Differential effects of cholesterol and budesonide on biophysical properties of clinical surfactant.

INTRODUCTION: Corticosteroids have been widely used in clinical medicine as a first-line therapy to modify the inflammatory response in many pulmonary and systemic diseases. Inhaled and intratracheally administered corticosteroids have a particular interest in that their use allows the clinician to circumvent systemic steroid side effects. However, it is vital that corticosteroids delivered via the lungs not interfere with surface activity of the pulmonary surfactant lining layer. RESULTS: We found differential effects of cholesterol and budesonide on the biophysical properties of a cholesterol-free clinical surfactant preparation, Curosurf. At a low concentration up to 1%, both steroids play a similar role of fluidizing the surfactant film. However, when steroid concentration is increased to 10%, cholesterol induces a unique phase transition that abolishes the surface activity of the Curosurf film. By contrast, 10% budesonide simply fluidizes the film, thus having only limited effects on surface activity. DISCUSSION: Together with those of a previous study using a cholesterol-containing surfactant, our findings suggest that cholesterol-free surfactant preparations may be more advantageous than cholesterol-containing preparations as a carrier of budesonide because a larger amount of the drug may be delivered to the lungs without significantly compromising the surface activity of pulmonary surfactant. METHODS: Langmuir balance was used to study the effect of cholesterol and budesonide added at different concentrations on surface activity of Curosurf. Atomic force microscopy (AFM) was used to reveal their effects on the interfacial molecular organization and lateral structure of Curosurf films.

Self-assembled amyloid-like oligomeric-cohesin Scaffoldin for augmented protein display on the saccharomyces cerevisiae cell surface.

In this study, a molecular self-assembly strategy to develop a novel protein scaffold for amplifying the extent and variety of proteins displayed on the surface of Saccharomyces cerevisiae is presented. The cellulosomal scaffolding protein cohesin and its upstream hydrophilic domain (HD) were genetically fused with the yeast Ure2p N-terminal fibrillogenic domain consisting of residues 1 to 80 (Ure2p(1-80)). The resulting Ure2p(1-80)-HD-cohesin fusion protein was successfully expressed in Escherichia coli to produce self-assembled supramolecular nanofibrils that serve as a novel protein scaffold displaying multiple copies of functional cohesin domains. The amyloid-like property of the nanofibrils was confirmed via thioflavin T staining and atomic force microscopy. These cohesin nanofibrils attached themselves, via a green fluorescent protein (GFP)-dockerin fusion protein, to the cell surface of S. cerevisiae engineered to display a GFP-nanobody. The excess cohesin units on the nanofibrils provide ample sites for binding to dockerin fusion proteins, as exemplified using an mCherry-dockerin fusion protein as well as the Clostridium cellulolyticum CelA endoglucanase. More than a 24-fold increase in mCherry fluorescence and an 8-fold increase in CelA activity were noted when the cohesin nanofibril scaffold-mediated yeast display was used, compared to using yeast display with GFP-cohesin that contains only a single copy of cohesin. Self-assembled supramolecular cohesin nanofibrils created by fusion with the yeast Ure2p fibrillogenic domain provide a versatile protein scaffold that expands the utility of yeast cell surface display.

Biophysical interaction between corticosteroids and natural surfactant preparation: implications for pulmonary drug delivery using surfactant a a carrier.

Intratracheal administration of corticosteroids using a natural pulmonary surfactant as a delivery vehicle has recently received significant attention in hopes of treating premature newborns with or at high risk for chronic lung disease. As a new practice, both the surfactant preparation used as the carrier and the corticosteroid delivered as the anti-inflammatory agent, and their mixing ratios, have not been standardized and optimized. Given the concern that corticosteroids delivered via a pulmonary surfactant may compromise its surface activity and thus worsen lung mechanics, the present study was carried out to characterize the biophysical interaction between a natural surfactant preparation, Infasurf, and two commonly used inhaled corticosteroids, budesonide and beclomethasone dipropionate (BDP). Based on surface activity measurements by the Langmuir balance and lateral film structure studied by atomic force microscopy, our findings suggest that when Infasurf is used as a carrier, a budesonide concentration less than 1 wt% of surfactant or a BDP concentration up to 10 wt % should not significantly affect the biophysical properties of Infasurf, thus being feasible for pulmonary delivery. Increasing corticosteroid concentration beyond this range leads to early collapse of the surfactant film due to increased film fluidization. Our study further suggests that different affinities to the surfactant films are responsible for the different behavior of budesonide and BDP. In addition to the translational value in treating chronic lung disease, this study may also have implications in inhaled steroid therapy to treat asthma.

