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Physical Properties and Stability Mechanisms of Poly(Ethylene Glycol) Conjugated Liposome Encapsulated Hemoglobin Dispersions

Liposomes encapsulating hemoglobin (LEHs) surface-conjugated with 2000 and 550 Da poly(ethylene glycol) (PEG) were produced via extrusion through 400, 200 and 100 nm pore diameter membranes in two types of phosphate buffer with different ionic strengths. The lipid bilayers were composed of dimyristoyl-phosphatidylcholine (DMPC), cholesterol, dimyristoyl-phosphoethanolamine-PEG (DMPE-PEG), dimyristoyl-phosphatidylglycerol (DMPG), and α-tocopherol (in a 43:40:10:5:2 mole ratio). N-acetyl-L-cysteine was coencapsulated in order to suppress hemoglobin (Hb) oxidation. Various physical properties of PEG-LEH dispersions were determined: size distribution, encapsulation efficiency, P50 (partial pressure of O2 where half of the oxygen binding sites are saturated with O2), cooperativity coefficient, and encapsulated methemoglobin (MetHb) level. In order to study the stabilization mechanism of these dispersions, the effective bending constant (KB) and the spontaneous radius of curvature (R0) of PEG-LEHs were extracted by fitting a mathematical model describing the size distribution of a liposome dispersion to the experimentally measured size distributions [1]. We observed that liposome dispersions extruded in phosphate buffer (PB) were more monodisperse than liposomes extruded in phosphate buffered saline (PBS), and higher molecular weight PEG promoted the formation of narrower size distributions. Moreover, extrusion in PB and lipid conjugation with higher molecular weight PEG imparted higher bilayer rigidity (high KB), and stabilized the liposome dispersions by the spontaneous curvature mechanism, whereas the other liposome dispersions were stabilized by thermal undulations (low KB). The P50 and cooperativity coefficient of PEG-LEHs extruded in PBS and PB was comparable to that of human blood, and the encapsulated MetHb levels were less than 5%. The highest encapsulation efficiencies obtained were 27%–36% (82–109 mg Hb/mL) for LEH dispersions extruded in PBS and grafted with 2000 Da PEG. These dispersions yielded KBs’ ranging from 7 kT to 27 kT, which indicated that these dispersions were stabilized by spontaneous curvature. Whereas the same lipid combination extruded in PBS, however, instead conjugated with 550 Da PEG resulted in KBs’ ranging from 2 kT to 2.7 kT, which indicated that these dispersions were stabilized by thermal undulations. Thermal undulations permitted Hb leakage through the lipid bilayers, which in turn lowered the encapsulation efficiency to 1%–10.7% (3–32 mg Hb/mL). Taken together, the experimentally measured size distributions and encapsulation efficiencies of PEG-LEH dispersions can be readily explained through analysis of the magnitude of KB, which dictates the stability mechanism of the liposome dispersion.