Comparative structural and rheological analysis of model and clinical surfactants: role of protein-enriched multilayers and bulk supply

Pulmonary surfactant is essential for reducing the surface stress at the alveolar liquid-air interface, but the mechanisms leading to this reduction are still not fully understood. The occurrence of protein modulated multilayers below the interface has been reported, and they have been suggested to play a role in lung surfactant dynamics. However, evidence on their formation and their precise role in lowering the surface stress remains limited. In the present study, we combine neutron reflectometry using a Quadrotrough interfacial dilatational rheometer, with cryo-TEM imaging to investigate the conditions required for multilayer formation and their subsequent influence on interfacial properties. By comparing three model lipid systems with varying concentrations of hydrophobic surfactant proteins SP-B and SP-C, we demonstrate that a higher protein content is necessary for the formation of multilayers and the efficient re-spreading of lipids upon expansion. When comparing the model systems with the natural surfactant replacement Infasurf, we found that multilayers alone are insufficient to reduce the surface stress effectively. These results confirm that both the multilayers and the continuous bulk lipid-protein supply play a critical role in efficiently achieving a low surface stress through structural modifications.