History of Blood Substitutes
People have been interested in blood transfusions for hundreds of years. The Incas are thought to have performed the first blood transfusions. In 1616, William Harvey described how blood was circulated in the body. This discovery sparked many to try using different fluids to substitute for blood such as: beer, urine, milk, plant resins and animal bloods. In 1667, the first successful transfusion was recorded, but interest waned, for many blood transfusion recipients died. No progress occurred until the early 1900’s when Karl Landsteiner, an Austrian doctor, discovered the 4 blood types we use today. Doctors could be certain of blood matches, and this made blood transfusions not only safe, but also a routine procedure. During WWII, concern grew over a shortage of blood for wounded soldiers who could not be treated at hospitals. Research began for a blood substitute that would reduce this shortage after the Vietnam War. Today, the two most promising red cell substitutes are perfluorocarbon-based oxygen carriers (PFBOCs) and hemoglobin-based oxygen carriers (HBOCs).
In 1966, scientists synthesized an “oxygen therapeutic” that survived in mice. PFBOCs achieve oxygen delivery by using organic chemicals with high gas solubility. The perfluorinated carbons are chemically and biologically inert but are able to dissolve a large amount of gas. One of the problems with perfluorocarbons is that they are an oil-like fluid that does not mix well with water and cannot carry water-soluble salts and metabolic substrates. In order to be used as a red cell substitute, perfluorocarbons are mixed with fluids such as albumin or in physiologic electrolyte solutions. Today, most PFBOCs are mixtures of perfluorocarbons with emulsifying agents. Emulsifying agents are substances that help stabilize two seemingly unblendable things. PFBOCs utilize Puronic-68, egg yolk phospholipids and triglycerides as emulsifying agents.
Green Cross Corp. in Osaka Japan developed the first PFBOC with a mixture of perfluorodecalin and perfluorotripropylamine and using egg yolk phospholipids and Pluronic-68 as emulsifying agents. Their product was called Fluosol-DA. The problem with Fluosal-DA was that they dissolve less oxygen than pure liquids. It could only deliver 0.4 mL oxygen per 100 mL. In order to meet metabolic oxygen demand, the patients would have had to breathe in gas that was 100% oxygen which would lead to adverse effects due to oxygen toxicity.
More recently, Alliance Corp developed a mixture of perfluorooctyl bromide and egg yolk phospholipids as the emulsifying agent. Oxygent, their product, could deliver up to 1.3 mL oxygen per 100 mL, but this is still much lower than normal blood which could deliver blood at 5 mL oxygen per 100 mL. The advantage of Oxygent over Fluosal-DA is that it has a longer circulation time, and it can be excreted from the body in 4 days compared to Fluosal –DA which could take months.
HBOCs are oxygen carriers that use purified human, animal or recombinant hemoglobin. The first major clinical study with purified hemoglobin resulted in nephrotoxicites, poisonous effects on the kidney. The hemoglobin used was found to have ethrocyte membrane stromal lipids as well as bacterial endotoxins. To side-step these problems, stroma-free hemoglobin were developed that also was free of endotoxins, and it did reduce nephrotoxicity but new problems arose. The stroma-free hemoglobin was found to have too high of an affinity for oxygen which affected oxygen being delivered to tissue. Today, many new processes have been developed that fix these problems.
Stabilized Hemoglobin (Tetrameric Hemoglobin)
One way to fix the problem that stroma-free hemoglobin has a high affinity towards oxygen is to use functional DPG analogs such as pyridoxal-5’-phospoate that attach to the DPG pocket. DPG stands for 2,3-diphosphoglycerate which is a substance made in red blood cells that helps control the movement of oxygen to body tissue. The more DPG in the cell the more oxygen delivered to the tissue. The less DPG; the less oxygen delivered to tissues.
