(124h) Effect of Cholesterol Nanodomains On Monolayer Morphology and Dynamics

Zasadzinski, J. A., University of Minnesota
Squires, T. M., University of California, Santa Barbara
Choi, S. Q., University of Minnesota
Kim, K., Univerisity of California, Santa Barbara

Cholesterol segregates into 10 – 100 nm diameter nanodomains in dipalmitoylphosphatidylcholine (DPPC) monolayers and reduces the surface viscosity of the monolayer by an order of magnitude for each wt% cholesterol in the film. The nanodomains consist of 6:1 lipid:cholesterol “complexes” that segregate to and lengthen lipid domain boundaries, consistent with a reduced line tension λ. The surface viscosity of the monolayer, ηs, decreases exponentially with the area fraction of the complex at fixed surface pressure, similar to simple polymer blends.  However, ηs increases exponentially with surface pressure at fixed cholesterol content, as predicted by a free area model that relates ηs to monolayer compressibility and collapse pressure. The elastic modulus, G’, initially decreases with cholesterol fraction, consistent with the decrease in λ expected from the line-active in analogy to 3-D emulsions.  However, increasing cholesterol further causes a sharp increase in G’ between 4 and 5 mol% cholesterol due to an evolution in the domain morphology, so that the monolayer is elastic rather than viscous. Understanding the effects of small mole fractions of cholesterol should help resolve the role of cholesterol in human lung surfactants and may give clues as to how cholesterol influences raft formation in cell membranes.

   We find using a combination of fluorescence optical microscopy, atomic force microscopy, Langmuir isotherms and a novel micron scale monolayer rheometer that minute fractions of cholesterol lead to dramatic changes in lipid monolayer morphology and have equally dramatic effects on monolayer dynamic properties. One wt% cholesterol reduces the surface viscosity of DPPC monolayers by an order of magnitude, two wt% reduces surface viscosity by two orders of magnitude.  We can relate these changes in surface viscous and elastic properties to characteristic features of the isotherms and monolayer morphology, and hence to the molecular organization of the films.  Our findings suggest a possible role for cholesterol in lung surfactant (LS), a lipid-protein monolayer necessary to reduce the surface tension in the lung alveoli. At present, even the existence of cholesterol in native LS is questioned, as the lung lavage required to harvest LS inevitably causes blood and cell debris to be co-extracted, potentially contaminating LS with cholesterol. This lack of consensus over the role of cholesterol is reflected in the composition of replacement lung surfactants for neonatal respiratory distress syndrome (NRDS), which occurs in 20,000-30,000 premature births each year. Survanta and Curosurf, two clinically approved animal extract replacement surfactants for treatment of NRDS, have all cholesterol removed after harvesting. Infasurf, the third clinically approved surfactant, retains 4-5 wt% cholesterol. Resolving this controversy is difficult, as there is little information on the effects of small mole fractions of cholesterol on the organization and dynamics of phospholipid monolayers at the molecular, monolayer or cell membrane scale (mitochondrial membranes have ~ 5 mol% cholesterol, endoplasmic reticulum ~ 10 mol%).  Cholesterol is also implicated in the formation of stable, 10 – 100 nm “rafts” within the plasma membrane of cells, which may serve as platforms for organizing proteins responsible for cell signaling or membrane trafficking. The local viscous, elastic and line tension properties of the rafts relative to their surrounding membrane likely have important implications for their formation, stability and function.

   AFM images show that cholesterol segregates into line-active nanodomains of a 6:1 DPPC:cholesterol complex that lowers surface viscosity by orders of magnitude, by introducing sources of free area for the film.  Rheology and isotherm measurements for films with ≥ 5 mol% cholesterol suggest a fundamentally different molecular organization that at lower cholesterol fractions; a reasonable hypothesis is that cholesterol  begins to directly intercalates into the DPPC lattice at higher mole fractions, while being excluded at lower mole fractions. The small fractions of cholesterol significantly change the spreading and other dynamic properties of replacement lung surfactants, which consist of 50 – 70 mol% DPPC.  Upon exhalation, i.e. at small alveolar area and high surface pressure, the exponentially increasing surface viscosity should minimize the Marangoni flow that would otherwise occur due to surface tension gradients between the alveoli and the bronchi.  At the ~1s time scales common for respiration, the elastic resistance to flow that occurs with increasing cholesterol should also oppose any Marangoni flow.  Upon inhalation, and large alveolar area, lower surface pressures are associated with exponentially lowered viscosity, as would be needed to quickly cover an expanding alveolar interface.  DPPC films with >5 mol% cholesterol have a significant elastic component over the ~1s time scales common to respiration.  If a film is primarily viscous over this time scale, microstructural elements in the film have time to relax and re-arrange.  Films that are primarily elastic over this time scale, on the other hand, store deformational energy and work to “undo” the deformations imposed previously. On exhalation, the surfactant films may store deformation energy elastically,  making it easier for the alveoli to re-expand on inhalation. This complex relationship between surface viscosity, surface pressure, and cholesterol content may make for an optimal rheological response to minimize the energy of breathing.  At high surface pressures, a high surface viscosity coupled with an elastic response may slow the kinetics of three-dimensional buckling that occurs on monolayer collapse.  Understanding the effects of cholesterol will enable these ideas to be tested by providing a simple way to alter monolayer mechanical properties by orders of magnitude with only subtle changes in lung surfactant composition and pressure-area isotherms.