Poly(methyl methacrylate)-supported polydiacetylene films: unique chromatic transitions and molecular sensing.

Polydiacetylenes (PDAs) constitute a family of conjugated polymers exhibiting unique colorimetric and fluorescence transitions, and have attracted significant interest as chemo- and biosensing materials. We spin-coated PDA films upon poly(methyl methacrylate) (PMMA), and investigated the photophysical properties and sensing applications of the new PDA configuration. Specifically, the as-polymerized blue PDA layer underwent distinct transformations to purple, red, and yellow phases, which could be quantified through conventional color scanning combined with application of image analysis algorithms. Furthermore, we recorded a reversible red-purple PDA transition that was induced by ultraviolet irradiation, a phenomenon that had not been reported previously in PDA film systems. We show that distinct color and fluorescence transitions were induced in the PMMA-supported PDA films by amphiphilic substances-surfactants and ionic liquids-and that the chromatic transformations were correlated to the analyte structures and properties. Overall, this study presents a new chromatic PDA film system in which noncovalent interactions between the PMMA substrate and spin-coated PDA give rise to distinct chromatic properties and molecular sensing capabilities.

Oxidized phosphatidylcholines in membrane-level cellular signaling: from biophysics to physiology and molecular pathology.

The oxidation of lipids has been shown to impact virtually all cellular processes. The paradigm has been that this involvement is due to interference with the functions of membrane-associated proteins. It is only recently that methodological advances in molecular-level detection and identification have begun to provide insights into oxidative lipid modification and its involvement in cell signaling as well as in major diseases and inflammation. Extensive evidence suggests a correlation between lipid peroxidation and degenerative neurological diseases such as Parkinson's and Alzheimer's, as well as type 2 diabetes and cancer. Despite the obvious relevance of understanding the molecular basis of the above ailments, the exact modes of action of oxidized lipids have remained elusive. In this minireview, we summarize recent findings on the biophysical characteristics of biomembranes following oxidative derivatization of their lipids, and how these altered properties are involved in both physiological processes and major pathological conditions. Lipid-bearing, oxidatively truncated and functionalized acyl chains are known to modify membrane bulk physical properties, such as thermal phase behavior, bilayer thickness, hydration and polarity profiles, as manifest in the altered structural dynamics of lipid bilayers, leading to augmented membrane permeability, fast lipid transbilayer diffusion (flip-flop), loss of lipid asymmetry (scrambling) and phase segregation (the formation of 'rafts'). These changes, together with the generated reactive lipid derivatives, can be further expected to interfere with lipid-protein interactions, influencing metabolic pathways, causing inflammation, the execution phase in apoptosis and initiating pathological processes.

Oxidized phosphatidylcholines promote phase separation of cholesterol-sphingomyelin domains.

Lipid lateral segregation in the plasma membrane is believed to play an important role in cell physiology. Sphingomyelin (SM) and cholesterol (Chol)-enriched microdomains have been proposed as liquid-ordered phase platforms that serve to localize signaling complexes and modulate the intrinsic activities of the associated proteins. We modeled plasma membrane domain organization using Langmuir monolayers of ternary POPC/SM/Chol as well as DMPC/SM/Chol mixtures, which exhibit a surface-pressure-dependent miscibility transition of the coexisting liquid-ordered and -disordered phases. Using Brewster angle microscopy and Langmuir monolayer compression isotherms, we show that the presence of an oxidatively modified phosphatidylcholine, 1-palmitoyl-2-azelaoyl-sn-glydecero-3-phosphocholine, efficiently opposes the miscibility transition and stabilizes micron-sized domain separation at lipid lateral packing densities corresponding to the equilibrium lateral pressure of ∼32 mN/m that is suggested to prevail in bilayer membranes. This effect is ascribed to augmented hydrophobic mismatch induced by the oxidatively truncated phosphatidylcholine. To our knowledge, our results represent the first quantitative estimate of the relevant level of phospholipid oxidation that can potentially induce changes in cell membrane organization and its associated functions.

Oxidized phosphatidylcholines facilitate phospholipid flip-flop in liposomes.

Lipid asymmetry is a ubiquitous property of the lipid bilayers in cellular membranes and its maintenance and loss play important roles in cell physiology, such as blood coagulation and apoptosis. The resulting exposure of phosphatidylserine on the outer surface of the plasma membrane has been suggested to be caused by a specific membrane enzyme, scramblase, which catalyzes phospholipid flip-flop. Despite extensive research the role of scramblase(s) in apoptosis has remained elusive. Here, we show that phospholipid flip-flop is efficiently enhanced in liposomes by oxidatively modified phosphatidylcholines. A combination of fluorescence spectroscopy and molecular dynamics simulations reveal that the mechanistic basis for this property of oxidized phosphatidylcholines is due to major changes imposed by the oxidized phospholipids on the biophysical properties of lipid bilayers, resulting in a fast cross bilayer diffusion of membrane phospholipids and loss of lipid asymmetry, requiring no scramblase protein.

