Monolayer kinetic model of formation of ß-cyclodextrin-ß-carotene inclusion complex.

The carotenoids are sensitive molecules and their chemical integrity must be preserved from pro-oxidant elements which could affect and decrease their physiological benefits. The encapsulation based on the inclusion of the carotenoids into cage molecules is a promising approach for preserving over time of the intrinsic properties of the carotenoids. It is well known that cyclic oligosaccharide ß-cyclodextrin (CD) as a cage molecule possesses strong inclusion ability to ß-carotene (C) and as a result of the hydrophobic interactions forms an inclusion complex. In the present paper a monolayer kinetic model was established with the notion to extract more information about the influence of the molecular structure and organization to the interfacial interactions between the interacting species as well as about the role of the specific areas, which are often underestimated in previously studied dispersed systems. We developed the monolayer kinetic model for the formation of the inclusion CD-C complex by applying an experimental approach for following the kinetics by means of measuring the decrease of the surface area (ΔA) versus time (t) at constant surface pressure (π) and the decrease of surface pressure (π) versus time (t) at constant surface area (A). We also visualized by AFM the state of the monolayers at the initial and end points of the kinetic process. The values for the degree (d) and constant (Ka) of the association were estimated and compared with those from the studies of dispersed systems.

Properties of mixed monolayers of clinical lung surfactant, serum albumin and hydrophilic polymers.

It is now established that the surface activity of the clinically used lung surfactant is reduced by serum proteins and can be restored by adding the hydrophilic polymers. The mechanisms of lung surfactant inactivation by serum proteins and restoring effect by the hydrophilic polymers remain not completely understood. In this paper the state and rheological dilatational properties of surface films formed from clinical lung surfactant Exosurf, Survanta, Curosurf and Alveofact in the presence of serum albumin (BSA) and hydrophilic polymers polyvinylpyrrolidone (PVP), polyethylene glycol (PEG) and Dextran were studied. The obtained results suggest that the lung surfactant and BSA mixtures spread at air-water interface form a DPPC/BSA mixed monolayers with lower content of DPPC. The presence of hydrophilic polymers PVP, PEG and Dextran restore the DPPC content in the surface film. The effectiveness of the DPPC spreading and formation of better compacted film increases in order Exosurf, Survanta, Curosurf, Alveofact. The obtained results are in accordance with the generally admitted ideas about the mechanisms of serum protein inactivation and restoring effect of hydrophilic polymers based on the previously studies of the lung surfactant adsorption rate.

Savinase proteolysis of insulin Langmuir monolayers studied by surface pressure and surface potential measurements accompanied by atomic force microscopy (AFM) imaging.

The mechanism of the enzymatic action of Savinase on an insulin substrate organized in a monolayer at the air-water interface was studied. We followed two steps experimental approach classical surface pressure and surface potential measurements in combination with atomic force microscopy imaging. Utilizing the barostat surface balance, the hydrolysis kinetic was followed by measuring simultaneously the decrease in the surface area and the change of the surface potential versus time. The decrease in the surface area is a result of the random scission of the peptide bonds of polypeptide chain, progressively appearance of amino acid residues, and their solubilization in the aqueous subphase. The interpretation of the surface potential data was based on the contribution of the dipole moments of the intact and broken peptide groups which remain at the interface during the proteolysis. An appropriate kinetic model for the Savinase action was applied, and the global kinetic constant was obtained. The application of the AFM revealed the state of the insulin monolayers before and after the Savinase action. The comparison of the topography of the films and the roughness analysis showed that insulin Langmuir-Blodgett (LB) films transferred before the enzyme action were flat, while at the end of hydrolysis, roughness of films has increased and the appearance of 3D structures was observed.

Hydrolysis of mixed monomolecular films of tricaprylin/dilauroylphosphatidylcholine by lipase and phospholipase A2.

