Gel Beads of Alginate from Laminaria Hyperborea Leaf - Stability related to size and polymer distribution
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Alginate microbeads have through many years of research shown great potential as an immunoisolation system for the entrapment of insulin-producing cells. However, one of the main challenges in the use of alginate gels as an immunoprotective barrier is the destabilisation of the gel network under physiological conditions. Alginate is a binary heteropolymer containing 1,4-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues, known for its gel forming properties in the presence of divalent cations. The traditional polycation layer used for the stabilisation of alginate gel beads has been associated with cellular overgrowth and complement activation, which has urged the investigation of novel alginate gel beads with both high mechanical stability and biocompatibility. The basis of this master s thesis is the investigation of the stability of Laminaria hyperborea leaf alginate gel beads, which have previously been shown to be highly biocompatible (Tam et al., 2011) yet unstable under physiological conditions; and to provide a strategy to increase the stability of the gel beads. The stability of the alginate beads was systematically investigated with respect to size, polymer distribution and polymer leakage, and was ultimately compared to the widely studied and mechanically stable L. hyperborea stipe alginate. The gelling conditions and alginate bead formulations were varied, where the stability related to size was studied through consecutive saline treatments and quantified by light microscopy. The polymer distributions and dimensions of various alginate beads were examined after treatment in different washing solutions, through fluorescence labelling of the alginates and subsequent visualisation by confocal laser scanning microscopy (CLSM). In addition, the amount of leaked alginate from saline-treated alginate beads was assessed, and the chemical compositions and average molecular weights of the leaked alginates were determined by 1H NMR and SEC-MALLS, respectively. Consecutive saline treatments of leaf alginate beads gelled in calcium and/or barium revealed a strong stabilising effect related to size upon the inclusion of barium as cross-linking ion. Furthermore, the presence of non-gelling ions in the alginate and gelling solutions during the preparation of the gel beads showed a slight destabilising effect on bead size during saline treatments. The addition of free G-blocks to leaf alginate formulations suggested an insignificant effect related to the swelling properties of the gel beads upon consecutive treatments in saline. The investigations on the polymer distributions of leaf alginate beads gelled in calcium or barium revealed inhomogeneous polymer distributions for all gelling conditions. The presence of non-gelling sodium ions, in the gelling or washing solutions, had a disrupting effect on the initial polymer distributions of the gel beads. The use of barium as cross-linking ion conserved parts of the inhomogeneous polymer distribution in washing solutions containing low concentrations of calcium, specifically Hank s balanced salt solution and 0.9 % NaCl with 2 mM CaCl2. It was further established that the initial inhomogeneous polymer distributions could not be recovered with low calcium concentrations once lost during saline wash. Treatment in mannitol completely preserved the initial polymer distribution and size of the leaf alginate beads, but was excluded as a viable washing solution due to bead destruction. The extension of the gelling time of alginate beads and the varying of barium concentration in the gelling solutions showed insignificant effects in preserving the initial polymer distributions upon treatment in washing solutions. The addition of free G-blocks (10 mg/ml) to leaf alginate gelled in 20 mM BaCl2 might suggest a high degree of preservation of the inhomogeneous polymer distribution after treatment in 0.9 % NaCl with 2 mM CaCl2. Upon treatment in saline, barium-alginate beads were found to leach M-enriched alginate with low-molecular weight averages. The leached alginate (7.0 % (w/w) of the starting material) from leaf alginate beads was found to be significantly larger than for the corresponding stipe alginate beads (0.8 % (w/w) of starting material). Throughout the study, the stipe alginate beads exerted higher stability with respect to size, polymer distribution and polymer leakage compared to leaf alginate beads. However, the stability of leaf alginate was improved through: i) the use of barium as cross-linking ion instead of calcium, ii) the inclusion of low concentrations of calcium in the washing solutions and iii) possibly the addition of free G-blocks to the alginate bead formulations at high barium concentrations. The addition of free G-blocks showed promising results in conserving the inhomogeneous polymer distribution in leaf alginate beads after washing. However, the fluorescence image collection obtained by CLSM holds strong uncertainties, and should be investigated further.