Adverse biophysical effects of hydroxyapatite nanoparticles on natural pulmonary surfactant.

Inhaled nanoparticles (NPs) must first interact with the pulmonary surfactant (PS) lining layer that covers the entire internal surface of the respiratory tract and plays an important role in surface tension reduction and host defense. Interactions with the PS film determine the subsequent clearance, retention, and translocation of the inhaled NPs and hence their potential toxicity. To date, little is known how NPs interact with PS, and whether or not NPs have adverse effects on the biophysical function of PS. We found a time-dependent toxicological effect of hydroxyapatite NPs (HA-NPs) on a natural PS, Infasurf, and the time scale of surfactant inhibition after particle exposure was comparable to the turnover period of surfactant metabolism. Using a variety of in vitro biophysicochemical characterization techniques, we have determined the inhibition mechanism to be due to protein adsorption onto the HA-NPs. Consequently, depletion of surfactant proteins from phospholipid vesicles caused conversion of original large vesicles into much smaller vesicles with poor surface activity. These small vesicles, in turn, inhibited biophysical function of surfactant films after adsorption at the air-water interface. Cytotoxicity study found that the HA-NPs at the studied concentration were benign to human bronchial epithelial cells, thereby highlighting the importance of evaluating biophysical effect of NPs on PS. The NP-PS interaction mechanism revealed by this study may not only provide new insight into the toxicological study of nanoparticles but also shed light on the feasibility of NP-based pulmonary drug delivery.

On the low surface tension of lung surfactant.

Natural lung surfactant contains less than 40% disaturated phospholipids, mainly dipalmitoylphosphatidylcholine (DPPC). The mechanism by which lung surfactant achieves very low near-zero surface tensions, well below its equilibrium value, is not fully understood. To date, the low surface tension of lung surfactant is usually explained by a squeeze-out model which predicts that upon film compression non-DPPC components are gradually excluded from the air-water interface into a surface-associated surfactant reservoir. However, detailed experimental evidence of the squeeze-out within the physiologically relevant high surface pressure range is still lacking. In the present work, we studied four animal-derived clinical surfactant preparations, including Survanta, Curosurf, Infasurf, and BLES. By comparing compression isotherms and lateral structures of these surfactant films obtained by atomic force microscopy within the physiologically relevant high surface pressure range, we have derived an updated squeeze-out model. Our model suggests that the squeeze-out originates from fluid phases of a phase-separated monolayer. The squeeze-out process follows a nucleation-growth model and only occurs within a narrow surface pressure range around the equilibrium spreading pressure of lung surfactant. After the squeeze-out, three-dimensional nuclei stop growing, thereby resulting in a DPPC-enriched interfacial monolayer to reduce the air-water surface tension to very low values.

Comparative study of clinical pulmonary surfactants using atomic force microscopy.

Clinical pulmonary surfactant is routinely used to treat premature newborns with respiratory distress syndrome, and has shown great potential in alleviating a number of neonatal and adult respiratory diseases. Despite extensive study of chemical composition, surface activity, and clinical performance of various surfactant preparations, a direct comparison of surfactant films is still lacking. In this study, we use atomic force microscopy to characterize and compare four animal-derived clinical surfactants currently used throughout the world, i.e., Survanta, Curosurf, Infasurf and BLES. These modified-natural surfactants are further compared to dipalmitoyl phosphatidylcholine (DPPC), a synthetic model surfactant of DPPC:palmitoyl-oleoyl phosphatidylglycerol (POPG) (7:3), and endogenous bovine natural surfactant. Atomic force microscopy reveals significant differences in the lateral structure and molecular organization of these surfactant preparations. These differences are discussed in terms of DPPC and cholesterol contents. We conclude that all animal-derived clinical surfactants assume a similar structure of multilayers of fluid phospholipids closely attached to an interfacial monolayer enriched in DPPC, at physiologically relevant surface pressures. This study provides the first comprehensive survey of the lateral structure of clinical surfactants at various surface pressures. It may have clinical implications on future application and development of surfactant preparations.