Effect of cholesterol on electrostatics in lipid-protein films of a pulmonary surfactant.

We report the changes in the electrical properties of the lipid-protein film of pulmonary surfactant produced by excess cholesterol. Pulmonary surfactant (PS) is a complex lipid-protein mixture that forms a molecular film at the interface of the lung's epithelia. The defined molecular arrangement of the lipids and proteins of the surfactant film gives rise to the locally highly variable electrical surface potential of the interface, which becomes considerably altered in the presence of cholesterol. With frequency modulation Kelvin probe force microscopy (FM-KPFM) and force measurements, complemented by theoretical analysis, we showed that excess cholesterol significantly changes the electric field around a PS film because of the presence of nanometer-sized electrostatic domains and affects the electrostatic interaction of an AFM probe with a PS film. These changes in the local electrical field would greatly alter the interaction of the surfactant film with charged species and would immediately impact the manner in which inhaled (often charged) airborne nanoparticles and fibers might interact with the lung interface.

Kelvin probe force microscopy in application to biomolecular films: frequency modulation, amplitude modulation, and lift mode.

Kelvin probe force microscopy (KPFM) is a powerful technique to visualize the differences of work function in metals and lateral surface potential distribution in thin organic films. Earlier we have shown that frequency modulated-Kelvin probe force microscopy has significant advantages in both sensitivity and resolution when applied to metal and inorganic interfaces in vacuum. High resolution, high sensitivity, and performance in ambient conditions are required in order to study biologically relevant samples. In this work we compared the resolution of frequency modulation (FM-KPFM), amplitude modulation (AM-KPFM), and lift modes KPFM for imaging the local electrical surface potential of complex biomolecular films and demonstrated that FM-KPFM mode has superior resolution for biological applications. The power of the method was illustrated on pulmonary surfactant films, revealing nm spatial resolution and mV potential sensitivity in ambient air. At this level of resolution this method can provide critical insight into the molecular arrangement and function of complex biosystems in a way that other KPFM modes cannot do. Based on the observed changes in the local surface potential we discovered that excess cholesterol produces nm size electrostatic domains and change the electric fields.

Effect of SP-C on surface potential distribution in pulmonary surfactant: Atomic force microscopy and Kelvin probe force microscopy study.

The air-lung interface is covered by a molecular film of pulmonary surfactant (PS). The major function of the film is to reduce the surface tension of the lung's air-liquid interface, providing stability to the alveolar structure and reducing the work of breathing. Earlier we have shown that function of bovine lipid extract surfactant (BLES) is related to the specific molecular architecture of surfactant films. Defined molecular arrangement of the lipids and proteins of the surfactant film also give rise to a local highly variable electrical surface potential of the interface. In this work we investigated a simple model of artificial lung surfactant consisting of DPPC, eggPG, and surfactant protein C (SP-C). Effects of surface compression and the presence of SP-C on the monolayer structure and surface potential distribution were investigated using atomic force microscopy (AFM) and Kelvin probe force microscopy (KPFM). We show that topography and locally variable surface potential of DPPC-eggPG lipid mixture are similar to those of pulmonary surfactant BLES in the presence of SP-C and differ in surface potential when SP-C is absent.