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.

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. (C) 2008 Elsevier B.V. All rights reserved.

This paper compares six phospholipidic monolayers at the water/chloroform interface by performing dilational rheological measurements with a drop tensiometer apparatus. The chosen lipids differ both in their headgroup structure and fatty acyl chain saturation or symmetry. The study concentrated on monolayers formed with DPPC, DPPE, DOPC, DOPE, POPC and POPE. Using a generalized Maxwell rheological model, transposed at the interface, the intimate intermolecular interactions between amphiphilic molecules are studied on and off the monolayer plane. The equilibrium and nonequilibrium phenomena are analyzed and, respectively, correlated with monolayer cohesion and with monolayer/sub-surface interactions. The purpose of this work is to gain further insights into the influences (as slight as they are) of the weak changes in phospholipid structure and on the behavior of the monolayers. The results, widely described, provide further details on nuances existing between very similar molecules, and likewise, on the synergies created between the different effects.

In this paper, we compared the efficiency of polymer films, made of a poly(ethylene glycol) (PEG2,000)/poly(d,l-lactide) (PLA50) mixture, or a PEG2,000-PLA50 copolymer, to prevent adsorption of a model protein, the hen egg-white lysozyme (HEWL), at the air–water interface. This was achieved by analyzing the surface pressure/surface area curves, and the X-ray reflectivity data of the polymer films spread on a Langmuir trough, obtained in absence or in presence of the protein. For both the mixture and the copolymer, the amount of protein adsorbed at the air–water interface decreases when the density of the polymer surface coverage increases. It was shown that even in a condensed state, the polymer film made by the mixture can not totally prevent HEWL molecules to adsorb and penetrate the polymer mixed film, but however, protein molecules would not be directly exposed to the more hydrophobic phase, i.e. the air phase. It was also shown that the configuration adopted by the copolymer at the interface in its condensed state would prevent adsorption of HEWL molecules for several hours; this would be due in particular to the presence of PEG segments in the interfacial film.

Bicompartmental structures, named “hand-bag like structures” (HBS), where phospholipid membranes stabilise both an oily and a water phase, were firstly observed in cationic emulsions thanks to cryomicroscopy. The emulsion formulation was found to play a crucial role: no HBS were observed neither in the absence of cationic lipid nor in the presence of DOTAP, while oleylamine and DSPE-PEG were found to increase the proportion of HBS compared to stearylamine. The HLB value and the unsaturation of the lipids acyl chains then appeared as some key parameters for HBS formation. Interfacial rheology measurements have given additional information on the interfacial film such as its cohesion (Ee), interactions with the adjacent phases (Ene) and relaxation time τ which was found to be the most discriminating parameter.τ was indeed significantly longer for the samples containing no HBS. This suggests that the ability of the interface to rearrange could play a role in HBS formation.

The dilatational properties, structure, and morphology of the surface films spread at the air–water interface from complex lipid/protein systems were studied by measuring the surface pressure–area and surface potential–area isotherms, the surface rheological properties, and AFM images. The commercially available lung surfactants Alveofact, Curosurf, Survanta, and Exosurf were used as examples.

In the present paper, different spectroscopic methods were applied to evaluate conformational changes of hen egg-white lysozyme (HEWL) in various solvents and in the presence of poly(ethylene glycol) (PEG). In citrate (0.007M, pH=6), or in Tris (0.1M, pH=7.4), no conformational change of the protein was measured across the range of concentrations tested. In addition, HEWL in ultra-pure water revealed no irreversible conformational change and no activity loss, at least at low concentrations (< or =0.2mg/ml). Whereas PEG can induce a reorganization of water molecules, no change of the secondary and tertiary protein conformations was observed in the presence of PEG. In addition, in the presence of PEG of various molecular weights, no change of enzymatic activity of the HEWL was observed across the range of concentrations tested.

In order to obtain information on the structure and the stability of lipid nanoparticles, interfacial films, made from their spreading, were studied at the air/water interface. The related results were compared with the spreading of their individual components at the same interface. The interfacial behaviour of the particles indicated a nanocapsule structure with an oily core surrounded by a surfactant layer. Unlike liposomes or high density lipoproteins (HDL), no destruction leading to the release of their core was observed during their spreading and compression. Moreover, the sampling of the nanoparticle films at the air/water interface deposited on mica plate depicted by atomic force microscopy (AFM) showed entire nanocapsules. This stability is attributed to their surface cohesion related to their surfactant shell. Indeed, a partial particle surface “erosion” occurred leading to the spread of a certain amount of less attached hydrophilic surfactant molecules without releasing the nanocapsule lipid content. This study illustrates that the Langmuir balance can be used as a tool for studying the general organisation of lipid particles. Moreover, this method may allow the determination of the release profile of some encapsulated drugs.

