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Despite the many advantages of small interfering RNA (siRNA) inhalation therapy and a growing prevalence of respiratory pathologies, its clinical translation is severely hampered by inefficient intracellular delivery. To this end, we previously developed hybrid nanoparticles consisting of an siRNA-loaded nanosized hydrogel core (nanogel) coated with Curosurf®, a clinically used pulmonary surfactant (PS). Interestingly, the PS shell was shown to markedly improve particle stability as well as intracellular siRNA delivery in vitro and in vivo. The major aim of this work was to identify the key molecular components of PS responsible for the enhanced siRNA delivery and evaluate how the complexity of the PS coat could be reduced. We identified surfactant protein B (SP-B) as a potent siRNA delivery enhancer when reconstituted in proteolipid coated hydrogel nanocomposites. Improved cytosolic siRNA delivery was achieved by inserting SP-B into a simplified phospholipid mixture prior to nanogel coating. This effect was observed both in vitro (lung epithelial cell line) and in vivo (murine acute lung injury model), albeit that distinct phospholipids were required to achieve these results. Importantly, the developed nanocomposites have a low in vivo toxicity and are efficiently taken up by resident alveolar macrophages, a main target cell type for treatment of inflammatory pulmonary pathologies. Our results demonstrate the potential of the endogenous protein SP-B as an intracellular siRNA delivery enhancer, paving the way for future design of nanoformulations for siRNA inhalation therapy. Statement of significance Despite the therapeutic potential of small interfering RNA (siRNA) and a growing prevalence of lung diseases for which innovative therapies are needed, a safe and effective siRNA inhalation therapy remains non-existing due to a lack of suitable formulations. We identified surfactant protein B (SP-B) as a potent enhancer of siRNA delivery by proteolipid coated nanogel formulations in vitro in a lung epithelial cell line. The developed nanocomposites have a low in vivo toxicity and show a high uptake by alveolar macrophages, a main target cell type for treatment of inflammatory pulmonary pathologies. Importantly, in vivo SP-B is also critical for the developed formulation to obtain a significant silencing of TNFα in a murine LPS-induced acute lung injury model.

Surfactant protein C (SP-C) has been regarded as the most specific protein linked to development of mammalian lungs, and great efforts have been done to understand its structure–function relationships. Previous evidence has outlined the importance of SP-C palmitoylation to sustain the proper dynamics of lung surfactant, but the mechanism by which this posttranslational modification aids SP-C to stabilize the interfacial surfactant film along the compression–expansion breathing cycles, is still unrevealed. In this work we have compared the structure, orientation and lipid–protein interactions of a native palmitoylated SP-C with those of a non-palmitoylated recombinant SP-C (rSP-C) form in air-exposed multilayer membrane environments, by means of ATR-FTIR spectroscopy. Palmitoylation does not affect the secondary structure of the protein, which exhibits a full α-helical conformation in partly dehydrated phospholipid multilayer films. However, differences between the Amide I band of the IR spectrum of palmitoylated and non-palmitoylated proteins suggest subtle differences affecting the environment of their helical component. These differences are accompanied by differential effects on the IR bands from phospholipid phosphates, indicating that palmitoylation modulates lipid–protein interactions at the headgroup region of phospholipid layers. On the other hand, the relative dichroic absorption of polarized IR has allowed calculating that the palmitoylated protein adopts a more tilted transmembrane orientation than the non-palmitoylated SP-C, likely contributing to more compact, dehydrated and possibly stable multilayer lipid–protein films. As a whole, the behavior of multilayer films containing palmitoylated SP-C may reflect favorable structural properties for surfactant reservoirs at the air–liquid respiratory interface.

