Penetration of dissolved amphiphiles into two-dimensional aggregating lipid monolayers

The review demonstrates the recent theoretical and experimental progress in the understanding of penetration systems at the air-water interface in which a dissolved amphiphile (surfactant, protein) penetrates into a Langmuir monolayer. The critical review of the existing theoretical models which describe the thermodynamics of the penetration are critically reviewed. Although a rigorous thermodynamic analysis of penetration systems is unavailable due to their complexity, some model assumptions, e.g. the invariability of the activity coefficient of the insoluble component of the monolayer during the penetration of the soluble component results in reasonable solutions. New theoretical models describing the equilibrium behaviour of the insoluble monolayers which undergo the 2D aggregation in the monolayer, and the equations of state and adsorption isotherms which assume the existence of multiple states (conformations) of a protein molecule within the monolayer and the non-ideality of the adsorbed monolayers are now available. The theories which describe the penetration of a soluble surfactant into the main phases of Langmuir monolayers were presented first for the case of the mixture of the molecules possessing equal partial molar surfaces (the mixture of homologues), with further extension of the models to include the interesting process of the protein penetration into the monolayer of 2D aggregating phospholipid. This extension was based on a concept which subdivides the protein molecules into independent fragments with areas equal to those of the phospholipid molecule. Various mechanisms for the effect of the soluble surfactant on the aggregation of the insoluble component were considered in the theoretical models: (i) no effect on the aggregate formation process; (ii) formation of mixed aggregates; and (iii) the influence on the aggregating process via the change of aggregation constant, but without any formation of mixed aggregates. Accordingly depending on the mechanism, different forms of the equations of state of the monolayer and of the adsorption isotherms of soluble surfactant are predicted. Based on the shape of the experimental pi-A isotherms, interesting conclusions can be drawn on the real mechanism. First experimental evidence has been provided that the penetration of different proteins and surfactants into a DPPC monolayer in a fluid-like state induces a first order main phase transition of pure DPPC. The phase transition is indicated by a break point in the pi(t) penetration kinetics curves and the domain formation by BAM. Mixed aggregates of protein with phospholipid are not formed. These results agree satisfactorily with the predictions of the theoretical models. New information on phase transition and phase properties of Langmuir monolayers penetrated by soluble amphiphiles are obtained by coupling of the pi(t) penetration kinetics curves with BAM and GIXD measurements. The GIXD results on the penetration of beta-lactoglobulin into DPPC monolayers have shown that protein penetration occurs without any specific interactions with the DPPC molecules and the condensed phase consists only of DPPC.     

Penetration of surfactin into phospholipid monolayers: nanoscale interfacial organization

Atomic force microscopy (AFM) combined with surface pressure-area isotherms were used to probe the interfacial behavior of phospholipid monolayers following penetration of surfactin, a cyclic lipopeptide produced by Bacillus subtilis strains. Prior to penetration experiments, interfacial behavior of different surfactin molecules (cyclic surfactins with three different aliphatic chain lengths--S13, S14, and S15--and a linear surfactin obtained by chemical cleavage of the cycle of the surfactin S15) has been investigated. A more hydrophobic aliphatic chain induces greater surface-active properties of the lipopeptide. The opening of the peptide ring reduces the surface activity. The effect of phospholipid acyl chain length (dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine- (DPPC), and distearoylphosphatidylcholine) and phospholipid polar head (DPPC, dipalmitoylphosphatidylethanolamine and dipalmitoylphosphatidylserine) on monolayer penetration properties of the surfactin S15 has been explored. Results showed that while the lipid monolayer thickness and the presence of electrostatic repulsions from the interfacial film do not significantly influence surfactin insertion, these parameters strongly modulate the ability of the surfactin to alter the nanoscale organization of the lipid films. We also probed the effect of surfactin structure (influence of the aliphatic chain length and of the cyclic structure of the peptide ring) on the behavior of DPPC monolayers. AFM images and isotherms showed that surfactin penetration is promoted by longer lipopeptide chain length and a cyclic polar head. This indicates that hydrophobic interactions are of main importance for the penetration power of surfactin molecules.     

