Introduction

Blood is the most basic and essential component for maintaining life, supplies oxygen to the human body, and transports nutrients and waste. Red blood cells (RBCs) in the blood are essential components that supply oxygen to tissues and organs and release carbon dioxide to the outside of the body. Blood must be readily supplied in emergency situations such as surgery of seriously ill patients or during disasters, but blood is not always available. The current means to receive blood is only through allogeneic blood transfusions. However, blood supplies are insufficient to meet the demand due to low blood donation rates worldwide. Blood transfusions are currently the only means of obtaining blood; however, blood transfusions can cause immunological and non-immunological side effects and, rarely, infections, such as syphilis and hepatitis B virus (HBV) (Fölsch and Cassens 2009). In addition, some patients refuse blood transfusions due to religious reasons, which do not allow the use of allogeneic blood products (Gohel et al. 2005). In addition, Korea’s blood donation rate is 5.04% as of 2020, which is higher than the 3.15% of other high-income countries announced by the World Health Organization (WHO); however, the demand for blood exceeds the ability to supply it. It is speculated that this is due to a declining birth rate, a shortage of young donors, and an increasingly aging population (Kim 2022). In addition, through a study by the National Blood Collection and Utilization Survey (NBCUS), RBC collection and transfusion decreased by 3.0% and 6.1%, respectively, between 2015 and 2017, showing a decrease in blood donation and transfusion rates in the U.S. since 2008 (Jones et al. 2021).

Therefore, a blood supply source that can replace blood transfusions is needed. Artificial oxygen carriers (AOCs), which have been attracting attention as an alternative to blood transfusion, aim to improve the transport and release of oxygen to tissues. Therefore, AOCs can be used as an alternative to allogeneic blood transfusions or to improve tissue oxygenation and function in oxygen-poor organs (Kim and Lee 2009). AOCs also reduce the risk of disease transmission and avoid incompatibility issues as well as act as anti-ischemic agents in a variety of pathogenic conditions that inhibit tissue oxygenation (Baron 1999; Spahn 1999). Among AOCs with these characteristics, the most actively researched field is AOCs that replace RBCs. Research and development of RBC replacements have been centered on the U.S. and Japan. In particular, the Office of Technology Assessment (OTA) of the U.S. Congress presented items such as: (1) the oxygen dissociation curve and oxygen-carrying capacity must be similar to real RBCs; (2) the product is non-toxic and must have non-antigenic properties; (3) the circulation ability must be good; (4) the product must be able to circulate for a long time; and (5) the storage period should be long. These five items were suggested in the 1985 “Blood Policy and Technology” report (Congress et al. 1985). These RBC substitute AOCs are classified into hemoglobin-based oxygen carriers (HBOCs) and perfluorocarbon-based oxygen carriers (PFOCs) (Charache 1990).

First, HBOCs can have a variety of structures that can be made through several fabrication methods. In addition to the polymerization method, which combines hemoglobin with various polymer materials, the cross-linking method, which strengthens the bond between hemoglobin molecules, and the encapsulation method, which seals hemoglobin inside nanocapsules or microparticles, there are various manufacturing methods (Jansman and Hosta-Rigau 2018; Bialas et al. 2019). There are few limitations for each structure, and there are currently products such as Hemopure that are limited but clinically approved. On the other hand, the only way to manufacture PFOCs is through emulsion, and both first- and second-generation PFOCs have a long residence time in the body and are formulated with many limitations. In addition, there were previously clinically approved products, but sales were discontinued due to side effects (Table 1) (Gould et al. 1986; Kuznetsova 2003; AGENCY 2007; Chen et al. 2009; DeSimone et al. 2018; Ferenz and Steinbicker dentistry 2019). For this reason, PFOCs are considered to have many difficulties in development compared with HBOCs.