The mechanism of the hydrolysis by cutinase and the progressive fragmentation of lactic chains in diblock copolymers of PLA with various sizes attached to PEG were studied in a 2D monolayers model system. The hydrolysis kinetics was followed by measuring simultaneously the decrease of the surface area and evolution of the surface potential with time at barostatic conditions. The decrease of the surface area is due to the solubilization of the copolymers as well as of their hydrolytic products: detached PEG blocks and small soluble PLA fragments. The evolution of the surface potential detects the transient interfacial accumulation of charged insoluble PLA fragments. A kinetic model describing the enzymatic hydrolysis was developed and the values for the global hydrolytic kinetic constant were obtained without any fitting parameter. It was found that the global kinetic constant no practically depend on the length of the lactic and the presence of polyethylene–glycol chains.

A formulation process, based on the inversion phase of an emulsion, was used to prepare lipid nanocapsules. Triglycerides, lecithin, salted water and hydroxy stearate of poly(ethylene glycol) were used in the preparation. The amounts of each that allowed nanocapsules to be formed described a feasibility domain within a ternary diagram. The size distribution of various nanoparticulate carriers has already been shown to influence their applications. An experimental mixture design inside the feasibility domain has been used in order to approximate, through an empirical model, the influence of the quantitative composition of nanocapsules on their size distribution. Reduced cubic polynomial equations successfully modelled the evolution of responses in terms of particle average diameters and coefficients of variation. The results were presented using an analysis of response surface showing a scale of possible particle sizes between 20 and 95 nm and a coefficient of variation between 11 and 40%. Furthermore, this technique showed that the proportion of hydrophilic surfactant had a major influence on the average diameter and the size distribution of the particles decreasing when its proportion increased. On the contrary, the coefficient of variation and the average diameter slightly increase with the proportion of triglycerides. Such a tool offers major advantages to design the formulation of particles as a function of the required size distribution.

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.

The dilational rheological properties of interfacial films of poly(ethylene glycol) (PEG2000) and hen egg-white lysozyme (HEWL) were studied respectively at the dichloromethane (DCM)-water and air-water interfaces by means of the pendant drop method. In both cases, the observed interfacial behaviors were approached by a model corresponding to a two-dimensional viscoelastic solid. The interfacial layers were characterized by three physical constants: E-e, the equilibrium elasticity, E-ne, the nonequilibrium elasticity, and tau, the relaxation time. Because the interfacial dilational properties of the films were studied by using a ramp type perturbation approach or a sinusoidal variations approach, identical rheological physical constants values were obtained for PEG2000 and HEWL. From these studies, the interactions within the interfacial layer and those between the interfacial film and adjacent phases can be indirectly accessed and estimated.

This study proposed a method to understand the surfactant role in the first step of the formulation of a novel generation of lipidic nanocapsules. A dynamic rheological protocol was applied using a pendant drop tensiometer in order to determine the interfacial properties of the initial mixture implied in the first formulation step. The response, in terms of interfacial elasticities, described how this mixture led to monodisperse nanometer size range structures after a physico-chemical constraint.

According to our results concerning the behavior of lysozyme at interfaces, its secondary structure and its enzymatic activity, successful protein encapsulation would need to maintain a pH value far from the enzyme isoelectric point value during the formulation to reduce, in particular, the adsorption of lysozyme molecules at the created interfaces. Moreover, buffers or salt solution must be used in order to keep intact the native secondary conformation of lysozyme, and preserve its enzymatic activity.

Amiodarone is a drug that is widely used in the treatment of heart disease. To circumvent side effects, colloidal drug carriers have been designed to deliver the drug specifically to the site of action. For the purposes of in vitro characterization of such particles, difficult test systems are employed that usually require the quantitative separation of the drug carrier from the release medium before analysis. In this work, a Langmuir balance was used to characterize amiodarone release. Drug-loaded nanoparticles were prepared from a biodegradable polyester and assayed for their drug release kinetics. Simultaneously, nanoparticles were analyzed for their drug release by a standard procedure based on dialysis tubes combined with high-performance, liquid chromatography. The results obtained by the Langmuir balance experiments were compared with those obtained from high-performance liquid chromatography and were found to correlate well. The interexperimental variation was 4.4% for the Langmuir method (n = 4), and the interexperimental variation for HPLC was 2.9% (n = 3). The major advantage of this new method is the possibility diminishing significantly the required sample amount for the experiment, allowing drug detection in the lower nanomolar range. Moreover, the avoidance of prior nanoparticle separation from the release medium provides important progress of this technique. The Langmuir balance has proven its adaptability as a new sensitive tool for the characterization of amphiphilic drug release kinetics.