Penetration of sodium cetylsulfate into monolayers of 1,2-dipalmitoyl- and 1,2-dimyristoyl-phosphatidylethanolamine

The penetration of sodium cetylsulfate into monolayers of dipalmitoyl- and dimyristoyl-phosphatidylethanolamine was studied by the measurement of surface and penetration pressures using the vertical plate method of Wilhelmy. The penetration isotherms in two systems were investigated at different initial molecular areasA _M :System I: Sodium cetylsulfate/1,2-dipalmitoyl-phosphatidylethanolamine atA _M = 0.85; 0.75; 0.65; 0.55; 0.50; 0.46 and 0.44 nm² · molecule^–1.System II: Sodium cetylsulfate/1,2-dimyristoyl-phosphatidylethanolamine atA _M = 0.85; 0.75; 0.60 and 0.55 nm² · molecule^–1.(T=295 K; substrate 0.1 M NaCl)The penetration isotherms (F _t vs. logc _s ) increase linearly atF _t > 10 mN · m^–1 in system I and atF _t >25 mN · m^–1 in system II. The isotherms of both systems are shifted to lower surfactant concentrations with decreasing molecular area of spread monolayer. A maximum of the slopes (dF_t/d logc _s )occurs at A_M=0.50 nm² · molecule^–1. This behavior is also reflected in the dependenceG _p ⁰ (free standard penetration enthalpy) and _s (relative surface excess concentration of surfactant) onA _M . These changes are related to a different packing of the compounds in the binary penetrated monolayers.In the high pressure region both system are nearly identical. Differences in the low pressure region arise from the penetration into different monolayer states.Nomenclature _M effective cross sectional area of monolayer molecule - a _M partial molecular area of monolayer molecule - a _s partial molecular area of surfactant molecule in the penetrated film - a _s ⁰ molecular area of surfactant molecule at definite film pressure (eq. (3)) - A _M molecular area of theF/A-isotherm - A _N constant in equation (2) - A _K collapse area - b penetration coefficient in equations (2); (2 a) - c _s bulk concentration of surfactant - logc _s relative shift of penetration isotherm with regard to the adsorption isotherm at constantF _t - F film pressure of monolayer component in absence of surfactant - F _t total film pressure - F _p film pressure change due to penetration - F _p,max constant in equation (1) - G _p ⁰ free standard penetration energy - k Boltzmann constant - K constant in equation (1) - R gas constant - T temperature - x _s ⁰ mole fraction of surfactant in the penetrated film - _M surface concentration of monolayer molecules - _s relative adsorption of surfactant - _w ⁰ surface concentration of surfactant in monolayer-free surface - factor in equation (6 a) - surface tension     

Temperature dependence of poloxamer insertion into and squeeze-out from lipid monolayers

F68, a triblock copolymer of the form poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), is found to effectively seal damaged cell membranes. To better understand the molecular interaction between F68 and cells, we have modeled the outer leaflet of a cell membrane with a dipalmitoylphosphatidylcholine (DPPC) monolayer spread at the air-water interface and introduced poloxamer into the subphase. Subsequent interactions of the polymer with the monolayer either upon expansion or compression were monitored using concurrent Langmuir isotherm and fluorescence microscopy measurements. To alter the activity of the poloxamer, a range of subphase temperatures from 5 to 37 degrees C was used. Lower temperatures increase the solubility of the poloxamer in the subphase and therefore lessen the amount of material at the interface, resulting in a lower equilibrium spreading pressure. Additionally, changes in temperature affect the phase behavior of DPPC. Below the triple point, the monolayer is condensed at pertinent polymer insertion pressures; for temperatures immediately above the triple point, the monolayer is a heterogeneous mix of liquid expanded and condensed phase; for the highest temperature measured, the DPPC monolayer remains completely fluid. At all temperatures, F68 inserts into DPPC monolayers at its equilibrium spreading pressure. Upon compression of the monolayer, polymers are squeezed-out at surface pressures notably higher than those for insertion, with higher temperatures leading to a higher squeeze-out pressure. An increase in temperature decreases the solvent quality of water for the poloxamer, lowering solubility of the polymer in the subphase and thus increasing its propensity to be maintained within the monolayer to higher pressures.     

Penetration of bovine serum albumin into dipalmitoylphosphatidylglycerol monolayers: direct observation by atomic force microscopy

The penetration of bovine serum albumin (BSA) into dipalmitoylphosphatidylglycerol (DPPG) monolayers was observed using atomic force microscopy (AFM) and surface pressure measurements. The effects of surface pressure, amount of BSA and the addition of ganglioside GM1 (GM1) were investigated. The surface pressure of the DPPG monolayer was increased by the penetration of BSA, and the increase in surface pressure was greater in the liquid-expanded film than that in the liquid-condensed film. The AFM images indicated that BSA penetrated into the DPPG monolayer. The amount of BSA that penetrated into the DPPG monolayer increased with time and with the amount of BSA added. On the contrary, the AFM image showed that BSA penetration into the mixed DPPG/GM1 (9 : 1) monolayer scarcely occurred. GM1 inhibited the penetration of BSA into the DPPG monolayer.     

