Cross Linking Encapsulated Hemoglobin with Solid Phase Supports: Lipid Enveloped Hemoglobin Adsorbed to Surface Modified Ceramic Particles Exhibit Physiological Oxygen Lability

Abstract

To reduce hemoglobin toxicity, a cross linking agent is generally used. To preserve hemoglobin function, an allosteric modifier is generally used. Historically, the use of one has precluded the use of the other. A potential solution to this problem was investigated.

Hemoglobin A₀ was adsorbed irreversibly to carbohydrate coated nanosized diamond particles and then encapsulated in a standard mixture of phospholipids. Endotoxin free preparations with concentrations of bound hemoglobin near 10 g/dl were achieved with as little as 0.1 g/dl of free hemoglobin and remained stable over a 48 hour period. By transmission electron microscopy these particles appeared roughly spherical and measured approximately 75 nanometers well below that of alveolar capillary vessels. In shallow pH gradients, liquid electrophoresis demonstrated that such constructs exhibit Bohr effect behavior by the induction of a dramatic surface charge inversion at around pH 6.8. To evaluate oxygen lability, oxygen saturation trials were conducted in isosmolar physiological salts. Normal sigmoidal binding behavior of O₂ over a typical pO2 gradient could be modulated by systemic levels of pyridoxyl-5-phosphate. Constructs with a P₅₀ as low as 12 mm Hg could be increased to 37 mm Hg with the allosteric effector. Viscosity and bio distribution studies are to follow.

The use of solid phase nanocrystalline supports to cross link hemoglobin may reduce the toxicity of free hemoglobin while still enabling the use of allosteric modifiers.