Lanreotide self-assembly - role of salts and counterions

L’acétate de Lanréotide est un oligopeptide thérapeutique, analogue de la somatostatine. Ce peptide di-cationique, au-dessus d’une concentration critique d’assemblage (CAC), s’auto-assemble spontanément dans l’eau en formant des nanotubes supramoléculaires très bien ordonnés. Ces architectures spontanées sont directement utilisées dans la formulation à libération prolongée de ce peptide, la somatuline Autogel®. La compréhension des mécanismes d’assemblage ainsi que de l’influence des milieux sur ces assemblages peptidiques sont importants pour comprendre l’évolution au cours du temps de cette formulation après injection sous la peau. Les interactions électrostatiques entre peptides et contre-ions et les répulsions électrostatiques entre peptides jouent des rôles potentiellement importants (stabilisants ou déstabilisants) sur les assemblages finaux. Un premier objectif de ce travail était donc de comprendre comment la nature des sels (cations mono-, di- & multivalents, anions mono- & divalents ou encore des acides gras de longueurs de chaîne croissantes) influençait l’auto-assemblage du Lanréotide. Les cations mono, di- ou multivalents et les anions monovalents diminuent la CAC du Lanréotide mais ont peu d’effet sur la structure des assemblages. Les anions divalents provoquent l’emboîtement des nanotubes. Le nombre de nanotubes emboîtés dépend de la nature du dianion. Un second objectif de ce travail était de comprendre dans quelles conditions l’interaction électrostatique forte qui se met en place entre le Lanréotide et des membranes lipidiques chargées négativement pouvait être déstabilisée. Il avait été précédemment montré que le Lanréotide en contact avec ces membranes s’auto-assemble à leur surface à des concentrations très faibles et interagit jusqu’à saturer la surface membranaire. Au-delà de la saturation des membranes, le Lanréotide en excès en solution se comporte comme le peptide pur en solution. Nous montrons dans ce travail que des sels monovalents ne déplacent pas le Lanréotide de la surface des bicouches lipidiques, des sels divalents le déplacent partiellement et seuls des polymères fortement chargés sont capables de déplacer le Lanréotide entièrement de la surface membranaire. Ces observations confirment l’hypothèse qui avait été faite selon laquelle l’interaction entre Lanréotide et membrane ne peut s’expliquer par une interaction simple entre ion et contre-ion, mais plutôt comme une interaction entre polyelectrolytes. Ces travaux ont été réalisés en combinant ultrafiltration, spectroscopie ATR-FTIR, microscopie électronique (coloration négative ou après cryofracture), diffusion de rayons X pour obtenir des informations sur les concentrations critiques d’assemblage du peptide, sa conformation, la morphologie ainsi que la structure moléculaire et supramoléculaire des architectures auto-assemblées.Lanreotide acetate is a therapeutic oligopeptide, analog of somatostatin. This di-cationic peptide, above a critical assembly concentration (CAC), spontaneously self-assembles in water, forming very well ordered supramolecular nanotubes. These spontaneous architectures are directly used in the sustained release formulation of this peptide, somatulin Autogel®. Understanding the assembly mechanisms as well as the influence of media on these peptide assemblies is important for understanding the evolution over time of this formulation after injection under the skin. Electrostatic interactions between peptides and counterions and electrostatic repulsions between peptides play potentially important roles (stabilizing or destabilizing) on the final assemblies. A first objective of this work was therefore to understand how the nature of the salts (mono-, di- & multivalent cations, mono- & divalent anions or even fatty acids of increasing chain lengths) influenced the self-assembly of Lanreotide. Mono, di- or multivalent cations and monovalent anions decrease the CAC of Lanreotide, but have little effect on the structure of assemblies. The divalent anions cause the embedding of the nanotubes. The number of embedded nanotubes depends on the nature of the dianion. A second objective of this work was to understand under which conditions the strong electrostatic interaction that takes place between Lanreotide and negatively charged lipid membranes could be destabilized. It had previously been shown that Lanreotide in contact with these membranes self-assembles at their surface at very low concentrations and interacts to saturate the membrane surface. Beyond the saturation of the membranes, the excess Lanreotide in solution behaves like the pure peptide in solution. In this work, we show that monovalent salts do not displace Lanreotide from the surface of lipid bilayers, divalent salts partially displace it and only highly charged polymers are able to displace Lanreotide entirely from the membrane surface. These observations confirm the hypothesis that had been made that the interaction between Lanreotide and membrane cannot be explained by a simple ion-counterion interaction, but rather as an interaction between polyelectrolytes. This work was carried out by combining ultrafiltration, ATR-FTIR spectroscopy, electron microscopy (negative staining or freeze-fracture), X-ray scattering to obtain information on the critical assembly concentrations of the peptide, its conformation, morphology as well as the molecular and supramolecular structure of self-assembled architectures