Hydrophobic lung surfactant proteins B and C (SP-B and SP-C) are critical for normal respiration in vertebrates, and each comprises specific structural attributes that enable the surface-tension-reducing ability of the lipid–protein mixture in lung surfactant. The difficulty in obtaining pure SP-B and SP-C on a large scale has hindered efforts to develop a non-animal-derived surfactant replacement therapy for respiratory distress. Although peptide-based SP-C mimics exhibit similar activity to the natural protein, helical peptide-based mimics of SP-B benefit from dimeric structures. To determine if in vitro surface activity improvements in a mixed lipid film could be garnered without creating a dimerized structural motif, a helical and cationic peptoid-based SP-B mimic was modified by SP-C-like N-terminus alkylation with octadecylamine. “Hybridized” mono- and dialkylated peptoids significantly decreased the maximum surface tension of the lipid film during cycling on the pulsating bubble surfactometer relative to the unalkylated variant. Peptoids were localized in the fluid phase of giant unilamellar vesicle lipid bilayers, as has been described for SP-B and SP-C. Using Langmuir–Wilhelmy surface balance epifluorescence imaging (FM) and atomic force microscopy (AFM), only lipid-alkylated peptoid films revealed micro- and nanostructures closely resembling films containing SP-B. AFM images of lipid-alkylated peptoid films showed gel condensed-phase domains surrounded by a distinct phase containing “nanosilo” structures believed to enhance re-spreading of submonolayer material. N-terminus alkylation may be a simple, effective method for increasing lipid affinity and surface activity of single-helix SP-B mimics.

This review is focused on the evolution and function of alveolar proteins The lung faces physical and environmental challenges, due to changing pressures/volumes and foreign pathogens, respectively The pulmonary surfactant system is integral in protecting the lung from these challenges via two groups of surfactant proteins - the small molecular weight hydrophobic SPs. SP-B and -C, that regulate interfacial adsorption of the lipids, and the large hydrophilic SPs. SP-A and -D, which are surfactant collectins capable of inhibiting foreign pathogens Further aiding pulmonary host defence are non-surfactant collectins and antimicrobial peptides that are expressed across the biological kingdoms Linking to the first symposium session, which emphasised molecular structure and biophysical function of surfactant lipids and proteins, this review begins with a discussion of the role of temperature and hydrostatic pressure in shaping the evolution of SP-C in mammals Transitioning to the role of the alveolus in innate host defence we discuss the structure, function and regulation of antimicrobial peptides, the defensins and cathelicidins. We describe the recent discovery of novel avian collectins and provide evidence for their role in preventing influenza infection This is followed by discussions of the roles of SP-A and SP-D in mediating host defence at the alveolar surface and in mediating inflammation and the allergic response of the airways Finally we discuss the use of animal models of lung disease including knockouts to develop an understanding of the role of these proteins in initiating and/or perpetuating disease with the aim of developing new therapeutic strategies (C) 2010 Elsevier B V All rights reserved

The pulmonary surfactant system constitutes an excellent example of how dynamic membrane polymorphism governs some biological functions through specific lipid–lipid, lipid–protein and protein–protein interactions assembled in highly differentiated cells. Lipid–protein surfactant complexes are assembled in alveolar pneumocytes in the form of tightly packed membranes, which are stored in specialized organelles called lamellar bodies (LB). Upon secretion of LBs, surfactant develops a membrane-based network that covers rapidly and efficiently the whole respiratory surface. This membrane-based surface layer is organized in a way that permits efficient gas exchange while optimizing the encounter of many different molecules and cells at the epithelial surface, in a cross-talk essential to keep the whole organism safe from potential pathogenic invaders.The present review summarizes what is known about the structure of the different forms of surfactant, with special emphasis on current models of the molecular organization of surfactant membrane components. The architecture and the behaviour shown by surfactant structures in vivo are interpreted, to some extent, from the interactions and the properties exhibited by different surfactant models as they have been studied in vitro, particularly addressing the possible role played by surfactant proteins. However, the limitations in structural complexity and biophysical performance of surfactant preparations reconstituted in vitro will be highlighted in particular, to allow for a proper evaluation of the significance of the experimental model systems used so far to study structure–function relationships in surfactant, and to define future challenges in the design and production of more efficient clinical surfactants.

