HBOCs aim to mimic the oxygen and nutrient transport roles of RBCs which are applicable in various life-threatening conditions, including trauma, stroke, acute blood loss, hemorrhagic shock, and myocardial infarction [24]. Hemoglobin (Hb) is a tetrameric protein molecule, approximately 64 kDa in size, composed of two noncovalently bound αβ dimers with excellent ability to carry oxygen. However, free Hb has restrictions to clinical use due to several difficulties such as nephrotoxicity, easy oxidation, no longer circulation time in animals, and hypertension [25]. To address these challenges, diverse approaches for engineering Hb modifications have been suggested to create HBOCs where human, bovine, and recombinant Hb were used as raw ingredients. Hb is obtained through a process involving cell lysis, chromatography, sterile filtration, and low-heat sterilization which is carried out using outdated human or bovine RBCs [23, 26]. HBOCs is a semi-synthetic carrier system utilizing the natural oxygen carrier Hb which not only preserves the oxygen transport function of natural Hb but also mitigates the toxic effects associated with free Hb [27].

To date, various techniques have been devised to develop HBOCs through chemical modifications and stabilization of the Hb molecule. The goal of these modifications is to enhance oxygen release, employing methods such as intramolecular cross-linking to maintain the tetramer. Additionally, the high affinity of oxygen has been diminished through the use of 2,3-DPG analogs or combinations of intramolecularly cross-linked structures and oxygen affinity modifiers like bis-(3,5-dibromosalicyl)-fumarate (DBBF) and 2-nor-2-formylpyridoxal phosphate (NFPLP) [28, 29]. HBOCs are categorized into two main types: chemically modified HBOCs and encapsulated HBOCs where Hb is enclosed within a protective shell [30], as shown in Fig. 2a.

Potential artificial oxygen carriers. a Hemoglobin-based oxygen carriers (HBOCs) with Hb modifications mechanisms; Chemical modification: where Hb α and β subunits cross-linked intramolecularly with glycine, glutaraldehyde, O-raffinose etc., conjugated on the surface by maleimide-activated Polyethylene glycol, and intramolecularly polymerized with glutaraldehyde, o-raffinose etc.; Encapsulation strategies: where Hb encapsulated with liposomes, sub-micron size polymeric vesicles (polymersomes) made from amphiphilic block copolymers, and polydopamine (PDA). Reprinted with permission from [30]; b Perfluorocarbon-based oxygen carriers (PFOCs), (Schematic illustration of structural compositions for PFC emulsions, where PFCs (perfluorodecalin/ Bromoperfluoro-n-octane) are surrounded by lecithin phospholipid emulsifier). Reprinted with permission from [56], c Stem cells derived oxygen carriers (SCOCs); Differentiation of human iPSCs into RBCs derived from primary fibroblasts using Oct4/Klf4/Sox2/LIN28 through three steps: generation of iPSCs, HSCs, and finally RBCs. Reprinted with permission from [73], d Oxygen delivery mechanism of Oxygen micro/nanobubbles (OMNBs), (A) OMNBs disruption using ultrasound and (B) diffusion of oxygen across the concentration gradient. Reprinted with permission from [86]

The chemically modified HBOCs include cross-linked Hb including both intra and inter-molecularly, polymerized Hb, polyethylene glycol conjugated Hb, natural extracellular biopolymer Hb, genetically engineered recombinant Hb [13,14,15]. Despite decades of devoted improvement, the majority of chemically modified HBOCs have fallen short of achieving sufficient circulatory half-lives like > 12–18 h, which significantly restricts their usefulness for short-term utilization in cases of acute blood loss [31, 32]. Furthermore, they generally exhibited undesirable toxicities in the clinical trials, such as, vigorous microvascular vasoconstriction leading to end-organ damage, hypertensive emergencies, cardiovascular dysfunction like myocardial infarction, renal toxicity, renal failure, gastrointestinal distress, and unfortunately enhanced mortality [33,34,35,36].

