A microscopy study of model lung surfactant monolayers

The design of effective and inexpensive synthetic lung surfactant (LS) replacement formulations for the treatment of Respiratory Distress Syndrome (RDS) is currently limited by a lack of knowledge of the full roles and mechanisms of action of the individual LS lipid and protein components. In particular, the role of LS protein SP-B in the surface activity of LS, and the relationship between the amino acid sequence of the protein and its activity in LS mixtures is currently unknown. The goal of this work was to develop and use microscopy techniques to study model LS monolayers containing synthetic versions of SP-B protein in order to examine and elucidate their surface phase behavior.;We have used these techniques to discover several roles of SP-B protein in LS, and to relate the function of SP-B to its amino acid sequence. We found that SP-B protein interacts synergistically with the anionic components of LS to change their phase behavior to one more suitable for proper LS function. SP-B protein increases the collapse pressure of saturated anionic LS lipids, and shifts the collapse event to a more reversible and homogeneous process. We have conducted the first direct test of the "squeeze-out" hypothesis of LS function, and show that removal of unsaturated lipid from mixed saturated/unsaturated phosphatidyl-glycerol (PG) monolayers is prevented by the addition of SP-B protein. The protein also shifts the collapse mechanism of PG monolayers to a high pressure, reversible folding and unzipping process; this type of folding collapse event has not been observed before in mixed lipid and protein monolayers and may explain how LS monolayers can both achieve low surface tensions and respread rapidly from the collapsed state.;We have observed similar behavior (folding collapse, elimination of "squeeze-out" of unsaturated lipid by SP-B protein, etc.) in multicomponent synthetic LS monolayers based on the Tanaka formulation, which is known to be a close mimic of natural LS. These results contradict the "squeeze-out" hypothesis of LS function, and provide direct evidence that model LS monolayers can contain significant amounts of unsaturated and anionic lipid and protein components at low surface tensions. This systematic study of model LS monolayers may help in the understanding of the natural LS system and may also facilitate the design of synthetic replacement formulations for the treatment of RDS.;We have designed, constructed, and utilized several novel techniques for the study of lipid and protein monolayers. A combined fluorescence (FM), polarized fluorescence, and Brewster angle microscope/Langmuir trough assembly was designed and built for the specific purpose of studying LS monolayers. As demonstrated throughout this thesis, this system is ideal for studying the phase behavior of LS monolayers, as well as other lipid and protein monolayer systems in general. We have also developed a technique for depositing monolayers onto substrates for examination with atomic force microscopy (AFM) and infra-red spectroscopy which allows for the observation of the entire transfer process and the correlation of images obtained on the micron (FM) and molecular (AFM) scales.