Langmuir balance investigation of superoxide dismutase interactions with mixed-lipid monolayers

Higher than theoretical encapsulation efficiencies in liposomes of the cytoplasmic protein, superoxide dismutase (SOD), were previously observed. The high encapsulation of SOD led to the consideration of lipid-protein interactions and the embedding of SOD in the lipid bilayer. Difficulty in other methods such as dynamic scanning calorimetry due to cholesterol obscuring the measurements brought about the interest for a modified Langmuir monolayer relaxation study. A novel method was devised to distinguish between different lipid compositions that formed either a favorable or an unfavorable environment for SOD. Normalized monolayer relaxations with SOD were compared between mixed-lipid compositions containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol (Chol). Lipid-monolayer relaxation with and without SOD in the subphase was plotted over 30 min to determine if the protein was altering the lipid-monolayer relaxation. The monolayer relaxation with SOD was normalized to the monolayer relaxation without SOD over the 30 min period. The results indicated that lipid length and mole percent of cholesterol were important parameters that must be adjusted in order to support a favorable environment for SOD interaction with the lipid. It was determined that hydrophobic interactions were dominant over electrostatic forces; thus, SOD was embedding into the lipid monolayer. Additionally, this study was correlated to a previous liposome study and proved that lipid-protein interactions were the reason for the higher encapsulation efficiencies. The significance of this method is that it (1) provides a connection between lipid-protein interactions observed in monolayers and bilayers and (2) establishes a simple and effective manner to test lipid compositions for lipid-protein interaction that will aid in optimization of liposome encapsulation efficiency.