Despite the discontinuation of numerous HBOCs products, valuable insights can be gleaned from fundamental research analysis that has identified their limitations. For instance, the infiltration of modified Hb into blood vessel endothelium has been partially attributed to the vasoactivity of acellular HBOCs, leading to oxidative damage and nitric oxide (NO) consumption, which is a crucial paracrine signaling element essential for the underlying smooth muscle relaxation [37, 38]. Moreover, the delivery of oxygen carried by these permeable Hb initiates autoregulation of blood flow, accelerating the contraction of arteriolar vessels, which, in turn, elevates blood flow as well as hydrodynamic pressure [38, 39]. In order to mitigate the harmful impacts of blood substitutes arising from the inherent toxicity of hemoglobin, a new pharmacologic cross-linking blood substitute called HemoTech has been reported, which is both safe and efficacious. The cGMP manufacturing process of HemoTech incorporates an innovative and validated orthogonal technology platform designed for the efficient removal of endotoxin, prions, as well as both non-enveloped and enveloped viruses. The outcomes from preclinical and clinical investigations affirm that HemoTech demonstrates non-toxic characteristics which possesses vasodilatory properties, capable of mitigating vasoconstriction subsequent to hemorrhage [10, 40]. Significantly, certain polymerized Hb products like PolyHeme™, Hemopure™, Oxyglobin™ and Oxy-vita exhibit restricted NO sequestration. This is probably because their larger particle sizes hinder their ability to interact with the endothelium underneath [38, 41]. The surface alteration of Hb using inert polymers, such as polyethylene glycol (PEG) with molecular weight > 5 kDa seen in Hemospan, leads to larger particle sizes. This enhancement improves blood circulatory half-lives, oncotic properties, product viscosity, and, promotes heightened intravascular oxygen delivery with minimal vasoactivity [42]. The Phase III clinical trial of Hemospan was employed to prevent hypotension, but it was also terminated in 2013 [43]. Considering, like other chemically modified HBOCs, PEG-modified polymerized or conjugated Hb is unable to autonomously regulate the oxidative state of iron (Fe) in their heme groups. This deficiency leads to the irreversible transformation of Fe2+-containing Hb into Fe3+-containing methemoglobin (metHb) [44, 45]. In contrast to Hb, metHb has a reduced capacity to carry oxygen and exhibits a heightened affinity for oxygen. This characteristic impedes oxygen delivery at physiological oxygen tensions, consequently leading to the onset of adverse effects, including hypotension and bradycardia [46].

In the encapsulated HBOCs, Hb is encapsulated within a phospholipid bilayer capsule, which will resemble the RBC membrane. A phase 1 human trial of Hb vesicles was recently approved in Japan in 2022 [20]. The researcher groups created Hb vesicles (HbVs), which a cellular-structured HBOCs that encapsulate purified and concentrated Hb molecules within liposome (PEGylated phospholipid vesicles) with a mean particle diameter of 225–285 nm, utilize a lipid bilayer membrane to shield against the toxic effects of molecular hemoglobin, and mimicking erythrocytes in the process shown in Fig. 3a [20]. In phase 1 study, twelve healthy male volunteers were received single doses of HbVs across 3 cohorts 1, 2, and 3 (n = 4), respectively. Cohort 1, and 2 receiving 10, 50 mL dosages without premedication and cohort 3 receiving 100 mL dosage with premedication (Fig. 3b). To prevent volume overload in healthy volunteers, the trial’s maximum dose was limited to 100 mL. In cohorts 2 and 3, HbVs was infused at 1 mL/min for the first 10 min, then increased to 2.5 mL/min and performed different laboratory test, pharmacokinetic analysis. Several adverse effects happened, regarded as liposome-induced infusion reactions which is shown in Fig. 3c. The appearance of adverse effects shortly after starting HbVs administration and their spontaneous resolution within minutes without medication support this inference. In cohort 3, prioritizing subject safety, premedication with dexamethasone (6.6 mg IV), famotidine (20 mg Orally), and acetaminophen (500 mg Orally) was administered 1 h before HbVs dosing, a common practice for liposomal drugs. The first two subjects completed the 100 mL infusion without adverse effects, but the third developed back discomfort and a chest rash after 10 mL, leading to an immediate stop. No clinically significant changes were observed in vital signs, except for body temperature (Fig. 3d). All deviated laboratory results resolved without related symptoms, and the half-life in the bloodstream was about 8 h [20].