Table 1 Comparison of the existing PFOCs and HBOCs
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However, PFOCs are liquid at room temperature and can dissolve 40 to 70% of oxygen; therefore, their oxygen-carrying capacity is approximately three times greater than that of conventional blood. As PFOCs themselves are not smoothly dissolved in the blood, they are manufactured and used in an emulsified state. However, there is a great advantage in that the higher the concentration of oxygen contained in the production of PFOC, the better the oxygen transport ability. In addition, another advantage is that blood is not required when preparing a PFOC emulsion, and there is no risk of infection with PFOCs (Silverman 2004). Thus, it is difficult to conclude that all PFOCs have failed completely clinically; future PFOCs are judged to have high development potential.

Therefore, after discussing the previously developed PFOCs, this review paper presents a direction for the future development of PFOCs as artificial blood products. In order to overcome the abovementioned difficulties and develop artificial blood products utilizing the advantages of PFOCs, this study intends to provide more easily accessible information to identify the unmet need (Fig. 1).

Fig. 1
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Content flow of PFC-based RBC Substitute Artificial Oxygen Carriers: Considerations and Direction of Technology (Jägers et al. 2021; Mohanto et al. 2023)

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Perfluorocarbons(PFCs)

PFC compounds have historically been used in industrial and medical applications (Schmieder et al. 2015). Industrial and commercial applications include refrigerants, aerosol propellants, blowing agents, solvents and polymers, and PFCs have even been used in fire extinguishers. In medicine, fluoride-containing compounds have been used as orthopedic implants, vascular structure replacements, inhalation anesthetics, anti-inflammatory agents, synthetic drugs, and steroids (Goodman et al. 1984; Lowe 1991; Park et al. 2001; Spahn and Pasch 2001; Hill et al. 2002; Woo et al. 2008). PFCs are also the first synthetic compounds to be tested as oxygen carriers (Riess and Krafft 2006). Chemically, PFCs consist of carbon chains of carbon–fluorine bonds (Fig. 2); the carbon–fluorine bond energy is very high compared to the energy of carbon-hydrogen and other bonds. Furthermore, PFC liquids also have higher densities and lower boiling and melting points than water. The surface tension of PFCs is also lower than that of water, and the discovery that PFC fluids can greatly dissolve gases such as oxygen and carbon dioxide has led to evaluation as a medium for transporting respiratory gases (Wahr et al. 1996; Spahn 1999; Winslow 2000; Lowe 2001; Squires 2002). PFOCs can dissolve 50 times the amount of oxygen found in plasma. In addition, oxygen is supplied using an organic compound with high gas solubility. Linear PFCs, such as perfluorooctyl bromide (PFOB), dissolve oxygen better than cyclic PFCs, such as perfluorodecalin (PFD) (Fig. 2, Table 2) (Goorha et al. 2003; Winslow 2005; Kuznetsova 2022). Furthermore, intravenous infusion of a large amount of untreated PFCs causes spontaneous foaming in the lungs and leads to death; therefore, PFCs must be used after emulsification (Fig. 3) (Lanaro et al. 2014). At this time, since PFCs are hydrophobic and do not mix with water, they is emulsified by using a surfactant such as phospholipids (Krafft and Riess 1998; Silverman 2004; Vorob’ev 2009).

Fig. 2
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PFC chemical structure examples. A PFD, B PFOB. Drawn with ChemDraw® software

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Table 2 Physicochemical properties of PFCs (Winslow 2005; Kuznetsova 2022)
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Fig. 3
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PFC emulsification method. The O/W emulsion method, which is performed by putting the oil phase composed of PFCs and a surfactant into the water phase, is common (Jägers et al. 2021)

