Characterization of the in situ structural and interfacial properties of the cationic hydrophobic heteropolypeptide, KL₄, in lung surfactant bilayer and monolayer models at the air - Water interface: Implications for pulmonary surfactant delivery

This study examines the various equilibrium in situ secondary structures of the pharmaceutical heteropolypeptide, KL₄, in the solid state, in solution, and in the monolayer state alone and mixed with dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylglycerol (POPG). In situ surface circular dichroism spectroscopy, using a method first reported by Damodaran (Damodaran, S. Anal. Bioanal. Chem. 2003, 376, 182-188), of equilibrated KL₄, DPPC/KL₄, POPG/KL₄, and DPPC/POPG/KL₄ monolayers at the air - water interface was used to examine the in situ two-dimensional conformation of KL₄. Gravimetric vapor sorption by solid KL₄ was used to analyze the effects of water molecules on the conformation of KL₄ when confined as a monolayer at the surface of water. Solid-state KL₄ conformation was determined by X-ray powder diffraction (XRPD). The equilibrium interfacial and spreading properties were measured at 25 °C, 37 °C, and 45 °C using the Wilhelmy plate method and Langmuir film balance. Equilibrium phase transition temperatures were measured using differential scanning calorimetry (DSC). It was found that solid-state KL₄, which takes up very little water, exhibits β-sheet and α-helix secondary structures, whereas KL₄ in solution appears to exist only as an α-helix. KL₄ forms a stable, insoluble monolayer, exhibiting β-sheet and aperiodic structures. These structures provide KL₄, when confined in two-dimensions, the structural flexibility to maximize favorable cationic lysine-water interactions and favorable leucine - leucine hydrophobic and van der Waals interactions; while effectively "shielding" the leucine residues away from water. In DPPC/KL₄ monolayers, KL₄ retains its native β-sheet and aperiodic structures, consistent with phase separation of DPPC and KL₄, in bilayers and monolayers. In POPG/KL₄ monolayers, KL₄ exhibits an increase in aperiodic secondary structures (loss of β-sheet) to maximize favorable electrostatic interactions, consistent with the observed negative deviations from ideal monolayer mixing.