Multilayers at the surface of solutions of exogenous lung surfactant: Direct observation by neutron reflection

Pharmacy-grade exogenous lung surfactant preparations of bovine and porcine origin, dispersed in physiological electrolyte solution have been studied. The organization and dynamics at the air/water interface at physiological temperature was analysed by neutron reflection. The results show that a well-defined surface phase is formed, consisting of a multilayer structure of lipid/protein bilayers alternating with aqueous layers, with a repetition period of about 70 Å and correlation depths of 3 to > 25 bilayers, depending on electrolyte composition and time. The experimental surfactant concentration of 0.15% (w/w) is far below that used in therapeutic application of exogenous surfactants and it is therefore likely that similar multilayer structures are also formed at the alveolar surface in the clinical situation during surfactant substitution therapy. Lung surfactant preparations in dry form swell in aqueous solution towards a limit of about 60% (w/w) of water, forming a lamellar liquid-crystalline phase above about 34 °C, which disperses into lamellar bodies at higher water concentrations. The lamellar spacings in the surface multilayers at the air/water interface are smaller than those in the saturated limit even though they are in contact with much greater water concentrations. The surface multilayers are laterally disordered in a way that is consistent with fragments of Lα-phase lamellae. The near surface layers of the multilayer structure have a significant protein content (only SP-B and SP-C are present in the preparations). The results demonstrate that a multilayer structure can be formed in exogenous surfactant even at very low concentrations and indicate that multilayers need to be incorporated into present interpretations of in vitro studies of similar lung surfactant preparations, which are largely based on monolayer models.