Penetration of a GPI-anchored protein into phospholipid monolayers spread at the air/water interface

Mammalian alkaline phosphatases (AP) belong to glycosylphosphatidyl inositol (GPI) anchored proteins family, which are localised and clustered on the outer layer of the plasma membranes forming microdomains. Using Langmuir film and polarisation modulation infrared reflection absorption spectroscopy (PMIRRAS) techniques, the penetration process of the protein into a phospholipid monolayer have been studied at the air–buffer interface. The penetration of AP-GPI in distearoylphosphatidylcholine monolayers (DSPC) induces a more important surface pressure increase than in dioleoylphosphatidylcholine (DOPC) monolayer. However, the exclusion surface pressure rather similar for both lipids, 20.5 and 22 mN m⁻¹ for, respectively, DSPC and DOPC, indicates that the AP-GPI cannot, in similar conditions, insert by itself into bilayer membranes of either biological or mimetic origin. PMIRRAS suggests that the pure acyl chains perdeuterated DSPC (d₇₀-DSPC) interact with Mg²⁺ present into the buffer. AP-GPI inserts progressively into the d₇₀-DSPC monolayer changing the environment of phospholipid molecules. Amide I band exhibits helix and β-sheets components with a predominance of the helix. The shapes, intensities and positions of the amide I and II bands suggest for the helix an orientation perpendicular to the interface after a period of molecular reorganisation.     

Amphiphilic peptides as models for protein-membrane interactions: interfacial behaviour of sequential Lys- and Leu-based peptides and their penetration into lipid monolayers

Synthetic peptides constructed with doublets of hydrophobic residues tandemly repeated with doublets of positively charged residues, (Leu-Lys-Lys-Leu)_n, were used as models for the study of protein-membrane interactions. Their behaviour has been compared with that of their strictly alternating iso peptides, (Leu-Lys)_n. Both peptides present a random coil structure in pure water. In saline solutions, (Leu-Lys-Lys-Leu)_n peptides adopt an α-helical structure whereas (Leu-Lys)_n transit into a β-sheet structure. These peptides form multilayer assemblies on a pure water subphase but they are organized in monomolecular films on a saline aqueous subphase. The stability of these films increases with the peptide length. Structured peptides (α helices and β sheets) penetrate readily into lipid monolayers, whereas the penetration of unordered peptides is very slow. We have not observed any significant difference between the behaviour of a helices and β-sheet structures.     

Penetration of beta-lactoglobulin into monoglyceride monolayers. dynamics, interactions, and topography of mixed films

In this work we have analyzed the penetration of betalactoglobulin into a monoglyceride monolayer (monopalmitin or monoolein) spread at the air-water interface and its effects on the structural, dilatational, and topographical characteristics of mixed films. Dynamic tensiometry, surface film balance, Brewster angle microscopy (BAM), and surface dilatational rheology have been used, maintaining the temperature constant at 20 degrees C and the pH and ionic strength at 7 and 0.05 M, respectively. The initial surface pressure (mN/m) of the spread monoglyceride monolayer (pii(MONOGLYCERIDE)) at 10, 20, and the collapse point is the variable studied. Beta-lactoglobulin can penetrate into a spread monoglyceride monolayer at every surface pressure. The penetration of beta-lactoglobulin into the monoglyceride monolayer with a more condensed structure, at the collapse point of the monoglyceride, requires monoglyceride molecular loss by collapse and/or desorption. However, the structural, topographical, and dilatational characteristics of monoglyceride penetrated by beta-lactoglobulin mixed monolayers are essentially dominated by the presence of monoglyceride (either monopalmitin or monoolein) in the mixed film. In fact, monoglyceride molecules have the capacity to re-enter the monolayer after expansion and recompression of the mixed monolayer. Thus, monoglyceride molecular loss by collapse and/or desorption is reversible. The topography of the monolayer under dynamic conditions corroborates these conclusions.     