Phase 1 study of HbVs; a Schematic diagram of production of HbVs, b Study design of phase 1 with and without premedication, c Summary of adverse effects related with the administration of the HbVs, d Result of systemic systolic blood pressure. Reprinted with permission from [20]

A new biomimetic HBOC with high stability, high reactive oxygen species (ROS) scavenging ability is erythrocyte membrane-encapsulated Hb oxygen carrier which was produced by coating the PLGA (poly-lactide-co-glycolide) core of hemoglobin through the membrane of the erythrocyte which maintains its long circulation time, high biological safety, and low immunogenicity [47]. Furthermore, another HBOC is polydopamine (PDA)-encapsulated Hb in which PDA as a biocompatible coating on the surface of Hb- nanoparticles imparts antioxidation properties, preventing the oxidation of Hb to metHb. Simultaneously, it diminishes Hb leakage and alleviates the toxicity linked to free Hb [48]. Moreover, a new candidate, ErythroMer is a bio-synthetic, first-in-class, and nano-cyte blood substitute and it contains deformable, hybrid peptidic-lipid nanoparticle that carries a high payload of hemoglobin (Hb) per particle. This structure enables (a) context-sensitive control of oxygen capture and release, while (b) reducing adverse interactions between hemoglobin and nitric oxide (NO). ErythroMer remains in the early testing phases. Recent preclinical studies have demonstrated that it can effectively deliver oxygen in mice with 70% of their blood volume replaced by ErythroMer. Similarly, in rabbits, when half of their blood volume was removed, infusing ErythroMer successfully resuscitated the animals, much like real blood [49, 50].

Furthermore, the production of genetically engineered hemoglobin involves the use of recombinant DNA technologies. The altered human Hb genes are introduced using plasmids into Escherichia coli or Saccharomyces cerevisiae. The engineered human hemoglobin genes are subsequently expressed within E. coli to generate human Hb molecules [51]. Rather than relying on them, current efforts aim to derive recombinant Hb from plants, such as Nicotiana benthamiana [52]. Natural Extracellular Biopolymer Hb HBOCs produced from Lumbricus terrestris (LtEc) which is under research, whereas another HBOC Hemarina-M101 was approved for clinical use by the European Union for donor organ preservation. It could improve COVID-19 patients’ survival [53, 54]. Additionally, researchers are increasingly directing their focus toward fetal Hb, known for its greater stability compared to adult Hb. Fetal Hb provides advantages attributed to its lower oxidative reactivity compared to adult Hb, although, both of them produced similar yields of purified functional protein [55]. The summary of different HBOCs is listed in Table 1.

Perfluorocarbons (PFCs) are hydrocarbons in which the hydrogen atoms are entirely replaced with fluorine atoms, occasionally with additional halogens. They are transparent, chemically inert, and seemingly non-toxic liquids characterized by low boiling points and they exhibit insolubility in water and alcohol. Clinical applications necessitate the solubilization of PFCs using an emulsifying agent, particularly advantageous for individuals who reject blood or proteins derived from humans or animals [21]. PFCs can dissolve substantial quantities of gases. The solubility of gases in PFC liquids follows the order CO2 > > O2 > CO > N2, corresponding to the decreasing molecular volume of the solute. PFC emulsion can easily navigate through blood vessels obstructed in certain diseases due to their small sizes and help to enhance the oxygenation rate, where RBCs face challenges. [22]. It exhibits an impressive oxygen dissolution concentration, reaching around 40%-50%, which surpasses water’s capacity by 20 times and exceeds plasma by 2 times [23]. Additionally, it can dissolve 130–160 mL of carbon dioxide, two to three times more than water’s corresponding capacity [24]. Linear PFCs like perfluoro-octyl bromide exhibit greater effectiveness in dissolving O2 compared to cyclic molecules such as perfluorodecalin. Overall, the solubility of O2 in PFCs is inversely proportional to molecular weight and directly proportional to the number of fluorine atoms [22]. Along with PFOCs, PFC-based nanoparticles were engineered for multifunctional nanomedicines, such as bioimaging contrast agents and drug delivery vehicles for the diagnosis and treatment of various diseases [25]. In early efforts to create substitutes for RBCs, PFC emulsions emerged as the most advanced alternatives to donor RBC units, as shown in Fig. 2b [56].