Pridoxylated stroma-free hemoglobin nearly has normal oxygen affinity (p50 = 22-24 mmHg), but is dissociable into αß-dimers that are excreted. Hemoglobin can also be stabilized with an α-specific crosslinker bis-fumarate that produces cross-linked hemoglobin. The cross-linked hemoglobin demonstrated an oxygen affinity of p50 = 30mmHg and had a longer circulation time.
To increase the circulation time of HBOCs, stabilized hemoglobin can undergo intermolecular cross-linking with bi- or poly-functional groups. This process is also known as polymerization. Pyridoxal-5’-phosphate, a DPG analog, can be polymerized with glutaraldehyde to increase intravascular circulation half-time of over 30 hours in adult baboons.
A process to increase circulation time that uses ring-opened raffinose (o-raffinose) as a cross-linker has also eliminated the stabilization process that is need prior to cross-linking. The o-raffinose HBOC delivers 4.3 mL of oxygen per deciliter which is close to the normal oxygen delivery capacity.
Due to the limited availability of human stroma-free hemoglobin, low oxygen affinity bovine hemoglobin has been utilized as a starting material. Bovine hemoglobin has a natural low affinity to oxygen and is not DPG dependent. The major deterrent to using bovine hemoglobin is the potential of transmission of animal-borne diseases such as bovine spongiform encephalopathy (BSE).
To further increase the circulation time, hemoglobin can be linked to a macromolecule to increase its size. Human or bovine hemoglobin that is conjugated with polyethylene glycol is protected from renal excretion. The polyethylene glycol hemoglobin has a larger molecular size and has a higher viscosity.
Recently a product called Hemospan has been developed by introducing additional surface thiols with iminothiolane onto the hemoglobin. This process usually adds about 5 additional thiols, and it is then linked to polyethylene glycol-5000. Hemospan then requires no more purification steps. One would think Hemospan would not work, for it has a lower hemoglobin concentration, higher viscosity, higher oxygen affinity and higher colloidal oncotic pressure than most other HBOCs in development. Hemospan did demonstrate an improvement in microcirculatory blood flow and tissue oxygenation in animal studies, but it is nor known if it improves perfusion in the microcirculation of the critical organs. The increased viscosity could also increase the cardiac workload of the patient.
Hemoglobin Vesicles (Hemoglobin encapsulated, embedded and coated vesicles)
Hemoglobin and red cell enzymes encapsulated in nanometer size biodegradable polymer vesicles have been developed. The advantage of the encapsulated hemoglobin over lipid vesicles is that these polymer vesicles could be permeable to glucose and other molecules that are needed to reduce methemoglobin. Methemoglobin is a particular type of hemoglobin that is useless for carrying oxygen and delivering it tissues. The nanocapsules could maintain hemoglobin concentration at 15 g/dL and normal p50 (oxygen affinity).
The earliest types of encapsulated hemoglobin had a short circulation half-life and formed methemoglobin. The circulation half-life was improved by surface changes using negative surface charges, sialic acid analogs or polyethylene-glycol. These modifications improved the half-life to over 24 hours. Methemoglobin formation was reduced by reducing enzymes such as methemoglobin reductase system. In animal studies, results were mostly successful, but complement activation occurred in rats and pigs.
Hemoglobin aquosomes were developed by coating hemoglobin molecules on the sugar coated hydroxyapatite nanoparticles. In rats, no undesirable changes were reported.
With improvements in technology, native or modified hemoglobin can be produced from microorganisms such as E. coli or yeast, transgenic plants or animals.
Recombinant human hemoglobin was produced in E. coli and S. cerevisiae using an expression vector containing two mutant human globin genes. One was a low oxygen affinity mutant, and the other fused α-globins. These recombinant hemoglobin products advanced to clinical trials, but it was stopped due to vasoconstriction and other harmful effects.
Transgenic mice and pigs have been used to produce human hemoglobin. Human α and β globin gene constructs are injected into fertilized eggs. The embryo is then developed in a surrogate mother. The problem is that these red blood cells contain the animal’s own hemoglobin as well as human hemoglobin.
This picture illustrates the different processes HBOCs go through before use.