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These PFCs were discovered and developed in large quantities in connection with the Manhattan Project during World War II (Benner et al. 1947; Fowler et al. 1947). Under the lead of Leland Clark (1918–2005), the use of PFCs as hemoglobin substitutes for oxygen transport began. Later, in 1970, the concept of “synthetic blood” was first used in perfusion experiments on animals using perfluorinated liquids and Clark bubble-defoam heart–lung machines(Clark and Gollan 1966; Clark Jr et al. 1970a; Clark Jr et al. 1970b). Based on these experiments, the first patent appeared detailing the electrochemical process to prepare PFCs, and PFCs began to be considered as promising blood substitutes in the scientific community (Maugh 1973; Kosugi et al. 1976). In the wake of the preceding events, the first PFC-related perfusion and oxygenation-related brands began to emerge in 1976: Fluosol-DC and Fluosol-43 (Kosugi et al. 1976; Nováková et al. 1976; Charbe et al. 2022). Later, in 1990, the first human administration of PFCs in premature infants occurred (Fuhrman 1990; Kaplan et al. 1990). However, side effects and stability issues of PFOCs have arisen, and their toxicity to the lungs, liver, and spleen has been highlighted more than their oxygen delivery capacity. For this reason, since 2009, research related to PFCs, such as clinical trials, has not been actively conducted.

Perfluorocarbon-based oxygen carriers (PFOCs)

First-generation PFOCs

First-generation PFOCs include Fluosol-DA (Green Cross Corporation, Japan), Oxypherol (Fluosol-43, Green Cross Corporation, Japan), and Perftoran (Perftoran, Russia) (Tables 3, 4) (Faithfull 1994; Millard 1994; Lowe 1999; Riess 2001; Eckmann and Lomivorotov 2003; Durnovo et al. 2008; Castro and Briceno 2010; Riess and Krafft 2013; Mohanto et al. 2023).

Table 3 Comparison of first-generation PFOCs (Holloway et al. 1986; Mohanto et al. 2023)
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Table 4 Summary of clinical outcomes and side effects of first-generation PFOCs (Mohanto et al. 2023)
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Fluosol-DA

Fluosol-DA uses PFD and PFTPA as PFCs (Fig. 4A, B). Fluosol-DA is a regulated product in the US, Japan and Europe from 1989 to 1990 for clinical use as an oxygen transport adjuvant in the insertion of a balloon catheter during percutaneous coronary angioplasty (Lowe 1999). Initial studies using intravenous doses of 20–500 mL demonstrated no negative effects (side effects) on the heart, liver, kidneys, and hematological function. In addition, as a result of using an intravenous dose of 500–1500 mL in Phase 2 and 3 clinical trials without controls for patients with hemorrhage and carbon monoxide poisoning, Fluosol-DA was recommended for bleeding, emergency blood transfusion, cerebral ischemia, and carbon monoxide poisoning (Habler and Messmer 2000); however, when it was subsequently administered to patients with acute bleeding and severe anemia, no significant changes were observed in the oxygen extraction rate, oxygen consumption, cardiac index, or mean arterial pressure after infusion. This led to the conclusion that the amount of PFCs was insufficient to have a significant impact on oxygen content. For this reason, the FDA denied approval for use of Fluosol-DA as a substitute for RBCs (Gould et al. 1986).

Fig. 4
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Chemical structure of PFC used as the main component of first-generation PFOCs: A The main component of Fluosol-DA, perfluorodecalin (PFD), B The main component of Fluosol-DA, perfluorotripropylamine (PFTPA), C The main component of Oxypherol, perflurotributylamine (FTBA), D The main component of Perftoran, FDA, E The main component of Perftoran, perfluoromethylcyclohexylpiperidine (FMCP). Drawn with ChemDraw® software

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In the manufacturing process of Fluosol-DA, emulsion freezing is essential for transportation and storage. To use this frozen emulsion, it has a characteristic of poor stability because it must be mixed after thawing (Lowe 1999; Riess 2001). Thus, to manufacture a more stable emulsion, we added PFTPA. The Fluosol-DA manufactured in this way exhibited a significantly extended half-life, surpassing the ideal intravenous half-life of 7 days, reaching a much longer 65 days (Riess 1994, 2001). This prolonged half-life can be considered as a long-term half-life, which may lead to issues related to prolonged residence and potential toxicity. In addition, the Pluronic F-68 surfactant component has been associated with side effects including complement activation, leukocyte inhibition, hemodynamic effects, flu-like symptoms (myalgia and fever), transiently decreased platelet counts, and induction of anaphylaxis (Lowe 1999; Spahn 2000; Ness and Cushing 2007). Due to these characteristics, Fluosol-DA failed commercially (Riess and Krafft 2013). Finally, it was withdrawn by the FDA in 1994 due to difficulties in frozen storage and thawing before use (Jahr et al. 2007).

