonstrated improved tissue oxygenation and increased systemic oxygen consumption. Despite these promising results, the product failed to progress beyond Phase III clinical trials.⁹¹

Oxygent is a PFC product currently undergoing Phase III clinical trials, following its approval for clinical study in China in 2017.⁸⁶ Another formulation, Oxycyte, has shown potential for treating ischemic myocardium, sickle cell crises, traumatic brain injuries, and as a general blood substitute. A key advantage of Oxycyte lies in its minimal impact on normal coagulation parameters and platelet function. Nonetheless, its development was halted following the discontinuation of Phase II clinical trials in 2014 due to a lack of continued sponsorship.⁸³

More recently, an albumin-derived perfluorocarbon-based artificial oxygen carrier (A-AOC) has emerged as a novel advancement in the field. Utilizing nanoparticle technology, A-AOC exhibits high biocompatibility and has been hypothesized to coat nitrogen bubbles in circulation, thereby preventing their aggregation and facilitating safe transport through the bloodstream. These nanoparticles are believed to bind to nitrogen bubbles attached to the vascular endothelium and transport them to the lungs for elimination.⁸⁰ A-AOC is currently in the preclinical trial stage.⁵⁰

An overview of these PFC-based oxygen carriers, including their clinical indications and current status in clinical development, is presented in Table 4. These formulations collectively highlight the growing potential of PFCs in medical applications, particularly in the context of oxygen delivery and artificial blood substitutes.

Table 4.

Perfluorocarbon-based oxygen carriers: current products and clinical status.

Sr. no.	Product name	Manufacturer	Chemical composition	Surfactants	Clinical indication	Current status	References
1.	Fluosol-DA	Green Cross Corporation, Osaka, Japan	Perfluorodecalin and perfluorotripropylamine	Pluronic F-68, egg yolk phospholipid, potassium oleate	Hemorrhage, carbon monoxide poisoning, cerebral hypoxia, angioplasty, anemia	FDA approved its use in 1989; however, it withdrew its approval in 1994 due to side effects.	75, 84, 88
2.	Perftoran	Russian Academy, Moscow, Russia	Perfluorodecalin and perfluoromethylcyclohexylpiperidin	Proxanol, egg yolk phospholipid	Hemorrhagic shock and organ perfusion.	Approved for clinical use in Russia (1996), Republic of Kazakhstan (1995), Ukraine (2005), and the Kyrgyz Republic (2006). Between 2005 and 2010, Perftoran was marketed as Perftec in Mexico. Rebranded as Vidaphor in North America. Since its approval, it has been administered to over 35,000 patients worldwide	84, 81, 86
3.	Oxypherol (formerly Fluosol 43)	Green cross Crop., and Alpha therapeutic Corp., Los Angeles and blood product unit of Japan’s Green Cross Corp.	Perfluorotributylamine	Pluronic F-68	Organ perfusion, vasodilation, inflammation	Unknown	84
4.	Oxyfluor	Hemagen, Inc., St. Louis, Missouri	Perfluorodichlorooctane	Egg yolk phospholipid	Cardiopulmonary bypass in cardiac surgery and hemorrhage	It failed in Phase III trials	84, 85
5.	Oxycyte	Oxygen Biotherapeutics Inc., Morrisville, North Carolina	Tertbutylperfluorocyclohexane	Egg yolk phospholipid	Trauma, brain surgeries	Phase II clinical trials successfully completed in 2008. However, due to lack of patient assignment, further trials were suspended in 2014	83, 84
6.	Oxygent	Alliance Pharmaceutical Corporation, based in San Diego, California	Perfluorooctylbromide and perfluorodecylbromide	Egg yolk phospholipid	Cardiac surgery, orthopedic surgery, and bleeding disorders	Approved in China for clinical studies in 2017. Undergoing phase III trials.	84, 86, 92
7.	A-AOC	New development	Perfluorodecalin	HAS	Research is currently underway to determine	Undergoing preclinical trials	50, 83, 84, 92

A-AOC = albumin-derived perfluorocarbon-based artificial oxygen carrier, HAS = human serum albumin.