Penetration and interactions of the antimicrobial peptide, microcin J25, into uncharged phospholipid monolayers

Microcin J25 forms stable monolayers at the air-water interface showing a collapse at a surface pressure of 5 mN/m, 220 mV of surface potential, and 6 fV per squared centimeter of surface potential per unit of molecular surface density. The adsorption of microcin J25 from the subphase at clean interfaces leads to a rise of 10 mN/m in surface pressure and a surface potential of 220 mV. From these data microcin appears to be a poor surfactant per se. Nevertheless, the interaction with the lipid monolayer further increase the stability of the peptide at the interface depending on the mode in which the monolayer is formed. Spreading with egg PC leads to nonideal mixing up to 7 mN/m, with hyperpolarization and expansion of components at the interface, with a small excess free energy of mixing caused by favorable contributions to entropy due to molecular area expansion compensating for the unfavorable enthalpy changes arising from repulsive dipolar interactions. Above 7 mN/m microcin is squeezed out, leaving a film of pure phospholipid. Nevertheless, the presence of lipid at 10 and 20 mN/m stabilize further microcin at the interface and adsorption from the subphase proceeds up to 30 mN/m, equivalent to surface pressure in bilayers.     

Penetration of mercury-adsorbed phospholipid monolayers by polynuclear aromatic hydrocarbons

The penetration of phospholipid monolayers (dioleoyl lecithin) adsorbed on mercury by polynuclear aromatic hydrocarbons (PAH) is described. The PAH studied were anthracene, phenanthrene, pyrene, benzo[a]anthracene, fluoranthene and perylene. The penetration is monitored by measuring the differential capacitance of the monolayer; the uptake of PAH causes a potential shift (up to ?0.25 V) in the cathodic capacitance peaks. This occurs without displacement of the lipid from the mercury. The differential capacitance is measured by out-of-phase (90°) a.c. voltammetry and rapid cyclic voltammetry. The PAH permeate the mercury-adsorbed lipid layers from dilute aqueous solution; the order of affinity is benzo[a]anthracene > fluoranthrene = pyrene > anthracene = phenanthrene. The rates of penetration vary for the different compounds and depend on their water solubility.     

Density dependent friction of lipid monolayers

We measure frictional properties of liquid-expanded and liquid-condensed phases of lipid Langmuir-Blodgett monolayers by interfacial force microscopy. We find that over a reasonably broad surface-density range, the friction shear strength of the lipid monolayer film is proportional to the surface area (42-74 A2/molecule) occupied by each molecule. The increase in frictional force (i.e., friction shear strength with molecular area can be attributed to the increased conformational freedom and the resulting increase in the number of available modes for energy dissipation.     

The structure of percolating lipid monolayers

The lattice structure and in plane molecular organization of Langmuir monolayer of amphiphilic material is usually determined from grazing incidence X-ray diffraction (GIXD) or neutron reflectivity. Here we present results of a different approach for determination of monolayer lattice structure based on application of fractal analysis and percolation theory in combination with Brewster angle microscopy. The considerations of compressibility modulus and fractal dimension dynamics provide information on percolation threshold and consequently by application of percolation theory on the lattice structure of a monolayer. We have applied this approach to determine the monolayer lattice structures of single chain and double chain lipids. The compressibility moduli were determined from measured π-A isotherms and fractal dimensions from corresponding BAM images. The monolayer lattice structures of stearic acid, 1-hexadecanol, DPPC and DPPA, obtained in this way conform to the corresponding lattice structures determined previously by other authors using GIXD.     

Formation of dimers in lipid monolayers

In this work, we analyse theoretically the hypothesis that zwitterionic lipids form dimers in adsorption monolayers on water/ hydrocarbon phase boundary. A dimer can be modelled as a couple of lipid molecules whose headgroup lateral dipole moments have antiparallel orientation. Properties including surface pressure, chemical potentials and activity coefficients are deduced from a general expression for the free energy of the monolayer. The theoretical model is in a good agreement with experimental data for surface pressure and surface potential of lipid monolayers. The results favour the hypothesis about formation of dimers in equilibrium with monomers, with the amount of the species depending on the area per molecule and temperature. The reaction of dimerisation turns out to be exothermic with a heat of about 2.5kT per dimer. The results may be applied to the molecular models of membrane structures and mechanisms.     