Oxypherol(Fluosol-43)

Oxypherol uses FTBA as the PFC (Fig. 4C). Oxypherol showed significant effects as an anticoagulant and anti-inflammatory cytokine inducer (Ochikubo et al. 1999). Oxypherol with lidocaine resulted in more effective myocardial preservation in meeting myocardial oxygen demand during prolonged cardiac arrest in isolated rat hearts (Bito et al. 2000). Oxypherol administration prolonged xenograft survival using a model of guinea pig to rat liver transplantation. Although the onset of hyperacute rejection was delayed, all the rats died within 14 h (Tanaka 1999). Oxypherol has problems with an FTBA half-life in rats of 2.5 years, indicating long-term retention; therefore, its use was rejected in humans (Riess and Krafft 2006).

Perftoran

Perftoran used PFD and FMCP as the PFCs (Fig. 4D, E). Systemic administration of Perftoran after ischemia promoted faster and more complete structural regeneration during reperfusion (Kozhura et al. 2005). Intravenous injection of Perftoran increased the macroglobulin content in plasma and peritoneal exudate of rats with peritonitis and was effective in preventing adhesion formation after surgery (Yarema and Magomedov 2003). In addition, Perftoran was useful for the correction of structural and metabolic changes in the liver during atherosclerosis in rabbits (Leskova et al. 2003). Thus, it was approved for clinical use in Russia from 1995 to 1996 as a temporary intravascular oxygen carrier for hemorrhagic shock and perfusion of human organs (Lowe 1999; Kuznetsova 2003). Perftoran has also been approved for medical use in the Republic of Kazakhstan (1995), Ukraine (2005), Mexico (2005), and the Kyrgyz Republic (2006) (Castro and Briceno 2010).

There were no reports of specific technical or clinical problems with Perftoran, but problems have been reported in the FMCP used as the PFC for this product. The most common problem is the long-term half-life of 90 days. Furthermore, the relatively low surface activity reported for the floxamer can be problematic and decrease the stability of the emulsion (Riess 2001). Another problem with Perftoran is that its shelf life without freezing is approximately one month at 4–8 °C, which is too short to be used as a blood substitute.

As such, first-generation PFOC has problems such as low oxygen content, low oxygen transfer capacity, long half-life, and short shelf life. For this reason, clinical trials were suspended, or sales were suspended for in vivo clinical trials except for experimental purposes.

Second-generation PFOCs

Second-generation PFOCs were developed to overcome the difficulties of first-generation products, and Kusnetzova presented three main criteria for distinguishing generations (Kuznetsova 2003).

  1. 1.

    Content and nature of the fluorocarbon Phase: The content of PFCs contained in the emulsion of the second-generation products is 2 or 4 times higher than that of first-generation products.

  2. 2.

    Properties of emulsifiers: Second-generation products use natural phospholipids as an emulsifier instead of a water-soluble emulsifier (i.e., F-68) used in the first-generation product.

  3. 3.

    Storage conditions: Second-generation products are stored without freezing.

In addition, Reiss and Le Blanc et al. divided the desirable characteristics of second-generation PFCs into the following five categories and established them as standards: (1) oxygen-dissolving capacity, (2) rapid excretion and tissue retention, (3) reduction of severe side effects, (4) increased PFC content, and (5) large-scale production and availability. The three candidates selected according to the above five criteria were PFD, PFOB, and bis (perfluorobutyl) ethylene. Among them, PFOB used in Oxygent was preferred in clinical trials due to its safety and high excretion rate (Riess and Blanc 1982).