Gold nanoparticle self-assembly in saturated phospholipid monolayers.

Self-assembly of nanostructures on surfaces is a promising area in the emerging field of "bottom-up nanolithography". We describe a systematic analysis of hydrophobically capped gold nanoparticle (Au NP) assemblies created within monolayers of saturated phospholipids deposited at the air/water interface. We show that the Au NPs are segregated within the mixed monolayers, forming distinct configurations. Microscopy analysis reveals that organized Au NP aggregates, including wires, rings, and "doughnut-shape" structures, are observed only within condensed-phase monolayers comprising phospholipids exhibiting longer acyl side-chains. In these monolayers, the Au NPs are localized at the edges of the condensed phospholipid domains. In addition to the pronounced effect of the phospholipid phases at the air/water interface, NP organization was found to depend upon the hydrophobic capping agents of the particles. The Au nanostructures assembled at the air/water interface can be transferred onto solid substrates, suggesting that the self-assembly monolayer approach could be exploited for practical nanoelectronic and sensing applications.

Phospholipid-induced fibrillation of a prion amyloidogenic determinant at the air/water interface.

The peptide fragment 106-126 of prion protein [PrP(106-126)] is a prominent amyloidogenic determinant. We present analysis of PrP(106-126) fibrillation at the air/water interface and, in particular, the relationship between the fibrillation process and interactions of the peptide with phospholipid monolayers. We find that lipid monolayers deposited at the air/water interface induce rapid formation of remarkably highly ordered fibrils by PrP(106-126), and that the extent of fibrillation and fiber organization were dependent upon the presence of negatively charged and unsaturated phospholipids in the monolayers. We also observe that fibrillation was enhanced when PrP(106-126) was injected underneath preassembled phospholipid monolayers, compared to deposition and subsequent compression of mixed monolayers of the peptide and phospholipids. In a broader context, this study demonstrates that Langmuir systems constitute a useful platform for studying lipid interactions of amyloidogenic peptides and lipid-induced fibrillation phenomena.

Laser-modulated ordering of gold nanoparticles at the air/water interface.

Beam me up, Scotty! Laser irradiation of Langmuir monolayers of gold nanoparticles (NPs) and elaidic acid led to dramatic reorganization that was dependent on the laser power (see picture, scale bar = 100 microm). Variable-temperature experiments indicate that localized surface heating in an extremely small temperature range, induced by the laser beam, causes ordering of the NPs.

Gold nanostructures in diacetylene monolayer templates.

A new "bottom-up" approach for the construction of gold nanostructure assemblies through the use of diacetylene templates at the air/water interface is described. Condensed diacetylene Langmuir monolayers constrain Au nanoparticles (NPs) on the water surface. The Au NPs adopt distinct configurations, including nanowires and "nanoislets", depending upon the properties and molecular structures of the diacetylene monomers. The diacetylene/Au NP films can be transferred from the air/water interface onto solid substrates and further annealed without altering the surface organization, yielding stable organized gold nanostructures.

Self-assembly and lipid interactions of diacylglycerol lactone derivatives studied at the air/water interface.

Synthetic diacylglycerol lactones (DAG-lactones) have been shown to be effective modulators of critical cellular signaling pathways. The biological activity of these amphiphilic molecules depends in part upon their lipid interactions within the cellular plasma membrane. This study explores the thermodynamic and structural features of DAG-lactone derivatives and their lipid interactions at the air/water interface. Surface-pressure/area isotherms and Brewster angle microscopy revealed the significance of specific side-groups attached to the terminus of a very rigid 4-(2-phenylethynyl)benzoyl chain of the DAG-lactones, which affected both the self-assembly of the molecules and their interactions with phospholipids. The experimental data highlight the formation of different phases within mixed DAG-lactone/phospholipid monolayers and underscore the relationship between the two components in binary mixtures of different mole ratios. Importantly, the results suggest that DAG-lactones are predominantly incorporated within fluid phospholipid phases rather than in the condensed phases that form, for example, by cholesterol. Moreover, the size and charge of the phospholipid headgroups do not seem to affect DAG-lactone interactions with lipids.

Lipid/polydiacetylene films for colorimetric protein surface-charge analysis.

The distribution and organization of charges on a protein surface are fundamental properties which affect protein functions and interactions. We demonstrate a new approach for protein surface-charge analysis through modulating protein interactions with chromatic lipid/polydiacetylene (PDA) films. We show that visible and easily quantifiable blue-red transitions, induced on the film surface through electrostatic interactions between the negatively charged PDA and positive soluble species, constitute an effective means for characterizing protein surface charge. Specifically, protein-film interactions can be significantly modulated by complexation between the tested macromolecules and lipid-embedded multivalent calixarene ligands displaying charged residues, making possible protein discrimination based upon the abundance and organization of surface charge. The lipid/PDA film system, in conjunction with the calixarene-derived ligands, facilitates characterization of protein surface charges and identification of anomalous protein electrostatic properties.

