Tuning Supramolecular Structuring at the Nanoscale Level: Nonstoichiometric Soluble Complexes in Dilute Mixed Solutions of Alginate and Lactose-Modified Chitosan (Chitlac) Ivan Donati,* Massimiliano Borgogna, Esther Turello, Attilio Cesa ` ro, and Sergio Paoletti Department of Biochemistry, Biophysics, and Macromolecular Chemistry, University of Trieste, Via Licio Giorgieri 1, I-34127 Trieste, Italy Received November 14, 2006; Revised Manuscript Received February 14, 2007 Two oppositely charged polysaccharides, alginate and a lactose-modified chitosan (chitlac), have been used to prepare dilute binary polymer mixtures at physiological pH (7.4). Because of the negative charge on the former polysaccharide and the positive charge on the latter, polyanion-polycation complex formation occurred. A complete miscibility between the two polysaccharides was attained in the presence of both high (0.15 M) and low (0.015 M) concentrations of simple 1:1 supporting salt (NaCl), as confirmed by turbidity measurements; phase separation occurred for intermediate values of the ionic strength (I). The binary solutions were further characterized by means of light scattering, specific viscosity, and fluorescence quenching measurements. All of these techniques pointed out the fundamental role of the electrostatic interactions between the two oppositely charged polysaccharides in the formation of nonstoichiometric polyelectrolyte soluble complexes in dilute solution. Fluorescence depolarization (P) experiments showed that the alginate chain rotational mobility was impaired by the presence of the cationic polysaccharide when 0.015 M NaCl was used. Moreover, upon addition of calcium, the P values of the binary polymer mixture in 0.015 M NaCl increased more rapidly than that of an alginate solution without chitlac, suggesting an efficient crowding of the negatively charged alginate chains caused by the polycation. Introduction The study of the physicochemical parameters influencing the mixing of oppositely charged polyelectrolytes has been exten- sively tackled from both an experimental and a theoretical point of view. The first experiments performed in the field led to describe the liquid-liquid phase separation arising from the mixture of oppositely charged polyelectrolytes as coacervation. 1 Later, the term “associative phase separation” was suggested as more appropriate to describe such a phenomenon. The formation of aggregates involving biopolymers, 2 micelles, 3 and dendrimers 4 was reported and has prompted relevant research in the field encompassing the study of the influence of pH, ionic strength, charge density, 5,6 and the “critical” stoichiometry for coacervate formation in protein-polyelectrolyte and micelle- polyelectrolyte systems. 4,7 This extensive work has, on one hand, triggered the development of several interesting applications of the coacervation process (protein separation 8 and enzyme immobilization, 9 to name a few) and, on the other, contributed to establish the “phase boundaries” governing the liquid-liquid- phase separation and the precipitation of oppositely charged polyelectrolytes. Moreover, this extended analysis led one to propose different interpretations of the “electrostatically driven complexation” process (very recently reviewed by Dubin and co-workers in ref 10) and to recognize the entropic significance of the counterions released when associative phase separation occurs. 11 From a theoretical point of view, the first model developed to describe the formation of a concentrated complex coacervate phase was proposed by Voorn and Overbeek. 12 According to them, the process of coacervation of oppositely charged poly- electrolytes should be regarded as a “spontaneous phenomenon” resulting from the competition between the electrostatic attrac- tive forces and the entropic dispersing effects acting toward the separation of the polymeric chains. Within the assumptions of this model (electrostatic interactions described using the Debye- Hu ¨ckel theory, entropic term provided by the Flory-Huggins theory, unperturbed random coil configurations for the polyions, negligible solvent-solute interactions), a proper description of the albumin/acacia gum complex coacervation was achieved. 12 A different approach was developed by Veis and Aranyi 13 in their “dilute phase aggregate model”, where, besides taking into account the solvent-solute interactions, a notable contribution to the overall coacervation process is allocated to the confor- mational entropy gain arising from the rearrangements of the polymeric chains following the formation of the very first complexes. In fact, the main innovative contribution of this model, at variance with the Voorn-Overbeek one, is the description of complex coacervation of oppositely charged polyelectrolytes as a two-step process. First, a close approach driven by the electrostatic interaction occurs between oppositely charged polyelectrolytes to form coacervate solutions of low configurational entropy. Second, an entropically driven rear- rangement of the latter leads to the formation of complex coacervates. Thus, the Veis-Aranyi theory replaces the “spon- taneous and instantaneous” phenomenon depicted by Voorn and Overbeek with a process that can take much longer to occur. 10,14 The “coacervate solutions” have been later described as “soluble complexes”. 15-17 The latter require particular conditions to be formed, such as the presence of one component with weak ionic groups. In fact, the use of strong polyelectrolytes in aqueous solution leads to the formation of non-soluble polyelectrolyte complexes. As an example, even in highly dilute solution, sodium poly(styrenesulfonate) and poly(diallyldimethylammo- nium chloride-co-acryl-amide) form quasi-soluble particles. 18,19 * To whom correspondence should be addressed. Telephone: +39 040 558 2403. Fax: +39 040 558 3691. E-mail: idonati@units.it. 1471 Biomacromolecules 2007, 8, 1471-1479 10.1021/bm0610828 CCC: $37.00 © 2007 American Chemical Society Published on Web 04/07/2007