Second-generation PFOCs include Oxygent (Alliance Pharm, USA), Oxyfluor (HemaGen, USA), and Oxycyte (Oxygen Biothera, USA) (Tables 5, 6) (Faithfull 1994; Goodin et al. 1994; Remy et al. 1999; Cuignet et al. 2000; Riess 2001; Cabrales et al. 2004; Riess 2006; Riess and Krafft 2006; Riess and Krafft 2013; Mohanto et al. 2023).

Table 5 Comparison of second-generation PFOCs (Sen Gupta 2017; Mohanto et al. 2023)
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Table 6 Summary of clinical outcomes and side effects of second-generation PFOCs (Mohanto et al. 2023)
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Oxygent

Oxygent used PFOB and perfluorodecyl bromide (PFDB) as PFCs (Fig. 5A). In an anesthetized hemodilution canine model breathing 100% oxygen, intravenous administration of Oxygent and additional hemodilution was as effective as autologous RBC transfusion in maintaining tissue oxygenation (Habler et al. 1998a, b). Hyperoxic ventilation rapidly improved tissue oxygenation (Habler et al. 1998a, b). When myocardial oxygenation was supported by Oxygent after deep normovolume hemodilution in anesthetized dogs, the left ventricular myocardial contractile relaxation characteristics were improved (Keipert et al. 1994; Habler et al. 1997). In addition, the clinical results showed that fluid resuscitation with Oxygent increased local oxygen availability at the cell level, restored liver microcirculation and tissue oxygenation, and provided better hepatocellular energy metabolism than stored blood or pentastarch (Paxian et al. 2003). In a canine model of hemorrhagic shock, oxygen-supplemented fluid resuscitation under hyperoxia was more effective than conventional colloids. Use of Oxygent increased normal blood volume breathing supplemental oxygen. Likewise, the subcutaneous tissue oxygen tension of the bleeding rats was also increased (Rosen et al. 2006). In addition, Oxygent was able to prevent gastrointestinal ischemia in patients undergoing cardiac surgery with preserve gastrointestinal function after surgery (Frumento et al. 2002). Oxygent used with acute normovolume hemodilution was well tolerated and improved oxygen delivery in patients undergoing elective coronary artery bypass graft with cardiopulmonary bypass (CPB) (Hill et al. 2002). Furthermore, patients receiving high-dose PFCs showed increased cerebral blood flow and an increased number of total cerebral embolic loads (Hill et al. 2005). However, Mattrey suggested that the reported increase in cerebral embolism was due to interference generated by the PFCs in the automated transcranial Doppler ultrasound device used to detect the embolus (Mattrey 2007). Hill and Grocott argued for the validity of their results and suggested that they might explain the increased incidence of stroke in a follow-up Phase 3 trial of Oxygent, which was used to increase intraoperative blood thinning (Hill and Grocott 2007).

Fig. 5
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Chemical structure of PFC used as the main component of second-generation PFOCs: A The main component of Oxygent, perfluorooctyl bromide (PFOB), B The main component of Oxyfluor, 1,8-dichloroperfluorooctane (PFDCO), C The main component of Oxycyte, tertbutylperfluoro-cyclohexane (TBPCH). Drawn with ChemDraw® software

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A representative clinical side effect of Oxygent is macrophage activation associated with reticuloendothelial system (RES) uptake due to PFC particles. Headaches, back pain, and flu-like symptoms were also observed; all of the aforementioned symptoms were transient and reversible within 12 h, with a 15% reduction in platelet count (Riess 2001). However, the oxygen dose should be minimized as the particulate properties can easily overload the RES (Habler and Messmer 2000). This emulsion demonstrated significant oxygen delivery in both animal models and human clinical trials; however, Oxygent’s Phase 3 clinical trial was temporarily suspended due to safety concerns, including an apparent neurological imbalance, which has not been substantiated as being related to the product (Lambert et al. 2019; Jägers et al. 2021). Considering this neurological imbalance, an in-depth analysis of the secondary results of a Phase 2 cardiac surgery trial was conducted before deciding to continue with Phase 3 trials. This clinical trial was resumed in China by Double-Cranes Pharmaceutical (Eckmann and Lomivorotov 2003). However, this trial, which was a follow-up clinical trial, failed. This was attributed to simultaneous changes in both independent variables in the control versus test groups. Both patient groups first underwent autologous normovolemic hemodilution (ANH), but the Oxygent patients (1) then received a first dose of the test material, while the controls did not, and (2) also underwent a rapid additional one-liter blood withdrawal (or intraoperative autologous donation, IAD), which the control group did not incur, thus creating a double imbalance between the two groups. Hence, no causal link can be drawn between the observed bleeding and neurologic effects and PFC emulsion (Krafft and Riess 2021). Subsequently, the Phase 3 clinical trial examining the association of acute normovolemic hemodilution during cardiac surgery with Oxygent was not resumed (Castro and Briceno 2010; Lambert et al. 2019).