The purpose of this article was to describe the kinetics of the enzymatic action of one or more enzymes on mixture of substrates organized in 2D structures in order to mimic some situations existing in biological or industrial systems. Hydrolysis of the mixed monomolecular films of tricaprylin/dilauroylphosphatidylcholine (TC8/DiC12PC) by Thermomyces lanuginosus lipase (TLL) and phospholipase A2 (PLA2) was studied by measuring the decrease of the surface area and change of the surface potential at barostatic conditions. The decrease of the surface area detects the transition of the substrate into reaction products and their solubilization while the change of the surface potential detects the contribution of dipole moment of the molecules remaining at the interface during the hydrolysis. The kinetic models, describing the interfacial hydrolysis allowed us to estimate the values of the global kinetic constants for TC8 and DiC12PC hydrolysis, respectively. The role of interaction between all participants of the catalytic act in that complex catalytic system is shown. The catalytic activity of TLL and PLA2 is affected by the molecular environment in TC8/DiC12PC mixed monolayers.

Enzymatic proteolysis of alpha gliadin monolayer spread at the air-water interface.

The mechanism of the enzymatic hydrolysis under the proteolytic enzyme action of a plant protein alpha gliadin organized as a model monolayer system at the air/water interface was studied. The advantage of the monolayer technique is the ability to control and modify easily the interfacial organization of the molecules and the possibility to optimize the conditions for the hydrolysis. Enzymatic hydrolysis was studied by using a traditional barostat surface balance. The hydrolysis kinetic was followed by measuring simultaneously the decrease of the surface area and change of the surface potential with time. The decrease with time in film area is result of the random scission of the peptide bonds of polypeptide chain and their solubilization in the aqueous subphase. The interpretation of the surface potential data is based on the contribution of the dipole moments of the intact and broken peptide groups. An appropriate kinetic model describing the proteolytic action of a peptidase was applied and the global kinetic constant was obtained. The random scission of the protein chains gave kinetic constants comparable with those measured during the hydrolytic scission of polyester macromolecules but quite different to the values obtained with short-chain lipids.

Comparative study of lipolysis by PLA2 of DOPC substrates organized as monolayers, bilayer vesicles and nanocapsules.

The water-soluble lipolytic enzymes act at the interface of insoluble lipid substrates, where the catalytical step is coupled with various interfacial phenomena as enzyme penetration, solubilization of reaction products, loss of mechanical stability of organized assemblies of phospholipids molecule, etc. One biologically relevant example is the enzymatic hydrolysis of DOPC by PLA(2), which results in cleavage of phospholipids molecules into water insoluble lipolytic products, namely oleic acid and lysophospholipid. In general, the enzymatic activity depends on the substrate organization and molecular environment of the catalytic reaction. The lipolysis by phospholipase A(2) of dioleoylphosphatidylcholine substrates organized as monolayer, bilayers vesicles and lipid nanocapsules was studied by measuring the decrease of the surface area at constant surface pressure or increase of the surface pressure at constant area at air-water interface. A kinetic model describing the coupling of the catalytic act with corresponding interfacial phenomena was developed. By using the kinetic model the values for the global hydrolytic kinetic constants were obtained. The obtained value for the monolayer is five orders of magnitude higher than this obtained with small unilamellar vesicles and six orders of magnitude higher then those obtained with lipid nanocapsules. The comparison shows that the enzymatic catalytic act occurring in the lipid environment of the monolayer is more efficacious than at the vesicle and nanocapsules interfaces.

Action of Humicola lanuginosa lipase on mixed monomolecular films of tricaprylin and polyethylene glycol stearate.

The hydrolysis catalyzed by Humicola lanuginosa lipase (HLL) of pure tricaprylin (TC) or stearate of polyethylene glycol 1500 (PEG-St) as well as their mixtures spread as monomolecular films were studied. The catalytic transformation of the two substrates TC or PEG-St into their respective reaction products was detected by measuring simultaneously the decrease in the film area and the surface potential using the "zero order" trough at constant surface pressure. A kinetic model describing the enzymatic hydrolysis was developed. The surface concentrations of the two substrates and their respective reaction products as well as the values of the global kinetic constants of hydrolysis were determined. The experimentally obtained global kinetic constants of the catalytic action of HLL against TC and PEG-St present in mixed monolayers of TC/PEG-St are approximately the same as in the case of pure monolayers. These obtained results give some indications that the activity of enzyme is not significantly affected by the different molecular environments in the mixed monolayers.

