Simple method for preparing poly(ethylene glycol)-surface-conjugated liposome-encapsulated hemoglobins: physicochemical properties, long-term storage stability, and their reactions with O2, CO, and NO.

During the last few decades, liposome-encapsulated hemoglobin (LEH) dispersions have been investigated for use as red blood cell (RBC) substitutes. However, the process for formulating LEHs is cumbersome, and the composition of the lipid mixture is often complex. This work investigates a simple approach to formulating LEHs from a simple lipid mixture composed of high-phase-transition lipid distearoylphosphatidylcholine (DSPC) and cholesterol. To improve the circulation half-life and colloidal state of LEHs, the surfaces of unmodified LEHs were conjugated with poly(ethylene glycol) (PEG-LEHs). The results of this work show that PEG-LEH dispersions exhibited average diameters ranging from 166 to 195 nm that were colloidally stable for 4 to 5 months, hemoglobin (Hb) concentrations ranging from 9.6 to 14 g/dL, methemoglobin levels of less than 1%, oxygen affinities (i.e., P(50) values) ranging from 20 to 23 mm Hg, and cooperativity coefficients ranging from 1.4 to 2.2. The reactions of PEG-LEHs with physiologically important ligands, such as oxygen (O(2)), carbon monoxide (CO), and nitric oxide (NO), were also measured. It was observed that PEG-LEHs and RBCs exhibited retarded gaseous ligand binding/release kinetics compared to that of acellular Hb's. This result provides important insight into the pivotal role that the intracellular diffusion barrier plays in the transport of gases into and out of these structures. Collectively, our results demonstrate that the PEG-LEH dispersions prepared in this study show good potential as an RBC substitute.