Oxyfluor

Oxyfluor uses 1,8-dichloroperfluorooctane (PFDCO) as the PFC (Fig. 5B). Oxyfluor was used as an intravascular oxygen carrier in a porcine model of CPB and improved tissue oxygenation and total oxygen consumption, reduced some contribution of oxygen transported by RBCs, and increased the overall efficiency of oxygen transport (Briceño et al. 1999). Oxyfluor was also used in preclinical trials of resuscitation after hemorrhagic shock in a canine model. As a result, oxygen transport increased, and the total tissue oxygen supply was restored for 60 min (Goodin et al. 1994). In a clinical Phase 1a trial, the safety and tolerability of increasing doses (0.25, 0.50 and 1.00 ml PFDCO/kg) of Oxyfluor were evaluated in healthy male volunteers. As a result, subjects in the low-dose group (n = 6) were not affected by the treatment, but subjects in the mid-dose group (n = 6) showed flu-like symptoms. In addition, the subjects in the high-dose group (n = 5) also showed flu-like symptoms and a spurious decrease in platelet count. Subsequently, a clinical Phase 1b trial involving surgical patients who received pretreatment with dexamethasone was conducted. As a result of evaluating the safety and drug resistance of increased doses (1.0, 1.5, and 2.0 mL PFDCO/kg), patients were generally not affected by treatment. In addition, pretreatment with dexamethasone greatly reduced flu-like symptoms. However, the side effects of PFDCO, including decreased platelet counts, were transient and dose-dependent (Shaw and Richard 2006).

Safflower oil was added to see if it was possible to improve the stability of the emulsion and was evaluated with a triglyceride stabilization effect test. As a result, an improvement in emulsion stability with the addition of safflower oil was not clearly demonstrated (Riess 2001). As with Oxygent, similar clinical side effects including flu-like symptoms have been reported with Oxyfluor (Riess 2001). Phase 3 clinical trials were discontinued due to the suspension of funding from the giant pharmaceutical strategy partner (Spahn et al. 2002; Shaw and Richard 2006).

Oxycyte

The PFC used for Oxycyte was TBPCH (Fig. 5C), which delivered oxygen to tissues when combined with hyperoxic ventilation. There was no evidence of vasoconstriction or microvascular dysfunction in a hamster model of excessive hemodilution (Cabrales et al. 2004). Oxycyte used during isovolemic hemodilution, in which equal volumes of blood and colloids are infused to preserve isovolemia, produced higher arterial oxygen content and partial oxygen pressure without increasing regional cerebral blood flow (Yang et al. 2008). In addition, in a hamster window chamber model, it was confirmed that Oxycyte administered without stagnation in the ischemic area attenuated reperfusion injury after ischemia of the striated muscle. Pre-ischemic administration significantly reduced the functional capillary density after reperfusion and increased leukocyte activation (Cabrales et al. 2007). An infusion of Oxycyte saturated with oxygen prior to infusion increased brain tissue oxygenation in a model of selective middle cerebral artery occlusion in rats (Woitzik and Schiling 2007).