10.1.3. Limitations in the clinical use of PFCs

Despite their potential in medical applications, PFCs are associated with several limitations that hinder their widespread clinical adoption. One of the primary concerns is their prolonged residence time within the body, which can extend up to 90 days. This extended persistence facilitates uptake by the reticuloendothelial system, leading to accumulation in organs such as the liver and spleen, thereby increasing the risk of visceral toxicity.⁹³ Additionally, PFC emulsions’ relatively large particle size may provoke a complement activation-related pseudoallergy (CARPA) response, which can result in vascular occlusion, thrombosis, and pulmonary toxicity.^90,91

Furthermore, PFC formulations generally require stringent cold storage conditions due to their limited stability, thereby complicating handling and transportation. The efficiency of oxygen transfer can also be suboptimal in certain formulations. Moreover, the high concentrations of perfluorocarbon compounds and surfactants necessary for emulsification may present further safety risks to patients.⁹¹

To address these challenges, the use of biocompatible stabilizers in combination with auxiliary or mixed surfactant systems has been investigated as a strategy to enhance the physicochemical stability and clinical safety profile of PFC.⁸¹

10.2. Porphyrin-based AOCs

Natural Hb and myoglobin contain cyclic porphyrin structures with 4 pyrrole rings connected by methine bridges, enabling iron (Fe²⁺) chelation crucial for oxygen transport. Synthetic analogues, such as picket fence porphyrins, mimic this structure and aim to provide a protective environment for reversible oxygen binding.^94,95 However, early models faced issues with irreversible oxidation in aqueous solutions. Iron porphyrins were integrated into liposome bilayers to overcome this, creating stable lipid-heme systems with reversible oxygen-binding capability due to the hydrophobic liposome environment. These models demonstrated success in limited in vivo experiments.⁹⁶

Advancements include recombinant human serum albumin (rHSA) conjugated with iron porphyrins, showing improved oxygen transport, blood compatibility, and extended circulation, especially when combined with PEG. Further innovation led to HemoCD, a complex of Fe(II)-TPPS and cyclodextrin dimers, which exhibited enhanced oxygen affinity and half-life. A variant, FeIIPImCD, demonstrated even higher oxygen affinity. PEGylation of HemoCDs improved their stability in the bloodstream by reducing immune clearance. While promising for mimicking natural oxygen carriers, these synthetic systems require extensive preclinical validation trials. Overall, ongoing research in porphyrin-based models continues to explore efficient, reversible, and biocompatible oxygen delivery systems.⁹⁷

10.3. Micro/nanobubble-mediated oxygen transport

Micro/nanobubble-mediated oxygen transport represents a promising method for delivering oxygen under hypoxic conditions. This technique involves spherical vesicles, known as microbubbles and nanobubbles (MNBs), which encapsulate gas within shells composed of materials such as phospholipids, proteins, and polymers.⁹⁸ These bubbles are capable of transporting oxygen directly to deoxygenated erythrocytes, hypoxic tissues, and blood vessels, thereby addressing a critical issue in solid tumors where hypoxia often diminishes therapeutic efficacy. By enhancing oxygen levels, MNBs can improve the oxygen enhancement ratio (OER), increasing the sensitivity of tumor radiation therapy.⁹⁹ Their small size allows them to penetrate both major and minor blood vessels, making them versatile tools for drug delivery, oxygen transport, molecular imaging, and gene therapy.

Different shell materials provide distinct advantages. Lipid shells, commonly used in microbubbles, offer mechanical stability and facilitate oxygen permeability. Protein shells enhance stability and amphiphilicity, while polymer shells provide increased resistance to compression and expansion, though they may reduce echogenicity during ultrasound. The choice of shell material significantly influences the safety and efficiency of oxygen transport by minimizing interactions between the gas and surrounding tissues. Their ability to deliver oxygen and drugs, combined with their potential for molecular imaging, positions them as valuable tools in the development of innovative therapeutic strategies.¹⁰⁰

11. FUTURE OUTLOOK

The American Red Cross underscores a persistent and critical demand for blood and platelet donations in the United States, with a patient requiring these life-saving components approximately every 2 seconds. On a national scale, healthcare institutions utilize nearly 29,000 units of RBCs each day, leading to an annual transfusion of approximately 16 million blood components. Among the conditions driving this demand is sickle cell disease—an inherited hematologic disorder that affects an estimated 90,000 to 100,000 individuals in the country. Approximately 1000 infants are born with the condition each year. Given the chronic nature and severity of its complications, many individuals with sickle cell disease depend on regular blood transfusions as a cornerstone of their long-term care.¹⁰¹