To date, PFOCs can be categorized into five subclasses based on the primary PFC used in the emulsion product including perfluorodecalin-based PFOCs such as Fluosol-DA, Perftoran, Albumin-derived PFC-based AOC, PFOB (perfluorooctylbromide) -based PFOCs like Oxygent, perfluoro-dichloro octane- based PFOCs like Oxyfluor, tert-butyl perfluoro cyclohexane-based PFOCs such as Oxycyte, and Dodecafluoropentane (DDFPe)-based PFOCs [16]. The general properties of different PFOCs are listed in Table 2. Fluosol-DA demonstrated an oxygen-carrying capacity of only 7.2% (v/v), which is lower than that of human RBCs (17–20% v/v) and proved insufficient in critical situations like anemia with significant blood loss. Despite its tendency to accumulate in the liver and spleen, the substance could be cleared by the lungs for a specific duration [13]. Fluosol-DA’s development was impeded due to low durability and stability in blood vessels, along with the complexity of its configuration and use [26]. Oxyfluor™ and Oxygent™ exhibited enhanced lipophilicity, facilitating increased encapsulation of higher amounts of oxygen-enriched PFCs in these formulations. Oxyfluor™ was discontinued following early clinical trials due to severe adverse events associated with its effective dosage [27, 28]. Likewise, Phase III clinical trials of Oxygent™ were halted due to an enhanced occurrence of strokes among coronary bypass patients [13]. While the Russian-developed Perftoran™ has received approval for clinical use in Russia and Mexico and rebranded as Vidaphor™ by Fluor02 Therapeutics, Inc., USA, in North America and Europe, which has been safely administered to over 30,000 patients with only minor side effects, with aspirations for future FDA approval [29, 30]. Oxycyte is made up of submicron particles of 60% perfluoro(t-butylcyclohexane), with an egg phospholipid emulsifier. Phase II trial of oxycyte was completed in 2008 with severe non-penetrating traumatic brain injury patients, and initiated a Phase III trial in the USA to evaluate the cardiac surgery patients, but terminated in September 2014 due to lack of patient assignation [57, 58]. In 2018, ABL-101, the new code name of oxycyte, was reinstated in the Phase II trial in the UK specifically for acute ischemic stroke, launched by Aurum Biosciences Ltd, Glasgow, UK, a collaborative work. This clinical trial is groundbreaking as it employs a PFC nanoemulsion as a theranostic platform [59]. Dodecafluoropentane, a unique oxygen carrier with high affinity and transport capacity, exhibits notable safety effects on oxygen-reliant organs like the brain and heart [60, 61]. As a result, it has been formulated for addressing brain damage and hemorrhagic shock in medical treatment [62]. Presently, PFC development focuses on creating smaller and more stable emulsions. This effort aims to achieve necessary enhancements in biodistribution, circulatory properties, and clearance, exemplified by the creation of kinetically stable PFC nanoemulsions. Hence, researchers must seek a surfactant characterized by excellent biocompatibility when formulating novel PFOCs.

