Ripening of Catanionic Aggregates upon Dialysis Youlia Michina, † David Carrie `re,* ,† Clarisse Mariet, ‡ Me ´lanie Moskura, ‡ Patrick Berthault, § Luc Belloni, † and Thomas Zemb † CEA, IRAMIS, SCM, LIONS (Laboratoire Interdisciplinaire sur l’Organisation Nanome ´trique et Supramole ´culaire), CEA, IRAMIS, LPS (Laboratoire Pierre-Süe), and CEA, IRAMIS, SCM, LSDRM (Laboratoire Structure et Dynamique par Résonance Magnétique), F-91191 Gif-sur-YVette, France ReceiVed June 11, 2008. ReVised Manuscript ReceiVed October 3, 2008 We have studied the dialysis of surfactant mixtures of two oppositely charged surfactants (catanionic mixture) by combining HPLC, neutron activation, confocal microscopy, and NMR. In mixtures of n-alkyl trimethylammonium halides and n-fatty acids, we have demonstrated the existence of a specific ratio between both surfactant contents (anionic/cationic ≈ 2:1) that determines the morphology, the elimination of ions, and the elimination of the soluble cationic surfactant upon dialysis. In mixtures prepared with lower anionic surfactant contents, ill-defined aggregates are formed, and dialysis quickly eliminates the ion pairs (H + X - ) formed upon surfactant association and also the cationic surfactant until a limiting 2:1 ratio is reached. By contrast, mixtures prepared above the anionic/cationic 2:1 ratio form micrometer-sized vesicles resistant to dialysis. These closed aggregates retain a significant number of ions (30%) over 1000 hours, and dialysis is unable to eliminate the soluble surfactant. The interactions between surfactants have been estimated by measuring the partitioning of the CTA molecules between the catanionic bilayer, the bulk solution, and mixed micelles when they exist. The mean extraction free energy per CTA in the membrane has been found to increase by 1k B T to 2k B T as the soluble surfactant is depleted from the bilayer, which is enough to stop the dialysis. The vesicles produced above the anionic/cationic 2:1 ratio are formed by frozen bilayers and are resistant to extensive dialysis and therefore show an interesting potential for encapsulation as far as durability is concerned. Introduction Mixtures of oppositely charged surfactants, also called catanionic mixtures, form a wide range of morphologies depending on their composition in water and surfactants, such as mixed micelles, 1 lamellar phases, 2,3 cubosomes, hexosomes, 4 and vesicles. 5 In particular, it is now accepted that the catanionic vesicles form spontaneously and are stabilized by the spontaneous curvature and the entropy of mixing of the two surfactants in the bilayer. 6 The potential of the catanionic vesicles for encapsulation and release of water-soluble compounds has been evaluated, and it has been found that the permeability of the vesicle walls is generally comparable, and sometimes significantly lower, than that of vesicles prepared from liposomes (data in Table 1 calculated from refs 7-13). An alternative and now well- established way to use catanionic mixtures for drug delivery is to use a pharmaceutically active compound directly as one of the amphiphiles involved in the ion pair. In this case, the solute can be considered to be encapsulated inside the bilayer. 14-16 In both contexts, it is important to understand how the ripening of catanionic aggregates may or may not occur. The simplest case is to consider a release driven by purely entropic phenomena (i.e., that the catanionic aggregate is in contact with an infinite volume of solvent). Despite the usual decrease in the critical micelle concentration of the catanionic mixtures as compared to that of the single-surfactant solutions, 17 this situation is eventually expected to lead to a significant loss of surfactant from the aggregate although it has been neglected in previous studies using the dialysis of catanionic mixtures. 12,18 In the first strategy (encapsulation inside a vesicle), loss of surfactant will determine the possible disruption of the vesicle or undesirable surfactant release. 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