Strategies for Stabilising Gel Beads of Alginate from Laminaria Hyperborea Leaf by using Strontium as Gelling Ion
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Alginate microbeads have shown great promise as immunoisolating material for the encapsulation of living cells, such as insulin-producing cells for the treatment of diabetes. However, one of the main challenges related to the use of alginate as an immunoisolating material is destabilisation of the gel network under physiological conditions. Alginate is a linear binary polymer composed of 1,4-linked alpha-D-mannuronic acid (M) and beta-L-guluronic acid (G), varying in sequences and blocks along the chain. Alginate forms gels in the presence of divalent ions, such as calcium. A strategy for stabilising alginate beads under physiological conditions is by the use of alginates with a high guluronic acid content rather than alginates with a high mannuronic acid content. However these beads are shown not to be biocompatible and are associated with cellular overgrowth on the capsule surface. Alginates isolated from Laminaria hyperborea leaf have previously shown to be highlybiocompatible but unstable under physiological conditions. Hence, the main goal of this master thesis was to investigate the stability of L. hyperborea leaf alginate using strontium as gelling ion, and provide a strategy to increase the stability of the beads. The widely studied and mechanically stable L. hyperborea stipe alginate was included for comparison. Stability was investigated in terms of size, polymer distribution and polymer leakage. In addition, the mechanical properties of alginate gel beads were investigated in terms of gel strength. The stability related to size was investigated through consecutive saline treatments and quantified by light microscopy. The polymer distributions of various alginate gel beads were examined before and after treatment with saline, through fluorescence labelling of alginates and subsequently visualization of alginate beads using a confocal laser scanning microscope (CLSM). In addition, characterization of leaked alginate from saline-washed beads in terms of chemical composition and average molecular weight were determined by 1H NMR and SEC-MALLS, respectively. Mechanical propertieswere investigated for alginate gel beads as a force at a certain distance of compression. Consecutive saline treatments of alginate gel beads showed strong stabilising effect on size using strontium as cross-linking ion compared to calcium. Decreasing the strontium concentration from 50 mM to 20 mM did not affect size stability, but lowering the concentration further to 10 mM had a stabilising effect on size. Increasing the alginate concentration, especially by adding high-G stipe alginate or G-blocks, had a further stabilising effect on size for strontium alginate beads. Investigation of polymer distributions of leaf alginate beads and leaf alginate beads with added stipe alginate, gelled in calcium or strontium, revealed inhomogeneous polymer distributions in gelling solutions. For the leaf alginate beads washed with saline, disrupting effect of non-gelling sodium was seen on polymer distribution. The addition of stipe alginate to leaf alginate beads using strontium as gelling ion, revealed a preserved inhomogeneous polymer distribution of the stipe alginate withinthe bead, especially when decreasing the strontium concentration from 50 mM to 20 mM.Upon saline treatment, beads gelled in calcium or strontium leaked alginate of a high M content with low average molecular weights. The highest molecular weight averages and lowest M-content were leaked from alginate beads gelled in calcium. Compression of alginate beads indicated higher gel strength of gel beads by increasing the alginate concentration and molecular weight. In addition, high molecular weight leaf alginate beads were compressed to a larger extent than the corresponding high molecular weight stipe alginate beads, indicating that stipe alginate beads had a higher gel strength. The use of strontium as gelling ion had insignificant effect on force or distance of compression compared to calcium. In general, the stability of L. hyperborea leaf alginate was improved by the use of strontium as gelling ion, by increasing the alginate concentration and by the addition of very low molecular weight stipe alginate.