Elsevier

Carbohydrate Polymers

Volume 147, 20 August 2016, Pages 234-242
Carbohydrate Polymers

Effects of added oligoguluronate on mechanical properties of Ca – alginate – oligoguluronate hydrogels depend on chain length of the alginate

https://doi.org/10.1016/j.carbpol.2016.04.014Get rights and content

Highlights

  • Young’s modulus E of Ca alginate-oligoguluronate gels depend on c of oligoguluronate.

  • Effects of oligoguluronate on E of Ca-alginate gels depend on alginate chain length.

  • Oligoguluronates in Ca-alginate gels perturb the network junction functionality.

Abstract

The effect of adding shorter alginate fragments highly enriched in α-l-guluronic acid (oligoG) on the Young’s modulus of the Ca-induced alginate hydrogels were determined using nanoindentation. Ca-alginate gels using two low and one high molecular weight alginate, with increasing amount of added oligoG, were prepared at constant 20 mM total Ca2+ by in situ release of the cation. Differences in the effect on the mechanical properties of increasing amount of oligoG to the various alginate samples were attributed to their different capability to support network connectivity by junction zone formation. Upon decreasing the fractional Ca-saturation of all the α-l-guluronic acid residues (G) present, Fsat, by increasing the oligoG concentration, the lower molecular weight alginates displayed the largest reduction in Young’s modulus. This was suggested to be due to the few sequences of α-l-guluronic acid residues making up potential zones engaging in network connectivity of this alginate. Similar trends were observed for a low molecular weight alginate with larger fraction of G. The higher molecular weight sample displayed less reduction of Young’s modulus associated with increasing concentration of oligoG. The consequences of reduction in effective, mean junction zone functionality and associated increase in sol fraction with added oligoG on the elastic properties thus depend on the chain length of the alginates. These finding suggest that effects of added oligoG on Ca-induced alginate gelation should connect the effect on junction zone formation to those mediating network connectivity.

Introduction

Alginate is a linear copolymer of (1→4) linked β-d-mannuronic acid and α-l-guluronic acid (denoted as M and G), which can be extracted from brown algae species or produced by bacteria such as Azotobacter vinelandii and several Pseudomonas species (Moe, Draget, Skjak-Braek & Smidsrod, 1995). The M- and G-residues occur along the alginate chains in G-blocks (neighboring G-residues), M-blocks (neighboring M-residues) or MG-blocks (regions of alternating residues) (Moe et al., 1995). The molecular weight, fraction of residues and sequence vary with the origin of the alginate with respect to different algae, parts of one algae species or even harvesting season (Moe et al., 1995). Additionally to widen the selection of naturally obtained alginate, further ex vivo enzymatic modification of alginate sequences are possible by applying mannuronan C5 epimerases converting mannuronate to guluronate residues (Bjerkan et al., 2004; Høidal, Ertesvåg, Skjåk-Bræk, Stokke, & Valla, 1999; Moe et al., 1995; Svanem, Skjåk-Bræk, Ertesvåg, & Valla, 1999).

Alginates form gels in aqueous solution in the presence of cations like Ba2+, Sr2+ or Ca2+ (Haug, Larsen, & Smidsrod, 1967; Haug & Smidsrod, 1970). Calcium fill the cavity-like structures between neighboring G-residues (Fang et al., 2008; Fang et al., 2007; Stewart, Gray, Vasiljevic, & Orbell, 2014) and then induce pairing between two G-residues from neighboring chains creating structure known as egg-box model (Grant, Morris, Rees, Smith, & Thom, 1973; Sikorski, Mo, Skjåk-Bræk, & Stokke, 2007; Stokke et al., 2000). Furthermore, at high ionic saturation of guluronic acid, the number of laterally associated chain segments can increase beyond two chains (Fang et al., 2008; Fang et al., 2007; Padoł, Maurstad, Draget, & Stokke, 2015; Stokke et al., 2000). The minimum number of consecutive G-residues required to support Ca2+ mediated junctions is reported to be 8 ± 2 and less for Sr2+ (Stokke, Smidsrød, Zanetti, Strand, & Skjåk-Bræk, 1993). A recent study reports that alternating MG sequences support Ca2+ induced gelation(Donati et al., 2005). Recently, differences in the microstructural properties of alginates gels prepared using processes referred to as macroscopic homogeneous and inhomogeneous Ca-alginate gels and dynamics within the alginate hydrogels have been reported (Larobina and Cipelletti, 2013, Schuster et al., 2014).