Oxycyte has been investigated in various animal models and in some clinical trials targeting traumatic brain and spinal cord injury patients. A Phase 2 clinical trial targeting traumatic brain injury patients was successfully completed in 2008 (Castro and Briceno 2010; Hill 2019). However, subsequent Phase 2 clinical studies on the safety and efficacy of Oxycyte, which began in 2009, were discontinued in 2014 due to sponsor discontinuation resulting from recruitment failures (Ferenz and Steinbicker 2019; Lambert et al. 2019).

In summary, the second-generation PFOCs reached the Phase 3 (2014) but were ultimately discontinued during this phase. However, for second-generation PFOCs, the possibility of long-term retention in the body is high when the PFC content is significantly increased compared to that of first-generation products. Long-term toxicity resulting from this process will be one of the main factors to be considered in the future development of novel PFOCs.

Problems with the current technology level

The cause of failure in the existing product manufacturing technology described above needs to be accurately identified, and a strategy to overcome it based on this can then be established. Therefore, herein, we discuss the problems of the first- and second-generation PFOC preparation technologies described above (Fig. 6).

Fig. 6
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Existing PFC-based technology

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First-generation PFOCs

First-generation PFOCs could not be developed by increasing the content due to the low purity and physical properties of PFCs due to synthetic technical problems. In addition, when prepared as an emulsion, a synthetic polymer-based surfactant such as poloxamer is used in the formulation. As such, first-generation PFOCs had lower oxygen delivery efficiency due to their low PFC content (less than 25%). Additionally, there were toxicity issues associated with the use of synthetic polymer-based surfactants, resulting in reported cases of discontinuation after clinical trials.

Second-generation PFOCs

Second-generation PFOCs have been developed with significantly improved purity and physical properties compared with first-generation PFOCs through the development of synthesis technology and processes. Second-generation PFOCs have been used for artificial RBC preparations, and their biocompatibility was increased using synthetic polymer-based surfactants and phospholipid-based surfactants, which are biomembrane structural materials. Due to these technical characteristics and efforts, a number of products that have entered the clinical stage have been reported compared with first-generation products. However, currently only one product is in the clinical trial stage, and the remaining products have been confirmed to be in a clinical discontinuation or failure state.

Development direction of PFOCs

The problems of the first- and second-generation PFOCs that have been discussed above can be divided into three categories: (1) large particle size, (2) long body residence time, and (3) high PFC content (second-generation).

Both the large particle size and high content of PFCs cause common problems leading to long residence times in the body. The large particle size causes accumulation in the liver due to uptake in the reticuloendothelial system (RES), and the long residence time in the body causes side effects such as a complement activation-related pseudoallergy (CARPA) reaction (Vasir et al. 2005).

The RES is composed of cells derived from monocytes capable of phagocytosis of foreign substances and particles. Although the most important function of the RES is phagocytosis, it also participates in cytotoxicity against tumor cells and has a function of regulating the immune system (Baas et al. 1994). Nanoparticles (< 1000 nm) can pass through all capillaries after being injected into the bloodstream, and in most cases, they are rapidly absorbed by macrophages of the RES (Kreuter 1994; Kreuter et al. 1995). In fact, PFOCs are stored in the phagocytes of the RES after intravenous injection of perfluorinated compound particles are removed from the blood (Miller et al. 1976). When RES uptake occurs with the use of PFOCs, PFC particles accumulate in the liver and cause intrahepatic toxicity. This is because the particle size of PFCs used in PFOCs is too large. To avoid RES uptake in the liver, the size of the nanoparticles should be < 100 nm (Vasir et al. 2005).

