Ali Assifaoui

Zinc-pectinate beads as an in vivo self-assembling system for pulsatile drug delivery

Zinc-pectinate beads are interesting drug carriers for oral delivery. In order to investigate their in vitro and in vivo release behaviour, ionotropic gelation was used to entrap theophylline into calcium- or zinc-pectinate beads. Beads were investigated in vitro for their particle properties, especially the release kinetic in different media, and their in vivo pharmacokinetic parameters were tested in rats. Particle size varied between 1.8 and 2.8mm and encapsulation rates between 27 and 30% for Ca- and Zn-pectinate beads, respectively. While Ca-pectinate beads revealed a relative fast disintegration, drug release profiles from Zn-pectinate beads were very much release medium-dependent. Especially, in the presence of phosphate ions, the release from Zn-pectinate beads was blocked at 20% and 40% of the total drug load when tested in phosphate buffer or simulated colonic medium. In vivo Zn-pectinate beads (t(max): 12.0 ± 0.1h) led to a significant lag time for the theophylline absorption compared to Ca-pectinate (t(max): 6.0 ± 2.8h) or free theophylline (t(max): 2.5 ± 2.1h). This delayed release was attributed to the formation of a zinc phosphate coating in vitro and in vivo inducing the retention of theophylline release. Zn-pectinate beads exhibit interesting properties due to its potential as pulsatile delivery system induced by the in situ formation of Zn phosphate, while Ca-pectinate was found to be of limited suitability for controlled release of theophylline.

Thermal Denaturation of Pea Globulins (Pisum sativum L.)—Molecular Interactions Leading to Heat-Induced Protein Aggregation

The heat-induced denaturation and aggregation of mixed pea globulins (8%, w/w) were investigated using differential scanning calorimetry (DSC), SDS-PAGE, and size-exclusion chromatography (SEC-HPLC). DSC data showed that the pea proteins denaturation temperature (T(d)) was heating-rate dependent. The T(d) value decreased by about 4 °C by lowering the heating rate from 10 to 5 °C/min. The SDS-PAGE analysis revealed that protein denaturation upon heating at 90 °C was mainly governed by noncovalent interaction. The SEC-HPLC measurements indicated that low-denatured legumin (≈350-410 kDa) and vicilin/convicilin (≈170 kDa) globulins were heat-denatured and most of their subunits reassociated into high-molecular weight, soluble aggregates (>700 kDa). The addition of N-ethylmaleimide slightly modified the aggregation route of pea globulins. However, partially insoluble macroaggregates were produced in the presence of dithiothreitol, reflecting the stabilizing effect of disulfide bonds within legumin subunits.

Structure of calcium and zinc pectinate films investigated by FTIR spectroscopy.

Calcium and zinc pectinate gels were prepared using a method which allowed calcium or zinc to diffuse from the cross-linking solution through a dialysis membrane to form a gel with amidated low-methoxyl pectin. The gel thus obtained was then dried, and the film structure was studied using FTIR spectroscopy as a function of the cation content (0%, 5%, 10%, and 15% w/v). Important consideration was given to the three functional groups (amide, carboxyl ester, and carboxylate groups) present in the pectin. When the zinc content was increased, the three wavenumber values corresponding to these three functional groups did not change significantly, while for calcium pectinate, the three wavenumber values were shifted profoundly when the amount of calcium ions changed. These results confirm that calcium ions could form stable interactions with carboxylate groups as described by the eggbox model [Grant, G.T.; Morris, E.R.; Rees, D.A.; Smith, P.J.C.; Tho, D. FEBS Lett.1973, 32, 195-198] while the lower coordination number of zinc does not permit a structured gel to develop.