Two small hydrophobic proteins, SP-B and SP-C, are responsible for rapid adsorption of pulmonary surfactant to the air/water interface. Despite their physiological importance, the number of protein molecules required to trigger an absorption event remains unknown. To investigate this issue, we varied the protein content of calf lung surfactant extract (CLSE) by dilution with protein-depleted surfactant lipids (neutral and phospholipids, N&PL). Vesicles of a constant size and of composition ranging between 100% N&PL and 100% CLSE were generated by probe sonication. Their adsorption kinetics to an air/water interface were monitored at different temperatures using a Wilhelmy plate to measure surface tension. When plotted versus protein concentration, the adsorption rates during the initial change in surface tension exhibit a diphasic behavior, first increasing rapidly and linearly between 0% and 25% CLSE, and then more slowly at higher concentrations. Direct linearity at low protein content (0-5% CLSE ratio) was confirmed at 37 degrees C. These observations argue against cooperative behavior, for which the adsorption rate would first rise slowly with the protein content, and then increase suddenly once the critical number of proteins on each vesicle is reached. The apparent activation energy E(a) and the free energy of activation DeltaG(0)*, calculated from the temperature dependence of adsorption, further support the view that at least the early stages of protein-induced surfactant adsorption proceeds through a sequence of events involving not several, but a single surfactant protein.

Many membrane-bound protein precursors, including cytokines and growth factors, are proteolytically shed to yield soluble intercellular regulatory ligands. The responsible protease, tumor necrosis factor-a converting enzyme (TACE/ADAM-17), is a transmembrane metalloprotease-disintegrin that cleaves multiple cell surface proteins, although it was initially identified for the enzymatic release of tumor necrosis factor-a (TNF-a). Mammalian lung growth and development are tightly controlled by cytokines and peptide growth factors. However, the biological function of the cell shedding mechanism during lung organogenesis is not understood. We therefore evaluated the role of TACE as a “sheddase” during lung morphogenesis by analyzing the developmental phenotypes of lungs in mice with an inactive TACE gene in both in vivo and ex vivo organ explant culture. Neonatal TACE-deficient mice had visible respiratory distress and their lungs failed to form normal saccular structures. These newborn mutant lungs had fewer peripheral epithelial sacs with deficient septation and thick-walled mesenchyme, resulting in reduced surface for gas exchange. At the canalicular stage of E16.5, the lungs of TACE mutant mice were impaired in branching morphogenesis, inhibited in epithelial cell proliferation and differentiation, and delayed in vasculogenesis. Embryonic TACE knockout mouse lungs (E12) branched poorly compared to wild-type lungs, when placed into serumless organ culture. Gene expression of both surfactant protein-C and aquaporin-5 were inhibited in cultured TACE-mutant embryonic lungs, indicating defects in both branching and peripheral epithelial cytodifferentiation in the absence of TACE protein. Furthermore, both the hypoplastic phenotype and the delayed cytodifferentiation in TACE-deficient lungs were rescued by exogenous addition of soluble stimulatory factors including either TNF-a or epidermal growth factor in embryonic lung culture. Thus, the impaired lung branching and maturation without TACE suggest a broad role for TACE in the processing of multiple membrane-anchored proteins, one or more of which is essential for normal lung morphogenesis. Taken together, our data indicate that the TACE-mediated proteolytic mechanism which enzymatically releases membrane-tethered proteins plays an indispensable role in lung morphogenesis, and its inactivation leads to abnormal lung development. © 2001 Academic Press

Surfactant secretion is a critical regulated process in the metabolism of pulmonary surfactant. Presumably, because this process is vital to the survival of the organism, there are several independent pathways for stimulating secretion which work through different cell surface receptors and signaling mechanisms. In addition, there is apparent homeostatic regulation in that two components of surfactant, namely SP-A and dipalmitoylphosphatidylcholine, can inhibit secretion. Although secretion of surfactant has been studied for over two decades, there remains some important issues to be resolved. In vivo secretion can be stimulated by hyperventilation or even a single large breath. However, we do not know the biochemical mechanism for this physiologically important form of stimulation. In vitro, we know many of the proximal events in signaling, but we do not know how the lamellar bodies move within a cell or the docking mechanism at the plasma membrane. Many investigators have demonstrated that SP-A will inhibit secretion in vitro, but the mechanism is not known. Finally, there is a route of secretion of SP-A independent of lamellar bodies, but we do not know details of this pathway nor its regulation.