Human serum albumin (HSA) is a protein derived from human blood, possessing amphipathic properties, high biocompatibility, low toxicity, and minimal immunogenicity. Consequently, HSA is anticipated to offer a safer and more efficient emulsification process for developing perfluorocarbon (PFC) oxygen carriers. Wrobeln and colleagues employed HSA as an emulsifying agent to encase perfluorodecalin, creating a capsule containing perfluorocarbon [63]. The human erythrocyte membrane serves as a natural biological material containing several glycans and proteins on the surface, and retention time in the body is 120 days, which is employed as a natural carrier for long-circulating drugs. Moreover, it possesses excellent biocompatibility, nonimmunogenicity, and biodegradability [64]. Applying this functionalization method, some researchers prepared biomimetic PFC oxygen carriers.

A new novel oxygen self-enrichment biomimetic carbon–oxygen carrier was created which comprises two components: (1) an oxygen-carrying segment containing perfluorotributamine loaded with the near-infrared dye indocyanine green; (2) the external covering constituted the RBC vesicles extracted from the RBC membrane which helps to prevent uptake by macrophages, thereby extending the circulation time in vivo [64]. Gao and colleagues acquired nanoparticles by enclosing PFC within the biocompatible polymer poly (D, L-lactide-co-glycolide), PLGA. Subsequently, they coated the surface of these nanoparticles with the RBC membrane. This process resulted in nanoparticles characterized by a high oxygen-carrying capacity and an extended blood circulation time, referred to as PFC@PLGA-RBC membrane nanoparticles. For In vivo radiotherapy treatment of PFC@PLGA-RBC membrane nanoparticles, 4T1-tumor-bearing mice received a 200 µL IV injection of PFC@PLGA-RBCM. After 24 h, they were exposed to 8 Gy X-ray radiation. Mice provided with PFC@ PLGA-RBCM without X-ray exposure (PFC@PLGA-RBCM group), mice treated with X-ray radiation without nanoparticle (RT group), and mice administered with only PBS were used as control groups (Fig. 4a). PFC@PLGA-RBCM alone did not affect tumor growth, but merged with X-ray radiation, it significantly impeded tumor growth more effectively than radiation alone shown in Fig. 4b. 14 days post-treatment, mice were euthanized to evaluate tumors; average tumor weight was lowest in the group administered with both RT and PFC@PLGA-RBCM, significantly less than with RT alone (Fig. 4c). In the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling assay, significant damage and high tumor cell apoptosis were found in the PFC@PLGA-RBCM + RT group, with minimal damage in control groups. Additionally, Hematoxylin & Eosin-stained images revealed no inflammation or organ damage in major organs 14 days after PFC@PLGA-RBCM + RT treatment, suggesting no in vivo toxicity (Fig. 4d) [65]. Furthermore, human erythrocytes, with their biconcave structure supported by ankyrin, possess a flexible membrane that allows them to change shape, facilitating passage through narrow capillaries. Fu et al. featured a micron-sized, concave, and highly deformable core–shell PFC-based oxygen carrier following human RBC structure, called cDFCs whereas its core was PFOB, and poly(lactide-co-caprolactone) utilized as a thin and extremely deformable elastic shell [66].

In vivo radiotherapy treatment of PFC@PLGA-RBC membrane nanoparticles. a Experimental design of animal model; b Tumor growth curves of different groups; c Weight curves of tumor of different groups, d Stained tumor slices collected from 24 h post treatment of mice. Reprinted with permission from [65]

Recent advancements in stem cell technologies have enabled the production of RBC-like cells from diverse cultured sources, mimicking the natural biogenesis process of human erythrocytes. In 2006, Takahashi and Yamanaka discovered a novel cell source capable of differentiating into cell types from endoderm, ectoderm, or mesoderm lineages [67]. These ex vivo-generated RBC oxygen carriers hold remarkable potential for advancing the creation of an unlimited source of RBC units for transfusion [68]. It is produced through induced hematopoietic stem cells of different origins which closely resemble natural RBC in terms of their physicochemical characteristics and biological functions. These carriers hold promise for fulfilling the objective of extended oxygen delivery to patien