Effect of Ca 2+ , Ba 2+ , and Sr 2+ on Alginate Microbeads Y Ä rr A. Mørch,* ,§ Ivan Donati, ‡ Berit L. Strand, § and Gudmund Skja ˚ k-Bræk § Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7491 Trondheim, Norway, and Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Via Licio Giorgieri 1, 34127 Trieste, Italy Received January 4, 2006; Revised Manuscript Received March 7, 2006 Microcapsules of alginate cross-linked with divalent ions are the most common system for cell immobilization. In this study, we wanted to characterize the effect of different alginates and cross-linking ions on important microcapsule properties. The dimensional stability and gel strength increased for high-G alginate gels when exchanging the traditional Ca 2+ ions with Ba 2+ . The use of Ba 2+ decreased the size of alginate beads and reduced the permeability to immunoglobulin G. Strontium gave gels with characteristics lying between calcium and barium. Interestingly, high-M alginate showed an opposite behavior in combination with barium and strontium as these beads were larger than beads of calcium-alginate and tended to swell more, also resulting in increased permeability. Binding studies revealed that different block structures in the alginate bind the ions to a different extent. More specifically, Ca 2+ was found to bind to G- and MG-blocks, Ba 2+ to G- and M-blocks, and Sr 2+ to G-blocks solely. Introduction Alginate microcapsules have for many years been used as immune barriers for cell transplantation where the alginate gel protects the transplant from the host immune system. Microen- capsulation thus provides a potential way to overcome the need for immunosuppressive drugs. Although a variety of cells have been proposed and used for gel immobilization, 1-3 major interest has been focused on employing alginate microcapsules for the encapsulation of Langerhans islets as a potential treatment for Type 1 diabetes. Alginate, the main component of these capsules, is a polysaccharide mainly isolated from brown algae. It is a linear copolymer consisting of guluronic (G) and mannuronic (M) acid forming regions of M-blocks, G-blocks, and of alternating structure (MG-blocks). 4 Divalent cations such as calcium, barium, and strontium bind preferentially to the G-blocks in the alginate in a highly cooperative manner, 5 thereby forming a gel. Alginate’s affinity toward the different divalent ions has been shown to decrease in the following order: Pb > Cu > Cd > Ba > Sr > Ca > Co, Ni, Zn > Mn. 6,7 Since the composition and block structure varies greatly in different types of alginates, it follows that both the gel and ion-binding properties of alginate is influenced by the choice of alginate material and cross-linking ion. Traditionally, calcium has been used as the gel-forming ion in the production of gel beads for bioencapsulation purposes. However, Ca-alginate beads are sensitive toward chelating agents such as phosphate and citrate and nongelling agents such as sodium and magnesium ions. In physiological solution, this ion replacement results in osmotic swelling of the beads inevitably leading to increased pore size and destabilization and rupture of the gel. 8 To increase the stability and reduce the permeability of Ca-alginate gel beads, a polycation layer is often added to the alginate gel core, following the original microencapsulation protocol of Lim and Sun. 9 Most polycations are, however, toxic to cells, 10 and the most commonly used polycation for encapsulation purposes, poly-L-lysine (PLL), is the main capsule component responsible for the fibrotic overgrowth often seen on implanted alginate-PLL-alginate microcapsules. 11 Recently, much focus has therefore been on developing new methods omitting the polycation treatment, including the use of epimerized alginate 12 or covalently cross- linked alginate 13 as core material. Another method recently exploited by many groups giving promising results both in allo- and xenograft transplantation 14-18 as well as in biocompatibility studies 19 is the use of Ba-alginate instead of the traditional Ca-alginate. In principle, barium ions (Ba 2+ ) will, because of their higher affinity toward alginate, form stronger gels with alginate compared to calcium. 20 Nevertheless, the affinity is highly dependent on the alginate composition, and limited work has been done on characterizing the effect of different divalent ions on gel bead properties. Since barium is an inhibitor of K + channels in biomembranes at concentrations greater than 5-10 mM, 21 the concentration should be kept at a minimum when used in vivo. Strontium, on the other hand, is a nontoxic divalent cation which has been scarcely tested for encapsulation purposes. The aim of this study was to compare the effects of various gelling ions (Ca 2+ , Ba 2+ , and Sr 2+ ) on two alginates of different composition widely used for encapsulation purposes. We have studied the influence of Ca 2+ , Sr 2+ , and Ba 2+ on microbead stability, permeability, and gel strength and on the distribution of alginate in gel beads. In addition, the binding of these divalent ions to both natural and enzymatically modified alginates of different composition was studied. Materials and Methods Alginates. High molecular weight sodium alginate samples isolated from Macrocystis pyrifera (high-M alginate) and Laminaria hyperborea stipes (high-G alginate) were provided by Sigma Chemicals (U.S.A.) and FMC Biopolymer (Norway), respectively. High molecular weight * Corresponding author. Tel: (+47)73591689. Fax: (+47)73591283. E-mail: yrrm@ntnu.no. § Norwegian University of Science and Technology. ‡ University of Trieste. 10.1021/bm060010d CCC: $33.50 © xxxx American Chemical Society PAGE EST: 9.5 Published on Web 04/12/2006