Ascorbyl palmitate interaction with phospholipid monolayers: Electrostatic and rheological preponderancy

Ascorbyl palmitate (ASC₁₆) is an anionic amphiphilic molecule of pharmacological interest due to its antioxidant properties. We found that ASC₁₆ strongly interacted with model membranes. ASC₁₆ penetrated phospholipid monolayers, with a cutoff near the theoretical surface pressure limit. The presence of a lipid film at the interface favored ASC₁₆ insertion compared with a bare air/water surface. The adsorption and penetration time curves showed a biphasic behavior: the first rapid peak evidenced a fast adsorption of charged ASC₁₆ molecules to the interface that promoted a lowering of surface pH, thus partially neutralizing and compacting the film. The second rise represented an approach to the equilibrium between the ASC₁₆ molecules in the subphase and the surface monolayer, whose kinetics depended on the ionization state of the film. Based on the Langmuir dimiristoylphosphatidylcholine + ASC₁₆ monolayer data, we estimated an ASC₁₆ partition coefficient to dimiristoylphosphatidylcholine monolayers of 1.5 × 10⁵ and a ΔG_p = − 6.7 kcal·mol^− 1. The rheological properties of the host membrane were determinant for ASC₁₆ penetration kinetics: a fluid membrane, as provided by cholesterol, disrupted the liquid-condensed ASC₁₆-enriched domains and favored ASC₁₆ penetration. Subphase pH conditions affected ASC₁₆ aggregation in bulk: the smaller structures at acidic pHs showed a faster equilibrium with the surface film than large lamellar ones. Our results revealed that the ASC₁₆ interaction with model membranes has a highly complex regulation. The polymorphism in the ASC₁₆ bulk aggregation added complexity to the equilibrium between the surface and subphase form of ASC₁₆, whose understanding may shed light on the pharmacological function of this drug.     

Atypical surface behavior of ceramides with nonhydroxy and 2-hydroxy very long-chain (C28–C32) PUFAs

Unique species of ceramide (Cer) with very-long-chain polyunsaturated fatty acid (VLCPUFA), mainly 28–32 carbon atoms, 4–5 double bonds, in nonhydroxy and 2-hydroxy forms (n-V Cer and h-V Cer, respectively), are generated in rat spermatozoa from the corresponding sphingomyelins during the acrosomal reaction. The aim of this study was to determine the properties of these sperm-distinctive ceramides in Langmuir monolayers. Individual Cer species were isolated by HPLC and subjected to analysis of surface pressure, surface potential, and Brewster angle microscopy (BAM) as a function of molecular packing. In comparison with known species of Cer, n-V Cer and h-V Cer species showed much larger mean molecular areas and increased molecular dipole moments in liquid expanded phases, which suggest bending and partial hydration of the double bonded portion of the VLCPUFA. The presence of the 2-hydoxyl group induced a closer molecular packing in h-V Cer than in their chain-matched n-V Cer. In addition, all these Cer species showed liquid-expanded to liquid-condensed transitions at room temperature. Existence of domain segregation was confirmed by BAM. Additionally, thermodynamic analysis suggests a phase transition close to the physiological temperature for VLCPUFA-Cers if organized as bulk dispersions.     

Differential effects of human SP-A1 and SP-A2 variants on phospholipid monolayers containing surfactant protein B

Surfactant protein A (SP-A), the most abundant protein in the lung alveolar surface, has multiple activities, including surfactant-related functions. SP-A is required for the formation of tubular myelin and the lung surface film. The human SP-A locus consists of two functional SP-A genes, SP-A1 and SP-A2, with a number of alleles characterized for each gene. We have found that the human in vitro expressed variants, SP-A1 (6A²) and SP-A2 (1A⁰), and the coexpressed SP-A1/SP-A2 (6A²/1A⁰) protein have a differential influence on the organization of phospholipid monolayers containing surfactant protein B (SP-B). Lipid films containing SP-B and SP-A2 (1A⁰) showed surface features similar to those observed in lipid films with SP-B and native human SP-A. Fluorescence images revealed the presence of characteristic fluorescent probe-excluding clusters coexisting with the traditional lipid liquid-expanded and liquid-condensed phase. Images of the films containing SP-B and SP-A1 (6A²) showed different distribution of the proteins. The morphology of lipid films containing SP-B and the coexpressed SP-A1/SP-A2 (6A²/1A⁰) combined features of the individual films containing the SP-A1 or SP-A2 variant. The results indicate that human SP-A1 and SP-A2 variants exhibit differential effects on characteristics of phospholipid monolayers containing SP-B. This may differentially impact surface film activity.     

Diffusion and Partitioning of Fluorescent Lipid Probes in Phospholipid Monolayers

The pressure-dependent diffusion and partitioning of single lipid fluorophores in DMPC and DPPC monolayers were investigated with the use of a custom-made monolayer trough mounted on a combined fluorescence correlation spectroscopy (FCS) and wide-field microscopy setup. It is shown that lipid diffusion, which is essential for the function of biological membranes, is heavily influenced by the lateral pressure and phase of the lipid structure. Both of these may change dynamically during, e.g., protein adsorption and desorption processes. Using FCS, we measured lipid diffusion coefficients over a wide range of lateral pressures in DMPC monolayers and fitted them to a free-area model as well as the direct experimental observable mean molecular area. FCS measurements on DPPC monolayers were also performed below the onset of the phase transition (Π < 5 mN/m). At higher pressures, FCS was not applicable for measuring diffusion coefficients in DPPC monolayers. Single-molecule fluorescence microscopy and differential scanning calorimetry clearly showed that this was due to heterogeneous partitioning of the lipid fluorophores in condensed phases. The results were compared with dye partitioning in giant lipid vesicles. These findings are significant in relation to the application of lipid fluorophores to study diffusion in both model systems and biological systems.     

Force-field dependence on the liquid-expanded to liquid-condensed transition in DPPC monolayers

We have investigated using molecular dynamics simulations, the influence of the interaction cut-off and water model on the surface-pressure area isotherms of dipalmitoylphosphatidylcholine monolayers, where the phospholipids and the water molecules are modelled atomistically. We find that both the cut-off employed and the water model, influence the pressure area isotherms and the location of the liquid-expanded to liquid-condensed transitions. The combination of the Berger's et al. force field, with the TIP4P/2005 water model, and a long cut-off for the pair interactions ( ≥ 1.7 nm) provides a more accurate prediction of the surface pressure–area isotherm and reproduces the liquid condensed–liquid expanded transition observed in the experiments at 310 K.     

Mimicking SP-C palmitoylation on a peptoid-based SP-B analogue markedly improves surface activity

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.