Ionotropic alginate hydrogels have found widespread applications due to their versatility, biocompatibility, nontoxicity and mild gelation conditions (Davis, Volesky, & Mucci, 2003; Lim and Sun, 1980, Miralles et al., 2001). In the food industry, alginates are used as thickeners, stabilizers, film formers and gelling agents (Donati & Paoletti, 2009; Moe et al., 1995). In the biomedical and pharmaceutical industries alginate is used for controlled drug delivery, cell entrapment and immunological isolation barriers (de Vos, Faas, Strand, & Calafiore, 2006; Draget, Ostgaard, & Smidsrod, 1989). The understanding of gelation kinetics and mechanical properties of alginate hydrogels is essential in order to be able to control its properties and applicability.

Use of oligoguluronates (oligoG; DP  20) in combination with a high Mw alginate at different mixing ratios, opens the possibility to control gelation kinetics and mechanical properties without changing chemical structure of the compound (Draget & Smidsrod, 2006; Jorgensen, Sletmoen, Draget, & Stokke, 2007). OligoG is obtained by hydrolysis of an alginate high in G residues followed by selective precipitation. OligoG can be prepared with more than 90% α-l-guluronic acid (Haug & Larsen, 1965). They can be considered as free G-blocks that compete for multivalent cations with G-blocks within the alginate chains. However, due to their low degree of polymerization (DP  20) and their lack of inherent elastically active segments, their mode of interaction appear not to introduce network connectivity on their own when mixed with high Mw alginates (Draget and Smidsrod, 2006, Jorgensen et al., 2007).

Previously, based on characterization by dynamic light scattering and rheology, it is reported (Draget and Smidsrod, 2006, Jorgensen et al., 2007, Liao et al., 2015, Padoł et al., 2015), that the gelation is slowed down when the concentration of oligoguluronates added to the alginate was increased. Additionally, by rheology measurements it was observed that alginate gel strength decreases at low to medium Ca2+ concentration and increases at high Ca2+ the concentration (Draget and Smidsrod, 2006, Jorgensen et al., 2007) when concentration of oligoguluronates added to the alginate increased. It is recently suggested that oligoG affect junction zone formation within Ca-induced alginate gelation depending on the Ca-induced fractional saturation of the G residues, Fsat (Liao et al., 2015). Thus, an inhibitory effect on Ca2+ mediated junction zone formation due to oligoG in the range 0.5 < Fsat < 1.2 was suggested to arise from Ca2+ induced associations within the oligoG fraction. The promotive effect of oligoG on the junction zone formation observed for Fsat > 1.2 was suggested to arise from Ca-oligoG duplexes integrated in the alginate network that bridge other junction zones formed in the high molecular weight chains.

In the present work we studied the effect of oligoguluronates addition on the mechanical properties (Young’s modulus) of Ca-induced alginate gels. Gels were prepared with and without additional free oligoG and measured by nanoindentation technique. These issues were addressed using colloidal probe –atomic force based nanoindentation to determine the elastic properties. This technique has been demonstrated to be applicable for the determination of spatially resolved mechanical properties of soft materials, and applied to various types of specimens such as hydrogels and cells (Han, Grodzinsky, & Ortiz, 2011; Lin & Horkay, 2008; Suriano, Credi, Levi & Turri, 2014). In the present study, this approach also benefits from the reduced requirements of sample as compared to bulk rheological characterization. It was hypothesized that Young’s modulus would decrease when oligoguluronates was added as a consequence of an expected competition for Ca2+ between the added oligoG and the G-blocks confined within in the alginate chains. Moreover, we explore the consequences of the oligoG induced perturbation of the mechanical properties of the alginate hydrogels made from different average alginate chain lengths.

Section snippets

Materials

Three alginates samples and one oligoguluronate sample were selected for the study. The alginates originated from Laminaria hyperborea. Two low molecular weight alginates with different G-content were selected. The third alginate sample was a higher molecular weight sample with a G-content close to one of the low molecular weight samples. The weight average molecular weights Mw and intrinsic viscosities were determined by SEC-MALLS (in 50 mM Na2SO4, 10 mM EDTA) as previously reported (

Results and discussion

Fig. 1 shows examples of force-distance data for nanoindentation of Ca-induced alginate hydrogels for the alginate sample Alg0.66114 at two different final concentrations (15.5 and 23.2 mg/ml) and with increasing concentrations of oligoG included in the preparations. These data depicts the approach data obtained for a chosen gel specimen. Such data (Fig. 1) provides a qualitative impression that highly reproducible force–distance curves can be obtained for alginate gels moulded between parallel

Conclusions

In the present work, we have established novel effects of adding oligomers of guluronate sequences with number average degree of polymerization of 19 to alginates on the resulting mechanical properties of the Ca—alginate hydrogels. While the previous reports focuses on chemical composition of the alginate, molar ratio of Ca2+ relative to total Ca-binding moieties in the blend and overall concentration, the present data extend this by showing that the molecular weight of the alginate is also

Acknowledgement

This work is support by The Research Council of Norway, contract number 1826595/I40.

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