STM observation of alkyl-chain-assisted self-assembled monolayers of pyridine-coordinated porphyrin rhodium chlorides

Alkyl-chain-assisted self-assembled monolayers of pyridine-coordinated porphyrin rhodium chlorides were observed at the solid-liquid interface by scanning tunneling microscopy (STM). The resolved images at a molecular level were obtainable in the pure solution of pyridine-coordinated porphyrin rhodium chloride with four triacontyl groups [Rh(C300PP)(Cl)(Py)]. In the case of pyridine-coordinated porphyrin rhodium chloride with four octadecyl groups [Rh(C18OPP)(Cl)(Py)], the STM images were not obtainable in the pure solution of Rh(C18OPP)(Cl)(Py) but obtainable in the mixture containing Rh(C18OPP)(Cl)(Py) and free porphyrin C18OPP. On the basis of the mixed self-assembled monolayer analysis, the apparent difference in the adsorption free energy between Rh(CnOPP)(Cl)(Py) and CnOPP (deltaGapp) was calculated. The calculated deltaGapp values for C18OPP and C30OPP mixed systems were quite different. The disadvantage of the adsorption free energy of Rh(C18OPP)(Cl)(Py) makes it difficult to obtain molecularly resolved images of Rh(C18OPP)(Cl)(Py), and the large adsorption energy due to the long alkyl chains enabled us to obtain molecularly resolved images of Rh(C30OPP)(Cl)(Py).     

Electrochemical behaviour of p-Si in methanol solutions of chlorides

The mechanism of anodic dissolutions of p-Si single crystals in CH₃OH–LiCl and CH₃OH–LiCl–HCl solutions was investigated by means of the following electrochemical methods: linear sweep voltammetry, the potentiostatic transient technique and XPS surface analysis. The dissolution of p-Si proceeds by a two-step mechanism with the creation of a Si(II) surface intermediate. At low anodic overvoltage the dissolution proceeds with the formation of porous silicon, probably through the reaction: 2Si(II)Si+Si(IV). Structural etching of the single crystals surface was observed at high anodic overvoltage (E>2 V). At this potential range, silicon dissolves with the formation of a Si(IV) soluble product. Electrolysis of the methanol solvent containing Si(IV) in the cell p-Si|CH₃OH–LiCl–Si(IV)|M, where M=Pt, Cu or 18/8 stainless steel, leads to the deposition of an amorphous organosilicon layer on the cathode. The analysis of the deposit performed by means of XPS, FTIR and SEM allows determination of the morphology and composition of the film. The layer consists of Si–OCH₃ compounds and can be created only in methanol solvent. The film is unstable in a humid atmosphere and undergoes transformation into a Si–OH layer.Contribution to the 3rd Baltic Conference on Electrochemistry, Gdansk-Sobieszewo, Poland, 23–26 April 2003Dedicated to the memory of Harry B. Mark, Jr. (28 February 1934–3 March 2003)     

An investigation into drug partitioning behaviour in simulated pulmonary surfactant monolayers with associated molecular modelling

Drug delivery to the body via the inhaled route is dependent upon patient status, device use, and respirable formulation characteristics. Further to inhalation, drug‐containing particles interact and dissolve within pulmonary fluid leading to the desired pharmacological response. Pulmonary surfactant stabilises the alveolar air‐liquid interface and permits optimal respiratory mechanics. This material represents the initial contacting surface for all inhaled matter. On dissolution, the fate of a drug substance can include receptor activation, membrane partitioning and cellular penetration. Here, we consider the partitioning behaviour of salbutamol when located in proximity to a simulated pulmonary surfactant monolayer at pH 7. The administration of salbutamol to the underside of the surfactant film resulted in an expanded character for the 2‐dimensional ensemble and a decrease in the compressibility term. The rate of drug partitioning was greater when the monolayer was in the expanded state (ie, inhalation end‐point), which was ascribed to more accessible areas for molecular insertion. Quantum mechanics protocols, executed via Gaussian 09, indicated that constructive interactions between salbutamol and integral components of the model surfactant film took the form of electrostatic and hydrophobic associations. The favourable interactions are thought to promote drug insertion into the monolayer structure leading to the observed expanded character. The data presented herein confirm that drug partitioning into pulmonary surfactant monolayers is a likely prospect further to the inhalation of respirable formulations. As such, this process holds potential to reduce drug‐receptor activation and/or increase the residence time of drug within the pulmonary space.     

PEG-covered lipid surfaces: bilayers and monolayers

In this review paper we survey the ways in which various micropipet techniques have been used to study the mechanochemical and interactive features of lipid bilayer vesicles and monolayer-coated gas bubbles. Special emphasis will be made on characterizing the barrier properties of grafted PEG layers and how a hierarchical approach that uses a short barrier and extended ligand allows us to start to mimic nature's own solution to the problem of ubiquitous repulsion and specific attraction. The information gained from such studies not only characterizes the membrane and other lipid surfaces and their intersurface interactions from a fundamental materials science perspective, but also provides essential materials property data that are required for the successful design and deployment of lipid-based carriers and other capsules in applications involving this so-called ‘stealthy’ surface.