Reorganization of lipid nanocapsules at air-water interface: Part 2. Properties of the formed surface film.

The state, electrical and dilatational rheological properties of surface films formed at air-water interface from lipid nanocapsules (LNC) with various compositions as well as model monolayers formed by the LNC constituents-Labrafac, Solutol and Lipoid are investigated. These nanocapsules constitute potential drug delivery systems where lypophilic drug will be loaded in their core. The study of the model Labrafac/Solutol (Lab/Sol) mixed monolayers shows behavior close to the ideal. Small negative deviations in the mean molecular areas a and dipole moments mu are observed. All studied monolayers have elastic behavior during the small continuous compressions. The comparison between the properties of surface films formed from LNC with those of the model monolayers confirms the idea developed in the kinetic study that the surface films formed after a rapid disaggregation of the unstable nanocapsule fraction (LNC I) contains mainly Labrafac and Solutol. The Labrafac molar part (xLab) in the formed Lab/Sol mixed layer is established.

Reorganization of lipid nanocapsules at air-water interface. I. Kinetics of surface film formation.

The size, the electrical properties and the behaviour at air-water interface of lipid nanocapsules (LNC) with various compositions were investigated. Two populations of LNC are presented in the suspension after the preparation: with (LNC II) and without (LNC I) phospholipid molecules. After the spreading at air-water interface, a rapid disaggregation of LNC I, located in the vicinity of interface, occurs leading to formation of surface film. The phospholipid molecules stabilize the structure of nanocapsules and LNC II are more stable at the interface in comparison with LNC I. The formation of a surface film was followed after by measuring the evolution of the surface pressure, relative surface area change and surface potential. A kinetic approach describing the various processes during the surface film formation was proposed. The corresponding kinetic constants were estimated.

Reorganization of lipid nanocapsules at air-water interface 3. Action of hydrolytic enzymes HLL and pancreatic PLA2.

The action of the hydrolytic enzymes humicola lanuginosa lipase (HLL) and pancreatic phospholipase A2 (PLA2) on monolayers formed from lipid nanocapsules (LNC) and model monolayers containing their components, Labrafac, Solutol and Lipoid, is studied by simultaneous measuring the changes in the film area and the surface potential in the "zero order" trough at constant surface pressure (pi). The kinetic models describing the hydrolysis by HLL of the Labrafac, Solutol and their mixtures have been proposed. By using the developed theoretical approach together with the experimental results the surface concentrations of the substrates, hydrolysis products and values of the global kinetic constants were obtained. The comparison between the global kinetic constants in the case of HLL hydrolysis of pure Labrafac, Solutol monolayers and those of the model mixed Labrafac/Solutol monolayers, shows that the rates of hydrolysis are of the same order of magnitude, i.e. an additively of the HLL enzyme action is observed. The composition of the mixed Labrafac/Solutol monolayer, formed after the interfacial LNC destabilization, was estimated.

Interfacial properties of adsorbed films made of a PEG2000 and PLA50 mixture or a copolymer at the dichloromethane-water interface.

