(141h) Inhibition Mechanisms in Acute Respiratory Distress Syndrome: LS Inactivation

Lung surfactant (LS) is a mixture of proteins and lipids that line the alveolar air–liquid interface, decreasing work of breathing as well as stabilizing alveoli against collapse via an efficient reduction of the interfacial surface tension. Exposure of LS monolayer to subphase containing lysolipids(LL), especially near critical micelle concentrations (CMC), increases the compression ratio required to achieve near-zero surface tension and prevents LS vesicles from adsorption, leading to LS inactivation. The inactivation probably accounts for ineffectiveness of the current replacement lung surfactant (RLS) treatment on acute respiratory distress syndrome (ARDS), and thus new resistant formulations are in great need for treating ARDS. 

A systematic mechanism understanding of LS inactivation is the first step towards engineering such formulation. As one major class of potential inhibitors, LL, such as lysopalmitoylphosphatidylcholine (LPPC), produced by an inflammation-induced enzyme sPLA2, was rarely studied with LS monolayers. LPPC forms Gibbs monolayer, desorbing and adsorbing readily; moreover, LPPC can exert two opposing effects on the initial LS adsorption rate and later bilayer-monolayer conversion rate on a dose-dependent behavior with a possible synergistic effect with palmitic acid (PA). Hence, the big challenge lies in the quantification of incorporated LPPC in the monolayer and separation of the two LS adsorption steps so that LPPC-inactived step can be quantitatively specified. To solve the problem, this paper will quantify material exchange of up to three species (including LPPC and LS vesicles) between bulk and interface by a ‘deconvolution’ method with a custom-built confocal microscope. Besides, two models, the Langmuir trough and pulsating bubbles will be combined for the first time to enable separation of the diffusion step from LS adsorption by controlling bubble size. On the other hand, potential inactivation resistant molecules will be examined, with a focus on LS components that promote adsorption including anionic lipids, SP-B and SP-C; polymers that may inhibit LPPC/PA incorporation , by osmotic stress such as PEG and HA; and also cholesterol, which showed dramatic effects on LS monolayer in our work. Effects of LPPC, PA and potential resistant molecules on LS monolayer will be investigated from multiple scales: molecular packing by grazing incidence synchrotron small-angle X-ray diffraction(GISAXS), 100-1000nm scale domain morphology from confocal microscopy, and macro-scale characterization of micro-rheological and surface tension properties along with quantified material information. 

The combination of these techniques with theories of adsorption, transport and electrostatic interaction may unravel the currently unclear lipid-lysolipid/protein/cholesterol interactions and their role in LS adsorption, thus benefit the engineering of synthetic RLS formulation design.