Silica-coated calcium pectinate beads for colonic drug delivery

The aim of this work is to develop novel organic-inorganic hybrid beads for colonic drug delivery. For this purpose, calcium pectinate beads with theophylline are prepared by a cross-linking reaction between amidated low-methoxyl pectin and calcium ions. The beads are then covered with silica, starting from tetraethyoxysilane (TEOS), by a sol-gel process. The influence of TEOS concentration (0.25, 0.50, 0.75 and 1.00 M) during the process is studied in order to modulate the thickness of the silica layer around the pectinate beads and thus to control the drug release. The interactions between the silica coating and the organic beads are weak according to the physicochemical characterizations. A good correlation between physicochemical and in-vitro dissolution tests is observed. At concentrations of TEOS beyond 0.25 M, the silica layer is thick enough to act as a barrier to water uptake and to reduce the swelling ratio of the beads. The drug release is also delayed. Silica-coated pectinate beads are promising candidates for sustained drug delivery systems.

Influence of low methoxyl pectin gel textures and in vitro release of rutin from calcium pectinate beads

This study described the preparation and characterization of low methoxyl pectin (LMP) gels and beads for controlled release applications. The rheological characterization of the various formulations was proposed. Then the mechanical and morphological characterizations of beads were determined. Finally, the controlled release studies taking rutin as a model drug was evaluated. The results showed that Youngs modulus values of non-amidated LMP gels decrease when adding up to 15% sorbitol. Calcium pectinate beads loaded with rutin are about 600 μm, oblong shaped with dense matrix. Beads containing sodium bicarbonate showed about 80% lower rutin encapsulation efficiency by increasing the pH of the cross-linking solution. The rutin loaded in non-amidated pectinate beads containing 15% sorbitol showed the best release efficiency and swelling behavior. Therefore, the gel texture affects the release rate of the active compounds encapsulated in calcium pectinate beads and can be used as a parameter to modulate drug release.

Binding of Divalent Cations to Polygalacturonate: A Mechanism Driven by the Hydration Water.

We have investigated the interactions between polygalacturonate (polyGal) and four divalent cations (M(2+) = Ba(2+), Ca(2+), Mg(2+), Zn(2+)) that differ in size and affinity for water. Our results evidence that M(2+)-polyGal interactions are intimately linked to the affinity of M(2+) for water. Mg(2+) interacts so strongly with water that it remains weakly bound to polyGal (polycondensation) by sharing water molecules from its first coordination shell with the carboxylate groups of polyGal. In contrast, the other cations form transient ionic pairs with polyGal by releasing preferentially one water molecule (for Zn(2+)) or two (for Ca(2+) and Ba(2+)), which corresponds to monodentate and bidentate binding modes with carboxylates, respectively. The mechanism for the binding of these three divalent cations to polyGal can be described by two steps: (i) monocomplexation and formation of point-like cross-links between polyGal chains (at low M(2+)/Gal molar ratios, R) and (ii) dimerization (at higher R). The threshold molar ratio, R*, between these two steps depends on the nature of divalent cations and is lower for calcium ions (R* < 0.1) than for zinc and barium ions (R* > 0.3). This difference may be explained by the intermediate affinity of Ca(2+) for water with respect to those of Zn(2+) and Ba(2+), which may induce the formation of cross-links of intermediate flexibility. By comparison, the lower and higher flexibilities of the cross-links formed by Zn(2+) and Ba(2+), respectively, may shift the formation of dimers to higher molar ratios (R*).

Structural studies of adsorbed protein (betalactoglobulin) on natural clay (montmorillonite)

In this work, the adsorption of a small globular protein (betalactoglobulin, BLG), on a natural montmorillonite clay (Mt) was investigated in acidic buffer (pH = 3). The combination of different characterization techniques such as zetametry, X-ray diffraction, transmission electronic microscopy, fluorescence and solid state nuclear magnetic resonance spectroscopies shed light on the interaction mechanism between the clay mineral and the proteins. For low BLG concentration, a slight increase of the interlayer spacing of the clay mineral was noticed as well as structural changes of the protein. In contrast, as the concentration of BLG increased, the adsorption led to a partial exfoliation of the clay mineral, accompanied with significant secondary structural changes of the protein characterized by a loss of β-sheet organization. Altogether, our results revealed an unexpected adsorption scheme where the increase of the BLG/Mt weight ratio of the hybrid material leads to a partial exfoliation of the Mt, but at the expense of the protein native structure.