Adsorption kinetics of films of poly(ethylene glycol) (PEG2000) studied by the dynamic pendant drop method showed that PEG2000 was more tensioactive at the dichloromethane (DCM)-water interface than at the air-water interface. When initially solubilized into DCM, PEG2000 segments would form an adsorbed layer with hydrophobic segments buried into the polymer chains turned toward the organic phase. Compression of this layer, accompanied by viscoelastic effects, led to expulsion of some hydrophilic tails toward the water phase. When initially dissolved in water, adsorption of PEG2000 segments led to an elastic PEG2000 layer organized on both sides of the interface. Results showed that when the PEG2000-PLA50 (poly(D,L-lactide)) copolymer film was adsorbed at the DCM-water interface, it resulted in a mixed layer exclusively turned toward DCM and its rheological properties were governed by PLA50. When adsorption at the DCM-water interface resulted from a physical mixture of PEG2000 and PLA50, rheological properties of the film were influenced by the initial localization of PEG2000 in the bulk phases. In the case of a mixed film formed by the adsorption of PLA50 from DCM and PEG2000 from water, results showed that PEG2000 segments totally pushed those of PLA50 away from the interface and exclusively influenced the behavior of the mixed film.

Kinetics of liposome disintegration from foam film studies: effect of the lipid bilayer phase state.

The kinetics of interfacial liposome breakdown is investigated in the thin liquid film microinterferometric set up of Scheludko et al. Suspensions of small unilamellar vesicles of dimyristoylphosphatidylcholine are studied at temperatures above and below the temperature of the main gel-liquid crystal first order phase transition. The experimentally established time traces of the velocity of thinning of foam films are used to estimate the kinetic (rate) constants of interfacial liposome disintegration. New and previously established data for other lipids are summarized and compared with results from kinetic measurements of lipid monolayer formation. The thin film experiments confirm the existence of interfacial liposomal aggregates. A change in the kinetic behaviour is observed, due to the 'melting' of the hydrophobic tails in the lipid aggregates. This may have various consequences of biological and pharmacological importance.

Kinetics of calcium-induced fusion of cell-size liposomes with monolayers in solutions of different osmolarity.

The effects of osmolarity, calcium concentration and cell-size liposomes in the subphase on the surface tension of phospholipid monolayers were investigated. The monolayers were spread from chloroform solutions of phosphatidic acid at air/water solution interface. The liposomes (of average diameter 3 micron) were formed from phosphatidic acid/egg lecithin (1:2) mixtures in water or 0.1 M water solutions of sucrose. For this system there were critical concentrations of calcium ions to produce a large reduction of the monolayer surface tension. The threshold calcium concentrations depended upon the sucrose concentration in the subphase. Without sucrose the threshold calcium concentration was 8 mM, while for isoosmotic sucrose solutions (0.1/0.1 M in/out of liposome) it was 14 mM. It sharply increased to 28 mM CaCl2 at sucrose concentration difference across the liposome membrane 0.02 M and decreased to 26 mM, 19 mM, and 18 mM with further increase of that difference to 0.04 M, 0.06 M, and 0.08 M, respectively. The rate of monolayer surface tension decrease was measured as a function of time at 30 mM CaCl2 and different sucrose concentrations in the subphase solution. The initial rates at first decreased with increasing the osmotic pressure and after that they increased. The minimum occurred at sucrose concentration gradient across the liposome membrane 0.02 M, i.e., at the point of maximum threshold calcium concentration required for large decrease of the monolayer surface tension. These facts may be explained by recent theories of dynamics of adhesion, instability and fusion of membranes modeled as thin films.

Surface pressure hysteresis of mixed lipid/protein monolayers: applications to the alveolar dynamics.

Experimental data on surface pressure-surface area hysteresis of mixed serum albumin/dipalmitoyl lecithin/sphingomyelin monolayers in the Langmuir trough are presented. Several possible physicochemical mechanisms of the hysteresis are discussed: Marangoni effect, surface pressure relaxations, bulk-to-surface diffusion interchange, and collapse. Depending on the concrete conditions each of these mechanisms can be important. Possible applications of these results to the alveolar dynamics are presented and discussed on the basis of the balloon model of the alveolus. The main conclusions of biological importance are that 1) the alveolar stability depends on the DPL/SM ratio as well as on the protein content. Under normal breathing conditions the surface pressure hysteresis is small and does not play a decisive role in the alveolar dynamics. 2) At large extent of compression the collapse predominates in determining the hysteretic behavior of the alveolar surface.