Model Lung Surfactant
Respiratory distress syndrome (RDS), the fourth leading cause of infant mortality in the United States, arises from an insufficiently developed lung surfactant (LS). Healthy LS, a mixture of lipids and proteins that coats the inner surface of the lungs, reduces the alveolar surface tension to a few millinewtons per meter and, thus, facilitates breathing by stabilizing the large surface area changes associated with respiration. In RDS the absence of key proteins in the LS reduces the surfactant collapse pressure (i.e., monolayer compressibility) and the ability of the monolayer to respread during the breathing cycle, resulting in labored breathing, reduced oxygen transport, and often death in those afflicted.
Despite a long history of research on the relationship between alveolar surface properties and healthy respiration, and in particular between a breakdown in surface properties and respiratory distress syndrome, RDS remains a leading cause of infant death in the United States. Although the infant mortality rate due to RDS decreased from 8.4% in 1989 to 4.7% in 1999 with the advent of surfactant replacement therapy, further reduction may be achieved through improvements in the replacement surfactant. Currently, the most effective replacement surfactants are derived from animal extracts. Due to cost and purity concerns inherent in administering animal extracts to humans, the development of a purely synthetic replacement surfactant is a desirable goal. Deeper knowledge of the protein-lipid interactions that give rise to favorable LS properties (such as compressibility and re-spreadability) will facilitate the design of more efficacious treatments. Furthermore, such fundamental knowledge may give rise to improved treatments for other pulmonary ailments as well.
Of the four surfactant proteins currently identified, studies have revealed the particular importance of surfactant protein B (SP-B) in proper respiratory function. Recent work has shown that a synthetic 25 amino acid fragment of SP-B (SP-B1-25) mimics many of the desirable properties of the native protein. A characteristic of SP-B1-25 that may be of particular relevance to healthy respiration is that as the weight fraction of SP-B1-25 is increased the percentage of disordered phase increases.
Previous studies on dipalmitoylphosphatidylcholine (DPPC) have shown that the addition of 10wt.% SP-B1-25 induces the formation of a network of interconnected LE domains at surface pressures in excess of 55mN/m. In palmitic acid monolayers, Brewster angle microscopy studies have revealed a direct correlation between increasing SP-B1-25 concentration and the fraction of LE phase. As the LE phase is more compressible than the LC phase, the phenomenon of SP-B1-25 induced formation of LE phase may be suggestive of a key role played by SP-B in natural, healthy surfactant. However, the mechanism by which this process occurs is not well understood. This phenomenon is the focus of the present study. Here we show evidence that the addition of 11wt.% SP-B1-25 to monolayers of palmitic acid induces large density fluctuations in the membrane on macroscopic length scales, a signature of critical behavior. The implications of this observation may be relevant to the design of novel treatments for RDS.
Below shows 80 micron by 80 micron confocal microscopy images of PA/*SP-B1-25 monolayers between 1.50wt.% and 50.00wt.% *SP-B1-25 following excitation at 514nm. The bright domains denote monolayer regions where fluorescence is observed. Monolayers containing between 1.50wt.% and 10.50wt.% *SP-B1-25 exhibit elliptical LE domains that exist in an expanse of dark LC phase. The LE domain areas increase with increasing *SP-B1-25 concentration, but in a qualitative sense the domain morphology remains constant. Dramatic evolution in the phase structure is evident in above 11 wt%. The semi-circular LC protrusions (10mm-30mm in dimension) along the LC/LE borders and the fully separated, round LC domains suggest that the sheet-like LC phase is transitioning into circular LC domains. Monolayers above 15 wt% are characterized by circular LC domains in an expanse of bright LE phase, a qualitative reversal in the LE-LC phase coexistence from the low wt% films.
The average percent of LC areas (gray symbols) are plotted against peptide concentration below. The gray error bars denote the standard deviation of the averaged LC areas. The LC area data exhibit a negative, bilinear dependence on *SP-B1-25 weight percent. In the region near the junction of the two fitted linear functions, the standard deviation values are anomalously large. The black symbols in the figure below illustrate the molar area dependence of the PA monolayers on *SP-B1-25 weight percent. Below 11.3wt.% *SP-B1-25, the molar area values increase monotonically with increasing *SP-B1-25 concentration. The intersection of the two linear functions at 11.3wt.% *SP-B1-25 suggests that the molar area varies continuously with *SP-B1-25 concentration, and that the first derivative of the order parameter, exhibits a discontinuity at 11.3wt.% *SP-B1-25. This discontinuity suggests that a second order phase transition occurs near a critical *SP-B1-25 concentration of 11.3wt.%.
The standard deviations of the molar area values are plotted against *SP-B1-25 concentration in the figure above. In the classical view of critical phenomena, the potential barrier separating two stable phase states gradually disappears as a critical point is approached. Thermal fluctuations, sufficient in the neighborhood of the critical point to spontaneously drive the system between the two phases, are manifest as divergent fluctuations in the order parameter. Systems undergoing first order phase transitions do not exhibit divergent order parameter fluctuations. The standard deviations of the averaged molar areas provide a quantitative measure of the fluctuations in the molar area. These fluctuations become anomalously large near 11wt.% *SP-B1-25.
It is interesting to consider the biological significance of critical behavior and its potential role in respiration. While some evidence suggests that healthy breathing and critical phenomena are related 19,5, understanding the role in the natural mechanism by which healthy LS maintains stability requires further study. Regardless, the development of effective, purely synthetic therapies for RDS is a desirable goal given the cost and uncertainty associated with using animal extracts. Designing synthetic surfactants that employ critical behavior as a means of stabilizing large surface area changes associated with breathing may be a useful concept in the effort to achieve this goal.
Flanders B. N. and Dunn R. C. "A near-field microscopy study of submicron domain structure in a model lung surfactant monolayer" Ultramicroscopy. 91, 245-51 (2002).
Flanders, B. N., Vickery, S. A. and Dunn, R. C., "Imaging of monolayers composed of palmitic acid and lung surfactant protein B" J Microsc. 202, 379-85 (2001).
Vickery, S. A. and Dunn, R. C., “Direct Observation of Structural Evolution in Palmitic Acid Monolayers Following Langmuir-Blodgett Deposition”, Langmuir, 17, 8204-8209 (2001).
Flanders, B. N., Vickery, S. A. and Dunn, R. C., “Imaging of Monolayers Composed of Palmitic Acid and Lung Surfactant Protein B”, J. Microscopy, 202, 379-385 (2001).