Colorimetric polymer films for predicting lipid interactions and percutaneous adsorption of pharmaceutical formulations.

PURPOSE: To develop and demonstrate a rapid and simple colorimetric film assay for evaluating lipid interactions of pharmaceutical compounds and gel formulations. METHODS: The colorimetric assay comprises glass-supported films of phospholipids and polydiacetylene, which undergo visible and quantifiable blue-red transformations induced by interactions with amphiphilic molecules applied in very small volumes on the film surface. The color transitions are recorded by scanning of the films, and quantified through a simple image analysis algorithm. RESULTS: We show that pharmaceutical molecules and gel formulations induce blue-red transformations after short incubation with the lipid/polydiacetylene (PDA) films. Colorimetric dose-response curves exhibit dependence upon the lipid affinity and extent of membrane binding of the pharmaceutical compounds examined. The colorimetric lipid/PDA film assay was employed for distinguishing the contributions of individual molecular components within gel formulations. CONCLUSIONS: The colorimetric data yield insight into the degree of lipid binding of the molecules tested. The film assay is particularly advantageous for analysis of semi-solid (gel or lotion) formulations, elucidating the lipid interaction characteristics of specific molecular components within the mixtures. The new colorimetric film assay constitutes a generic, rapid, and easily applicable platform for predicting and screening interactions of pharmaceutical compounds and complex formulations with lipid barriers.

Colorimetric detection and fingerprinting of bacteria by glass-supported lipid/polydiacetylene films.

Glass-supported films of lipids and polydiacetylene were applied for visual detection and colorimetric fingerprinting of bacteria. The sensor films comprise polydiacetylene domains serving as the chromatic reporter interspersed within lipid monolayers that function as a biomimetic membrane platform. The detection schemes are based on either visible blue-red transitions or fluorescence transformations of polydiacetylene, induced by amphiphilic molecules secreted by proliferating bacteria. An important feature of the new film platform is the feasibility of either naked-eye detection of bacteria or color analysis using conventional scanners. Furthermore, we find that the degrees of bacterially induced color transformations depend both on the bacterial strains examined and the lipid compositions of the films. Accordingly, bacterial fingerprinting can be achieved through pattern recognition obtained by recording the chromatic transformations in an array of lipid/PDA films having different lipid components.

Glass-supported lipid/polydiacetylene films for colour sensing of membrane-active compounds.

Glass-supported biomimetic lipid/polydiacetylene films were employed for colourimetric detection and analysis of amphiphilic and membrane-active molecules. The sensor films comprise lipid monolayers that constitute a biomimetic membrane platform, interspersed within polydiacetylene domains that function as the colour reporter. The optical detection scheme is based on visible blue-red transitions of polydiacetylene, induced by amphiphilic analytes interacting with the film. The colour transitions of the lipid/polydiacetylene films can be either detected by the naked eye, recorded spectroscopically, or registered through digital image analysis using conventional scanning devices. Digital image analysis, in particular, allows quantification of the colourimetric transformations. Detection threshold of micromolar concentration of a membrane-active cytolytic peptide is demonstrated.

Investigations of antimicrobial peptides in planar film systems.

Planar systems--monolayers and films--constitute a useful platform for studying membrane-active peptides. Here, we summarize varied approaches for studying peptide organization and peptide-lipid interactions at the air/water interface, and focus on three representative antimicrobial membrane--associated peptides-alamethicin, gramicidin, and valinomycin. Experimental data, specifically surface pressure/area isotherms and Brewster angle microscopy images, provided information on peptide association and the effects of the lipid monolayers on peptide surface organization. In general, film analysis emphasized the effects of lipid layers in promoting peptide association and aggregation at the air/water interface. Importantly, the data demonstrated that in many cases peptide domains are phase-separated within the phospholipid monolayers, suggesting that this behavior contributes to the biological actions of membrane-active antimicrobial peptides.

Microscopic visualization of alamethicin incorporation into model membrane monolayers.

Lipid interactions and cooperative assembly properties are fundamental determinants for the action of antimicrobial membrane-active peptides. Here we analyze the interactions and aggregation properties of alamethicin, an antimicrobial pore-forming peptide, with films formed at the air/water interface. Surface-area/pressure isotherms, Brewster angle microscopy, and fluorescence-confocal microscopy provided detailed information on the morphologies and structural properties of the peptide and its effect on the film components. The pressure-area analysis and microscopy experiments facilitated unprecedented visualization of the structural consequences of alamethicin association at the air/water interface, with pure phospholipid films, and within mixed phospholipid/polydiacetylene (PDA) films. The analysis exposed the kinetic features and the interplay between the peptide aggregates and film constituents. In particular, the results demonstrate the use of phospholipid/PDA film assemblies for studying membrane-peptide association and interactions within two-dimensional films.