Synthesis and characterization of a d-glucose sensitive hydrogel based on CM-dextran and concanavalin A

Abstract

A facile method is described for the preparation of d-glucose-sensitive hydrogel membranes based on cross-linking carboxymethyl dextran (CM-dextran) with the glucose binding lectin concanavalin A (ConA) using carbodiimide chemistry. The mesh size of these hydrogels is determined by both covalent cross-links and affinity interactions between ConA and terminal glucose moieties on the dextran chains. Competitive displacement of the affinity interactions by d-glucose leads to changes in both morphology and permeability. The carbodiimide coupling chemistry has the advantage that it introduces no potentially cytotoxic groups into the gels formed rendering them more suitable for potential in vivo applications. Infrared spectra of the hydrogels suggest that, in addition to direct cross-linking of CM-dextran chains, gels are also stabilized by formation of amide bonds between amine groups in the ConA and carboxyl groups in CM-dextran. This suggests that the mechanical properties of the gels can be modified by the inclusion of inert proteins to increase the density of covalent cross-links. The permeability of the hydrogels, as shown by protein diffusion, increases in response to changes in the d-glucose concentration of the external medium, causing competitive displacement of the affinity cross-links. Sequential addition and removal of external glucose in a stepwise manner showed that these permeability changes are reversible. Results obtained using isothermal titration calorimetry confirmed the competitive binding between ConA-dextran and d-glucose this was confirmed to be a biospecific effect by the observation that l-glucose was not bound by free or dextran coupled ConA. Scanning electron microscopy of samples prepared using cryofixation and cryofracturing techniques showed that observed changes in permeability correlate well with glucose dependent structural changes in the gel. The effects of varying pH and ionic strength show that ion exchange effects also influence diffusion rates. However, an advantage of the synthetic route described is that the intrinsic charge density of the hydrogels produced can be tailored to the specific interaction by varying the degree of carboxylic substitution on the original dextran. The ease of preparation and control of final gel properties allowed by this approach offers possibilities for a range of biotechnological applications including drug delivery and biosensors.