Jacques Gueguen

Gliadin nanoparticles for the controlled release of all-trans-retinoic acid

Abstract The general objective of this work was to study the feasibility of preparing small-sized carriers from vegetal macromolecules. For this purpose, gliadin (a vegetal protein fraction from wheat gluten) nanoparticles were chosen as drug carriers for all-trans-retinoic acid (RA). The systems were prepared by a desolvation method for macro-molecules, which enabled us to obtain gliadin nanoparticles of about 500 nm, with a yield close to 90% of the initial protein. All experiments were performed using environmentally acceptable solvents such as ethanol and water. Moreover, due to the low solubility of this protein in water and to its high hydrophobicity, nanoparticles from gliadin do not need any further chemical or physical treatment to harden them. Gliadin nanoparticles were quite stable over 4 days in phosphate-buffered saline (PBS), but were degraded rapidly over 3 h when incubated in PBS solution containing trypsin. However, chemical cross-linkage of nanoparticles with glutaraldehyde significantly increased their stability. Under our experimental conditions, the payload limit was 76.4 μg RA/mg nanoparticles (for an RA/initial protein ratio of 90 μg/mg), which corresponded to a RA entrapment efficiency of about 75% of added drug. Nevertheless, the entrapment efficiency was high (between 97 and 85%) for RA/initial protein ratios up to 90 μg/mg. Finally, the in vitro release profiles of RA-loaded gliadin nanoparticles showed a biphasic pattern. An initial burst effect (in which about 20% RA was released) followed by zero-order diffusion (release rate 0.065 mg RA/h) were observed.

Molecular basis of film formation from a soybean protein: comparison between the conformation of glycinin in aqueous solution and in films

Fourier transform infrared spectroscopy has been used to investigate the conformational changes of glycinin. a major storage protein of soybean seeds, upon film-forming. The results show that the secondary structure of glycinin is mainly composed of a beta-sheet (48%) and unordered (49%) structures. The amide I band of glycinin in film-forming conditions, i.e. in alkaline media and in the presence of plasticizing agent, reveals the conversion of 18% of the secondary structure of the protein from the beta-sheet (6%) and random coil (12%) to the alpha-helical conformation due to the helicogenic effect of the ethylene glycol used as the plasticizing agent. Conformational changes also occur upon the film-forming process leading to the formation of intermolecular hydrogen-bonded beta-sheet structures. Results obtained from other plant families indicate that, whatever the origin and conformation of protein, formation of films leads to the appearance of intermolecular hydrogen-bonded beta-sheet structures, suggesting that this type of structure might be essential for the network formation in films. Thus, it is hypothesized that, in the film state, intermolecular hydrogen bonding between segments of beta-sheet may act as junction zones in the film network. This study reveals for the first time that there is a close relationship between the conformation of proteins and the mechanical properties of films.

Influence of plasticizers and treatments on the properties of films from pea proteins

Abstract Films were prepared, by a casting technique, from an industrial pea protein isolate, formulated in an alkaline medium in the presence of a plasticizer. The aim of the present work was to improve the hydrophobic character and mechanical properties of the film, mainly the tensile strength. The influence of plasticizer type on these film properties (mechanical behaviour, hydrophobicity, water permeability), and on the protein structure was investigated. Using glycols as plasticizers, the increasing of the chain length failed to improve the characteristics of the films. The comparison of infrared spectra of protein (11S glycinin) in aqueous solution and in film state, showed that conformational changes which accompanied the formation of films were significant. The amide I band of the protein sample in film state reveals that, in addition to β -turn and random coil structures, the protein presents a large amount of intermolecular β -sheet, probably due to polypeptide chains interactions. Besides, the significance of physical and chemical treatments was studied, either on film-forming solutions or on the protein film itself. The use of hydroxyethylacrylate as plasticizer, coupled to an ultra violet treatment, was also investigated and was found to correlate well with increased tensile strength and hydrophobicity. Formaldehyde added in the film-forming solution did not alter significantly the film behaviour, whereas treatment by immersion of the protein films in an ethanol–formaldehyde mixture markedly increased both their tensile strength and hydrophobic character.