Design of polygalacturonate hydrogels using iron(II) as cross-linkers: A promising route to protect bioavailable iron against oxidation

We designed stable and highly reproducible hydrogels by external unidirectional diffusion of Fe2+ ions into aqueous solutions of polygalacturonate (polyGal) chains. The Fe2+ ions act as cross-linkers between the Gal units in such a way that both the molar ratio R ([Fe2+]/[Gal units] = 0.25) and the mesh size of the polyGal network at the local scale (ξ = 75 ± 5 Å) have constant values within the whole gel, as respectively determined by titration and Small Angle Neutron Scattering. From macroscopic point of view, there is a progressive decrease of polyGal concentration from the part of the gel formed in the early stages of the gelation process, which is homogeneous, transparent and whose Young modulus has a high value of ∼105 Pa, up to the part of the gel formed in the late stages, which is heterogeneous, highly turbid and has a much lower Young modulus of ∼103 Pa. Since the local organization of the polyGal chains remains identical all along the hydrogels, this macroscopic concentration gradient originates from the formation of heterogeneities at a mesoscopic length scale during the gelation process. In addition, X-ray Absorption Spectroscopy measurements remarkably reveal that Fe2+ ions keep their +II oxidation state in the whole gels once they have cross-linked the Gal units. These polyGal hydrogels thus protect iron against oxidation and could be used for iron fortification.

Specific binding of trivalent metal ions to λ-carrageenan

Carrageenans are a family of sulphated cell wall polysaccharides extracted from seaweeds and are widely used in different industrial sectors. Relative to κ-carrageenan (κ-car) and ι-carrageenan (ι-car), the ionic binding behavior of λ-carrageenan (λ-car) is far less studied. In this work, the interaction and binding behavior between λ-car and metal ions of different valency (Na+, K+, Mg2+, Ca2+, Fe2+, Fe3+, Al3+, Cr3+) have been investigated. In contrast to the non-specific interaction of the monovalent and divalent cations, specific binding has been identified between λ-car and Fe3+/Al3+. The specific binding could lead to either precipitation or gelation of λ-car, depending on the way of introducing Fe3+/Al3+ ions. Fe3+ and Al3+ exhibit the same binding stoichiometry of [M3+]/[repeating unit] = 1.0, with the former having a relatively larger binding constant. Cr3+, though having very similar physical properties with Fe3+/Al3+, is incapable of binding specifically to Cr3+. The phenomena could not be interpreted in terms of counterion condensation, and are rather attributable to a mechanism in which hexa-coordination of Fe3+/Al3+ and entropy-driven cation dehydration play crucial roles in driving the binding of the trivalent metal ions to λ-car.

Insights into gelation kinetics and gel front migration in cation-induced polysaccharide hydrogels by viscoelastic and turbidity measurements: Effect of the nature of divalent cations

Polysaccharide-based hydrogels were prepared by the diffusion of various divalent cations (X2+) into the polygalacturonate (polyGal) solution through a dialysis membrane. The diffusion of various divalent cations (Mg2+, Ca2+, Zn2+ and Ba2+) was investigated. The polyGal gel growth was studied as a function of the initial cation concentration by both viscoelastic and turbidity measurements. We have demonstrated for the first time that the determination of the spatiotemporal variation of turbidity during the gelation process allowed to study the gel front migration. For Ca-polyGal, Zn-polyGal and Ba-polyGal, the gel front migration was characterized by the presence of a peak at the sol/gel interface. This peak was not observed in the case of Mg-polyGal where the gel was not formed. The apparent diffusion coefficient of the gel front (Dapp) which was calculated from the evolution of this peak increased when the initial cation concentration was increased. Moreover, we have suggested a gelation mechanism based on the presence of a threshold molar ratio R* (=[X2+]/[Galacturonic unit]) in which some point-like crosslinks are precursors of the formation of dimers and multimers inducing the contraction of the gel and thus the formation of the gel front.