This paper describes the preparation of colloidal lipid particles containing magnetite from warm emulsions. A two step method was used to obtain the nanoparticles: (i) formulation of a transparent phase by heating a O/W emulsion (aqueous surfactant solution melted with a lipid phase, containing the ethyl oleate and soybean lecithin) in which modified lipophilic magnetite is incorporated, and (ii) preparation of the nanoparticles by dispersing the warm transparent phase in cold water (7°C) under mechanical stirring. The latter method gives spherical nanoparticles of a mean size of 62 nm measured by Photon Correlation Spectroscopy and Transmission Electronic Microscopy. The magnetite entrapment efficiency was determined by use of a magnetophoretic sedimentation method.

The role of the polyethylene glycol (PEG) on the mechanisms of hydrolysis of poly(d,l-lactide-co-glycolide) (PLAGA) in mixed monolayers at alkaline pH and under the enzymatic action of cutinase was studied by use of a Langmuir balance. The obtained decreases in the surface area at constant surface pressure are interpreted as a result of solubilization of PEG and the progressively obtained small soluble fragments of PLAGA during the interfacial hydrolysis. In the framework of the random scission mode of fragmentation, the values for the degree of completion of the hydrolysis and specific activity of enzyme were obtained numerically. A small surface dilution effect of the PEG in respect to the degradable polyester molecules PLAGA in their mixed monolayers was found.

The dilational properties of monolayers are analyzed using the classical linear approximation. In most cases, the observed interfacial behavior can be approached by a model corresponding to a two-dimensional viscoelastic solid. The monolayer is characterized by two dilational elasticity terms (E-e, equilibrium elasticity, and E-ne, nonequilibrium elasticity) and by one relaxation time (tau). These three physical constants are obtained from the responses of a ramp type perturbation, or from the responses (as a function of the frequencies) after sinusoidal area variations. Using axisymmetric drop shape analysis experiments. a dipalmitoyl phosphatidylcholine (DPPC) layer at the dichloromethane/water interface is characterized. Measurements of the surface pressure variations as the response to linear or sinusoidal variations of surface area are performed. Identical rheological physical constants (equilibrium elasticity, nonequilibrium elasticity, and relaxation time) are obtained using both methods. Dilational behavior of DPPC monolayer can be attributed to the molecular diffusion between the DPPC layer and the adjacent phases.

Amiodarone, a drug used in heart therapy, is poorly soluble in water at room temperature, but forms transparent phases much more concentrated than the critical micellar concentration (CMC), when crystals are heated (above 60 degrees C) in presence of water and cooled down to room temperature. These pseudosolutions were supposed to be made of a complex system of micelles. In order to better understand the effects of pH and ion species on the supramolecular organization of amiodarone, interfacial pressure measurements were performed at the air/water interface on a Langmuir trough. Monolayers spread from chloroformic solutions over non bufferered subphases were insoluble at basic pH (NaOH, pH 10) but soluble at acidic pH (HCl, pH 4). However, a higher ionic strength obtained by adding NaCl (0.15 N) or NaH(2)PO(4) (0.15 N) to the subphase stopped the amiodarone solubilization. On an acidic phosphate subphase (NaH(2)PO(4), pH 4.4, 0.15 N), abnormally high surface pressures (>1 mN/m) were measured for high molecular areas (80-200 Å(2)/molecule) suggesting a supramolecular organization of the surface film. Insoluble monolayers were also obtained when the amiodarone supramolecular pseudosolution was spread on neutral (NaH(2)PO(4), pH 6.25, 0.15 N) or acidic (NaH(2)PO(4), pH 4.4, 0.15 N) subphases. However, a great instability on basic subphase (phosphate buffer pH 8.8) indicated the breakage of the supramolecular structure during spreading. These results are discussed taking into account the amiodarone state of ionization and the electrostatic interactions with counterions. Combining the use of phosphate counterions and that of acidic pH opens new perspectives in the optimization of amiodarone intravenous formulations.

In this paper, we compared the interfacial behaviors of spread films of poly(ethylene glycol) (PEG2000), poly(D,L-lactide) (PLA50), and a mixture of PEG2000 and PLA50, to understand the properties of a PEG2000-PLA50 diblock copolymer at the air/water interface. This was achieved (i) by analyzing the surface pressure-surface area curves obtained on a Langmuir trough and (ii) by modeling the dilatational properties of the films according to a modified Maxwell model. The properties of the films composed of a mixture of PEG2000 and PLA50 showed the influence of PEG2000 before the nucleation transition of PLA50. Then, increasing the lateral compression of the mixed monolayer led to the expulsion of PEG2000 segments with presumably their irreversible desorption into the bulk. In the case of the copolymer, the covalently bound PEG2000 segments were segregated at low surface coverage forming a stable film with the PEG corona oriented toward the water phase. This situation was conserved until high surface coverage (10-15 mN/m). Afterward, compression led to the penetration of PEG2000 into the tridimensional layer oriented toward the air/phase. These results allow better understanding and modulation of the composition and the hydrophilic character of interfaces formed during emulsion processes and thus improvement of the control of the surface properties of drug delivery systems.