Legume seed protein extraction, processing, and end product characteristics

Because of the difficulties in growing soybean in many parts of the world, other leguminosae crops (fababean, pea, lentil, lupine, bean chickpea, cow pea, etc.) are now being studied as new protein sources. They generally have a high protein content and a satisfactory amino acid composition. The studies which have led to the development of industrial flow sheets for protein extraction have mainly been carried out on pea (Pisum sativum), fababean (Vicia faba), and lupine (Lupinus albus). The processes generally used are (1) pin-milling plus air-classification which when applied to starch-rich legume seeds (pea, fababean), results in concentrates (defined as having protein contents of 60–75%), and (2) wet processes which produce isolates (defined as having protein contents of 90% to 95%). By air-classification, concentrates having 68% and 65% protein can be obtained, respectively, from fababean (31% protein) and pea (21% protein). Isolates, prepared by extraction of the flour proteins with alkaline solution followed by acid precipitation, have a protein content (N × 6.25) generally between 90% and 96% and a protein recovery yield varying between 60% and 65%. As a rule, isolates resulting from ultrafiltered extracts have a higher protein content. From the nutritional evaluation of these two types of products, concentrates and isolates, it appears that wet processes are more efficient for eliminating antinutritional factors. α-galactosides and glycosides are present in isolates only in traces. As for trypsin inhibitors and haemagglutinins, only one third of the activities in the flour remained in the isolates (in dry processes, the residual levels of these antinutritional factors were higher). From the studies of their functional properties, it appears that isolates and concentrates from sources such as fababean and peas, produced by the new processes described, are to some extent complementary or equivalent in their functional properties to those of the soybean, particularly for emulsifying and foaming purposes. These observations should encourage the development of these new processes.

Biopolymeric colloidal carriers for encapsulation or controlled release applications

Biopolymers represent an interesting alternative to synthetic polymers in order to be used as structured carriers for controlled release and encapsulation applications. In particular, the ability of these carriers to entrap both hydrophilic and hydrophobic drugs may be very promising for many applications. In addition, the absence of chemical compounds and organic solvents used to produce biopolymeric matrices could be very interesting for some industrial applications. Simple or complex coacervation methods involving proteins or protein and polysaccharide mixtures were used to create new matrices dedicated to controlled release applications. Controlled release experiments with model compounds were conducted in order to evaluate the performance of such matrices. An alternative and promising research field deals with particles obtained from hydrogel systems. Totally transparent solid matrices resulting from the dehydration of new protein gels were formed and swelling capacities of these matrices were studied.

Effect of dissociation and conformational changes on the surface behavior of pea legumin

Abstract In order to improve knowledge of the influence of dissociation of pea legumin on the surface behavior, the influence of pH and the effect of succinylation on the kinetics of adsorption have been studied at the air/water interface. Ultracentrifugation and the Wilhemy plate method were used, respectively, for the characterization of the protein and the interfacial tension measurements. Dissociation results in an improvement in the ability of the protein to diffuse to the interface whereas the charge of the molecule influences the kinetic parameters of anchorage and conformational rearrangement in the occupied interfacial layer. These effects are discussed in relation first to the dissociation of the oligomeric 12S globulin into subunits, then to the unfolding of these subunits, and finally to the charge variations and location along the primary sequence of legumin.

