The formation of a crosslinked carboxyhemoglobin membrane at an organic-aqueous interface

A 30% carboxyhemoglobin solution was encapsulated with a membrane composed of crosslinked hemoglobin as a potential artificial red blood cell by adding a 25 microL droplet of 30% carboxyhemoglobin solution in phosphate buffer to a buffer-saturated toluene solution of a homobifunctional crosslinking agent. Disuccinimidyl glutarate, disuccinimidyl suberate, and terephthaldicarboxaldehyde proved successful in crosslinking hemoglobin at concentrations of 0.05%, 0.08%, and 4.3%, respectively (saturated levels in buffer-saturated toluene). Disuccinimidyl glutarate exhibited maximal crosslinking of 11.0% in approximately 30 minutes. Disuccinimidyl suberate and terephthaldicarboxaldehyde required at least 12 hours to form a viable sphere and crosslinked 39.4% and 37.8% of the hemoglobin in 18 hours. Membrane integrity was assessed by determining the permeability of uncrosslinked hemoglobin through the membrane as a function of time.