Lipid and Protein Effects on Monolayer Stability

Acute respiratory distress syndrome (ARDS) is characterized by increased vascular permeability, decreased lung compliance, massive atelectasis and loss of aerated tissue, which often leads to respiratory failure and 40% mortality. Effective treatments for ARDS have become even more necessary as ARDS in non-survivors of Covid- 19 may be as high as 90%. LS plays two key roles in healthy respiration: (1) it establishes a minimum surface tension 𝛾!"#~15 mN/m, and (2) it prevents the lung from collapsing due to the Laplace Instability, by ensuring 2𝐸∗(𝜔) > 𝛾, where 𝐸∗(𝜔) = 𝐴(𝜔)(𝜕𝛾⁄𝜕𝐴) is the dilatational modulus, which describes the change in 𝛾 with interfacial area, 𝐴 at a breathing frequency 𝜔. Lung lavage of ARDS patients shows decreased concentrations of lung surfactant and increased concentrations of phospholipase A2 (PLA2), fatty acids and lysolipids (LPC). PLA2 catalyzes the hydrolysis of double-chain phospholipids in pathogen membranes into insoluble fatty acid and soluble single-chain LPC that accumulates in the alveolar fluids. Notably, LPC forms micelles at exceedingly low micromolar (𝜇𝑀) critical micelle concentrations (CMC). By incorporating otherwise insoluble LS molecules, LPC micelles can effectively “scrub” lung surfactant (LS) from the alveolar interface. By replacing LS at the interface with LPC, 𝛾!"# increases from ~ 15 to ~ 40 mN/m; moreover, the dilatational modulus decreases so that 2𝐸∗(𝜔) < 𝛾, triggering the Laplace instability. Injured or inflamed areas with LPC concentration above CMC are thus susceptible to lung instability, decreased compliance and alveolar flooding as observed in ARDS. Moreover, replacement surfactants would suffer the same fate so long as LPC concentrations are above the CMC. In Aim 1, we will characterize how lysolipid micelles solubilize LS using dynamic light scattering as well as by adsorption to alveolar sized bubbles in the capillary pressure microtensiometer (CPM) built for this project. We will examine shorter and longer chain lysolipids with choline, ethanolamine or anionic glycerol headgroups to correlate lysolipid CMC with 𝛾 and 𝐸∗(𝜔). This hypothesis suggests that decreasing the LPC concentration in the alveolar fluids below the CMC may be key to successful ARDS therapeutics. Adsorption of LPC into protein- free liposomes that do not adsorb to the air-water interface can reduce the LPC below the CMC. Co-administered synthetic lung surfactants containing the LS protein SP-B or peptide mimics of SP-B we developed, adsorb rapidly to the air-water interface to replace LPC at the interface. In Aim 2, we will examine the coupling between monolayer domain morphology and interfacial curvature. Monolayer collapse determines the minimum surface tension and is fundamentally different on curved surfaces compared to the flat surface of the Langmuir trough. We will use new experimental and theoretical techniques to measure line tension, dipole density difference, and the Young’s modulus of LS monolayers to quantify how monolayers bend or fold at collapse and describe the effects of interfacial curvature. These first of their kind experiments and theory should determine an optimal lung surfactant composition resistant to LPC adsorption and provide a new way of stabilizing the ARDS lung.