Lung surfactant (LS) is a lipid-protein mixture that lines the alveolar air-liquid interface and acts to reduce the work required for breathing and helps maintain proper lung inflation. The lack or inhibition of LS can induce life-threatening respiratory conditions. Currently, clinical therapy strategies are in the form of replacement LS (RLS) obtained from animals with minor additives. The issue with animal derived RLS is that they have large variations in composition from batch to batch, a high cost of materials isolation and purification, and the possibility of viral or prion contamination. This motivates the development of a synthetic RLS. However, in order to define an ideal LS composition, it is necessary to understand the role that each component plays, as well as defining an optimal physiological value for the surface shear and dilatational viscosity. Due to the complexity of native RLS, 2 or 3 component mixtures are studied to elucidate the effects of trace components. In the present work, a tertiary mixture of DPPC, 1-hexadecanol (HD), and dihydrocholesterol (chol) is used as a model system to isolate the role of trace amounts of cholesterol on monolayer physical properties. Cholesterol is found in the alveolar fluids of patients with respiratory disorders, and some believe that cholesterol at elevated levels prevent LS from reaching near-zero surface tension by altering the lipid phase behavior and fluidity. The clinical lung surfactants Survanta and Curosurf have all cholesterol removed. However, small amounts of cholesterol are found in LS extracted from animals, suggesting that cholesterol might be an integral part of LS. Infasurf, also clinically used, retains small fractions of cholesterol. As such, there is a need to determine the role and function of cholesterol in LS systems.
It is found that the addition of trace amounts of chol does not significantly change the phase behavior nor prevent the monolayer from reaching near-zero surface tension values. It is found using confocal microscopy that the monolayer phase separates into coexisting solid-like domains composed of DPPC and HD, within a continuous fluid-like matrix composed of DPPC and chol. The addition of chol acts to significantly alter the size and shape of the solid domains and it is believed that chol modifies the line tension between the solid and liquid phases. Further, it is found that increasing the chol fraction reduces the solid domain area fraction, which correlates to orders of magnitude decrease in the shear surface viscosity as measured via an oscillating micro-button. Plausible physiological implications of the findings in this work are discussed along with commentary regarding recommendations for the use of cholesterol in future synthetic RLS formulations.