CARPA reactions are already well known in the field of nanotoxicity as drug formulations of nanoparticles also cause this reaction (Szebeni 2001, 2004, 2005, 2012, 2014; Szebeni et al. 2007, 2011, 2012). When the CARPA response is activated, a “blood stress” response occurs (Szebeni 2014). It is known that anaphylatoxin, an inducing factor of a CARPA reaction, can activate white blood cells and platelets, and this activation allows these cells to bind to each other and also to capillary endothelial cells. It involves microthrombi formation and circulatory obstruction, mainly in the pulmonary and coronary microcirculation. These changes are a major cause of hemodynamic changes in CARPA reactions and can lead to anaphylactic shock (Szebeni 2004). The injection-related reactions that can cause these CARPA reactions are due to the physicochemical properties of the nanoparticles (Szebeni 2005). Clearance of nanoparticles usually occurs via the RES as previously described, and high particle loads can result in RES overload, impairing the body’s ability to clear other particle species (Ghaghada et al. 2009). Therefore, efforts to reduce particle size will be required to avoid RES uptake and simultaneously suppress CARPA induction.

A long residence time should also be used to promote in vitro excretion using high vapor pressure PFCs, and the stability of emulsions should be secured using a biocompatible stabilizer.

In conclusion, addressing the unmet requirements outlined in Table 7 requires a strategic approach to PFOC development. This may involve initial screening studies on key components with a small hydrophile-lipophile balance (HLB) value for effective miscibility with lipophilic PFCs, aiming for effective blending with lipophilic PFCs. Additionally, exploring the use of auxiliary surfactants, integrating two or more surfactants for enhanced stability, could be a feasible consideration to improve stability.

Table 7 Unmet needs for existing PFOCs
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Afterwards, if the surfactant selection is completed, the selection of equipment in the process for making PFCs emulsifiers should also be considered for making small particle sizes. These devices include microfluidizer, which can be fabricated into small particles by applying high horsepower and high psi, and ultrasonicators, which use the collapse of cavitation bubbles to generate high-energy conditions, including shear and microjet, to make particles smaller.

If the small particle size is produced by the above method, it is considered that toxicity problems caused by long-term retention in the body will also be reduced.

Conclusion

Recently, Korea’s blood donation rate has decreased significantly due to the aftermath of COVID-19 and the decrease in the blood donation population due to the increased elderly population. The problem of an insufficient supply of allogeneic blood is not a problem unique to Korea. Currently, there is a decline in blood donations and blood transfusions worldwide. However, even under these circumstances, the volume of blood required due to important situations such as accidents and surgeries is steadily increasing. In addition, there is a need for medical blood reserves to prepare for the appearance of casualties in each country in the war between Russia and Ukraine. Therefore, the provision of a blood transfusion supply is essential in the military sector as well as in the civilian domain.

For the abovementioned reasons, artificial blood, artificial RBCs, and artificial RBC substitutes have been attracting attention again recently. Among artificial blood products, the development of artificial RBCs has stalled due to side effects such as toxicity in the body. As a result, there is no FDA-approved developed product. There are several products that have received conditional approval in Russia and Africa, but the fatality rate is close to 50%. However, PFOCs cannot be considered a complete failure because among the previously developed products as discontinuation of these products has occurred not only due to clinical side effects but also due to suspension of clinical trials due to sponsorship funding problems.

Second-generation PFOCs, such as Oxygent and Oxyfluor, are stable for 1 year under refrigerated or room temperature storage conditions, demonstrating the ease of storage in emergencies. Second-generation PFOCs also have the advantage of being able to penetrate small blood vessels and blocked arteries to deliver oxygen, making them a promising field of research. However, as mentioned earlier, these PFOCs have unmet needs, the most crucial of which is the need for small particle sizes (< 100 nm). The reason for requiring small particle sizes is that when PFOC emulsions are intravenously administered, their content is absorbed by the RES. Particles larger than 100 nm struggle to pass through the liver properly, leading to prolonged residence in the body and potential toxicity issues. Moreover, particles larger than 350 nm are retained in the lungs, which has been associated with CARPA reactions (Mayer and Ferenz 2019). Therefore, we must aim to manufacture particles as small as possible, ideally < 100 nm in size. Research into reducing particle size may start with exploring process methods or finding suitable surfactants that harmonize well with PFC. Effective PFOC development may be facilitated by developing blood substitutes while considering the unmet demands stemming from identifying the causes of failure in existing PFOC products.