Galactose-Substituted Alginate 2: Conformational Aspects Ivan Donati,* Anna Coslovi, Amelia Gamini, Gudmund Skjåk-Bræk, ‡ Amedeo Vetere, Cristiana Campa, ² and Sergio Paoletti Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Via Licio Giorgieri 1, I-34127 Trieste, Italy Received August 21, 2003 Galactose moieties have been introduced on the uronic groups of alginates from different sources via an N-glycosidic bond, thus affecting the net charge on the polymer chain. The modified polymers have been analyzed by means of viscosity and of high-performance size-exclusion chromatography combined with refractive index multiple angle laser light scattering (HPSEC-RI-MALLS) measurements. The latter technique enabled us to determine the molecular weight of the modified polymers, proving that the synthetic procedure did not affect the chemical integrity of the chain. The intrinsic viscosity and the radius of gyration data showed that the hydrodynamic properties of the polymer chain varied with the degree and the pattern of substitution. In the presence of a relatively low galactose content (up to 19%), a decrease of the hydrodynamic dimensions of the coil was experienced, while on increasing the degree of substitution (especially on GG diads) a re-extension of the chain was discovered. Measurements of intrinsic viscosity at different values of the degree of dissociation have demonstrated that this effect cannot be solely explained by the reduction of the charge density of the polymer. Rather, it implies the occurrence of conformational changes of the chain that are specific to the chemical nature of the site of substitution. These data have been supported by the values of the persistence length of the natural and modified polymers obtained with the Doty-Benoit equation. The chiro-optical properties of the modified polymers studied by means of circular dichroism (CD) spectroscopy confirmed that conformational variations occurred to the polymeric chain upon introduction of galactose residues. Introduction Polysaccharides constitute major components of that part of the biological scenery, which is often cumulatively called the “extracellular matrix” (ECM). Together with the whole matrix biopolymers, they augment the mechanical stability through the formation of a three-dimensional network, ensure appropriate dynamic response to stresses, and create highly swollen environments with controlled permeability. More- over, matrix biopolymers participate in the immunological “intelligence” network involved in cell/cell and guest/host specific interactions, control the tissue structure, regulate the function of cells, and allow the diffusion of nutrients, metabolites, and growth factors. 1,2 Therefore, polysaccharides from different sources (e.g., hyaluronate, alginate, and chitosan to mention only a few) represent appealing candi- dates to obtain three-dimensional scaffolds, typically hydro- gels, acting as analogues of the natural extracellular matrix. Alginates are a family of polysaccharides produced by brown algae 3 and bacteria. 4,5 Chemically, they are linear copolymers of 1 f 4-linked -D-mannuronic acid (M) and R-L-guluronic acid (G). The composition and sequential arrangement of the two residues varies with the species or the tissue from which they are isolated. 6 The monomers are arranged in a blockwise pattern along the chain with homopolymeric regions of M (M-blocks) and G (G-blocks) residues interspersed with regions of alternating structure (MG-blocks). Divalent cations such as calcium, strontium, and barium bind preferentially to the G-blocks in a highly cooperative manner, accounting for the formation of ionic hydrogels via the so-called “egg-box” model. 7 The rapid gel formation in the presence of millimolar concentrations of calcium ions, as well as the elucidation of the structure-function relationships, has established algi- nate as a very versatile material for preparing microcap- sules for cell therapy. Different cells have been suggested as candidates for gel immobilization including parathyroid cells for treatment of hypocalcemia, 8 dopamine-producing adrenal chromaffin cells for treatment of Parkinson’s dis- ease, 9 and endostatin-producing cells for treatment of brain tumors. 10 Major interest has been focused on insulin- producing cells for the treatment of type 1 diabetes, and alginate-poly-L-lysine capsules containing pancreatic islets of Langerhans have been shown to reverse diabetes in large animals. 11 It is important to underline that in all of the reported examples the main goal of the gel is to act as a barrier between the transplanted cells and the immune system of the host. No specific interaction between the polysaccharide * Corresponding author. Present address: Institute of Biotechnology- NTNU, Sem Sælands vei 6-8, N-7491 Trondheim, Norway. E-mail: donati@bbcm.units.it. ‡ On leave of absence from Institute of Biotechnology, Norwegian University of Science and Technology (NTNU), Sem Sælands vei 6-8, N-7491 Trondheim, Norway. ² Present permanent address: Bracco Imaging, CRM-TS, AREA Science Park, Building Q, SS14 Km 163.5, I-34012 Basovizza (TS). 186 Biomacromolecules 2004, 5, 186-196 10.1021/bm030063k CCC: $27.50 © 2004 American Chemical Society Published on Web 12/03/2003