On the use of a multi-site ion-exchange model to predictively simulate the adsorption behaviour of strontium and caesium onto French agricultural soils

International audienceIn case of nuclear accident, 90Sr and 134,137Cs are major radionuclides to account. In previous works (Appl. Geochem. 87, 167; ibid 93, 167), a database of ion-exchange parameters allowing the description of the Sr2+ and Cs+ adsorption on purified illite and smectite was developed for a multi-site ion-exchange (MSIE) model. In this study, the adsorption behaviours of Sr2+ and Cs+ were obtained with <150 μm fractions of French soil samples: a cambisol fluvic, a calcosol, and a cambisol typic. The <2 μm fractions of the soil samples were analysed by X-ray diffraction to estimate their clay minerals proportions that were then approximated to an illite/smectite mixture, in consistency with the CEC of the <150 μm fractions. The database was implemented with K-illite and -smectite parameters to account for the amendment of K+ in agricultural soils. The isotherms of Sr2+ and Cs+ on the three soils — at 0.033 mol kgw-1 CaCl2 (I = 0.1 mol kgw-1) and at the pH value of the water equilibrated with the soils — were then compared with simulations obtained using ion-exchange parameters from the database for the MSIE model. This simulation approach, based on the additive adsorption properties between severalreactive phases, allowed to describe satisfactorily the adsorption of Sr2+ and Cs+ in most cases. In order to highlight the limiting parameters of the modelling predictive ability, different treatments were made on soil samples. The removal of the natural organic matter did not change significantly the adsorption behaviour of either Sr2+ or Cs+. The removal of the exchangeable aluminium from the cambisol typic allowed a better simulation of the adsorption isotherm in the case of Sr2+. Finally, in the case of the calcosol, the satisfactory modelling of the decrease in adsorption of Sr and Cs using a synthetic CaCl2 pore water with increasing concentrations of KNO3 allowed to verify the robustness of the MSIE model and exchange parameters from the database

Adsorption of strontium and caesium onto an Na-illite and Na-illite/Na-smectite mixtures: Implementation and application of a multi-site ion-exchange model

International audienceThe application of a multi-site ion-exchange (MSIE) model to the prediction of contaminant behaviour onto soils or sediments requires a coherent adsorption database. Following the same approach exposed in preceding works on smectite (Siroux et al., Appl. Geochem. 87, 167; Wissocq et al., Appl. Geochem., 93, 167), a database is here implemented to allow modelling the adsorption of radionuclides $^{90}$Sr and $^{137}$Cs onto Na-illite, one of the major clay mineral encountered in the terrestrial environment. This database allows describing the adsorption properties of Sr$^{2+}$ and Cs$^+$ competing with major cations H$^+$$-$considered as the reference cation$-$and Na$^+$ onto an Na-illite. To obtain the adsorption site capacities and selectivity coefficients of illite sites for Na$^+$ towards H$^+$ , a saturation curve of Na$^+$ on Na-conditioned illite was acquired. Adsorption isotherms issued from literature have been reinterpreted using the MSIE model to obtain the adsorption properties of Sr$^{2+}$ and Cs$^+$ onto purified and Na-conditioned illite. Afterwards, the predictability and robustness of the database were verified simulating experimental adsorption of Sr$^{2+}$ and Cs$^+$ onto Na-illite/Na-smectite mixtures

Organic Nanoscrolls from Electrostatic Interactions between Peptides and Lipids: Assembly Steps and Structure

International audienceAn important aspect of cells is their shape flexibility that gives them motion but also a high adaptation versatility to their environment. This shape versatility is mediated by different types of protein−membrane interactions among which electrostatic plays an important role. In the present work we examined the interaction between a small dicationic peptide, that possesses self-assembly properties, and lipid model membranes. The peptide, lanreotide, spontaneously forms nanotubes in water that have a strictly uniform diameter. In the current work, we show that the interaction between the cationic peptide and negatively charged bilayers of lipids induces the formation of myelin sheath-like structures that we call nanoscrolls. By deciphering the different steps of formation and the molecular structure of the self-assembly, we show how electrostatics modify the spontaneous peptide and lipid way of packing