In light of the challenges associated with maintaining adequate blood supplies, AOCs have emerged as a promising innovation in transfusion medicine. These synthetic agents, designed to replicate the oxygen-transporting function of RBCs, offer significant potential as alternatives to conventional blood products—particularly in circumstances where blood availability is limited or compromised. AOCs are anticipated to play a vital role in trauma care, surgical procedures, and emergency medicine.

Their potential impact is especially evident during large-scale public health emergencies. For instance, during the COVID-19 pandemic, the demand for oxygen therapy far outpaced the availability of mechanical ventilation systems. In such scenarios, AOCs—such as the HemO2Life product—demonstrated their value by supplementing oxygen delivery in vivo, thereby easing the burden on overextended critical care infrastructure.

Moreover, in conflict zones and other high-risk environments where rapid access to compatible blood is not feasible, AOCs could provide a crucial solution for managing acute blood loss and preventing life-threatening oxygen deprivation. Their stability, extended shelf-life, and lack of need for blood typing make them particularly suited for deployment in austere or emergency settings.

Ongoing advancements in biomedical research continue to address existing limitations of AOCs, with a focus on improving their safety, biocompatibility, and oxygen delivery efficiency. As these technological barriers are progressively overcome, AOCs hold the potential to become an integral part of standard medical protocols for conditions such as anemia, severe trauma, and ischemic injury, ultimately reshaping the future of transfusion and critical care medicine.

12. CONCLUSION

Within the diverse landscape of AOCs, HBOCs have emerged as particularly promising candidates. Their ability to emulate the physiological oxygen transport mechanism of native Hb, while also participating in the modulation of key signaling molecules such as NO, CO₂, and CO, positions them at the forefront of AOC research. Although challenges such as NO scavenging, vasoconstriction, and molecular instability have been observed, innovative formulation strategies—most notably PEG conjugation and liposome encapsulation—are actively being employed to address these limitations and enhance clinical viability.

Perfluorocarbons (PFCs), another prominent class of AOCs, offer unique advantages including improved tissue oxygenation and microcirculatory flow due to their small molecular size, as well as the benefit of an unlimited synthetic supply. However, their inorganic nature, prolonged residence time in the body, and potential for immunogenic responses such as the CAPRA reaction warrant further scientific scrutiny to ensure safe long-term use.

In recent developments, oxygen carriers derived from stem cells—particularly those originating from hSPCs and iPSCs—have emerged as promising biologically compatible substitutes for traditional oxygen delivery systems. These innovative carriers possess the notable advantage of being sourced autologously or homologously, enabling more physiologically relevant oxygen transport. While the technology shows considerable promise, several technical and regulatory challenges must still be addressed, which are already elaborated elsewhere in this review.

Additionally, emerging platforms such as porphyrin-based systems and micro- or nanobubble-mediated oxygen delivery represent innovative directions in the AOC field. While these approaches are still in early stages of development, their unique mechanisms of action suggest valuable potential for future application. Rigorous preclinical evaluation and safety assessments will be essential to fully harness their capabilities for clinical use.

Collectively, the field of AOCs is undergoing rapid innovation, with each approach contributing valuable insights and possibilities. Continued interdisciplinary research and technological refinement promise to bring safer, more effective oxygen therapeutics closer to clinical reality.

13. LIMITATION OF THE ARTICLE

A key limitation of this review lies in its predominant focus on English-language publications available through open-access repositories, which may result in the exclusion of relevant studies published in other languages or housed within subscription-restricted databases. Moreover, given the accelerated pace of advancements in biomedical and technological research, the review may not fully encompass the latest developments, as the field continues to evolve rapidly.

AUTHOR CONTRIBUTIONS

The requirements for authorship as stated as per ICMJE criteria have been met by all. All authors contributed equally to the article’s preparation (participated in research design, writing of the paper, reviewing, in data analysis). Both authors proofread and collectively approved the manuscript for publication. Each author believes that the manuscript represents honest work.