Optimization and in vitro stability of legumin nanoparticles obtained by a coacervation method

Legumin (a storage protein from Pisum sativum L.) nanoparticles of about 250 nm were prepared by means of a pH-coacervation method and chemical cross-linking with glutaraldehyde. Non-stabilized nanoparticles or coacervates were obtained by mixing an aqueous solution of legumin with a buffer. The influence of some experimental parameters (pH, surfactant content and ionic strength) on the size and yield of coacervates was studied. These systems were then treated with glutaraldehyde. After 2 h, a concentration of at least 0.05 mg glutaraldehyde/mg legumin was necessary to stabilize these colloidal systems. No significant differences in size and percentage of yield were obtained between legumin nanoparticles cross-linked with different glutaraldehyde concentrations. Legumin nanoparticles were quite stable in pH conditions close to neutrality. On the other hand, nanoparticles stored under acidic conditions (pH 5.5; 37°C) showed a rapid degradation and this fact may be of interest for pharmaceutical applications like cutaneous or transdermal administration of drugs.

Preparation of lectin-vicilin nanoparticle conjugates using the carbodiimide coupling technique

Abstract This work describes the preparation and the in vitro activity of conjugates formed by Ulex europaeus lectin (UE lectin) and vicilin (storage protein from Pisum sativum ) nanoparticles. The lectin was fixed by coupling its amino groups to carbodiimide-activated carboxylic groups on the vicilin nanoparticles. The influence of the carbodiimide concentration and the reaction time was studied. Typically, the amount of bound lectin was calculated to be about 30 μg lectin/mg nanoparticles, which corresponded to a lectin coupling efficiency of about 21%. Furthermore, the activity and the specificity of these conjugates was tested with bovine submaxillary gland mucin (BSM). When the experiences were carried out in the absence of l -fucose (specific sugar for UE lectin), the UE lectin-vicilin nanoparticle conjugates showed three times more interaction with mucin than the control (vicilin nanoparticles). Moreover, the specificity of the lectin was maintained after coupling to vicilin nanoparticles because the presence of l -fucose inhibited the interactions between conjugates and mucin.

Conformational changes upon dissociation of a globular protein from pea : a Fourier transform infrared spectroscopy study

Fourier transform infrared spectroscopy shows that the secondary structure of legumin, a globular protein from pea seeds, is composed of 41% beta-sheets and 16% alpha-helices and furthermore reveals the presence of beta-turns. The conformation prediction from the analysis of the amino-acid sequence of legumin using hydrophobic cluster analysis reveals that the C-terminal part of the alpha-polypeptide is devoid of defined secondary structures, whereas the beta-polypeptide is highly ordered. Comparison with analogous 11S globulins from other plant families indicates that ordered domains are highly preserved, phenomenon that may be associated with the similarity of the quaternary structure of these proteins. The results also reveal the presence of a large hypervariable region, located at the surface of the protein, that could be at the origin of the different functional properties of the 11S type globulins. The step-by-step destruction of the quaternary oligomeric structure of the native protein is accompanied by conformational changes that depend on the dissociation conditions. Whereas acylation leads to a decrease of the alpha-helix content by 10% at the expense of the beta-sheet content, addition of sodium perchlorate results in the conversion of 10% of the protein secondary structure from beta-sheet to unordered. These observations provide further evidence of the existence of different monomeric states that differ from their secondary structure and, therefore, exhibit different surface-active properties.

Structural characteristics of a globular protein investigated by X-ray photoelectron spectroscopy: comparison between a legumin film and a powdered legumin

Films of legumin, a pea protein, were deposited onto a glass support using the Langmuir-Blodgett method, at various surface pressures. XPS study of these films show that their thickness increases with the deposition pressure. At the pressure limits of films stability, the thickness values (respectively 73 and 110 A) are close to the protein dimensions. Layered at low pressure, the oblate protein stands up when pressure increases. Furthermore, XPS study shows that the orientation of the external flexible loops depends on the obtention conditions. Thus, in the case of Langmuir-Blodgett films, hydrophobic residues are turned towards the external surface, and the hydrophilic ones towards the glass substrate. But, in the opposite, when protein is obtained by lyophilization, the hydrophilic residues are orientated outsides. It seems possible to determine by XPS the nature of the residues which give to the protein its reactivity, since they are located at its external surface.