Protein nanogelation using vitamin B6-bearing pullulan: effect of zinc ions

Nanogels are nanometer-sized hydrogel nanoparticles with three-dimensional networks of crosslinked polymer chains. We have previously reported a method for constructing protein nanogels using vitamin B-6 (pyridoxal)-bearing pullulan (PLPP) as a bio-crosslinker. To validate the potential for using this bio-crosslinker to prepare protein nanogels, we developed an improved method using metal ions to enhance the affinity between the protein and the bio-crosslinker. We investigated the binding of PLPP as a bio-crosslinker to a protein to produce protein nanogels using anionic bovine serum albumin (BSA) as a model protein. Ultraviolet-visible spectroscopic titration of PLPP with BSA showed that the binding of the PLPP to the BSA via the formation of Schiff bases was significantly enhanced in the presence of zinc ions because of the coordination of the Schiff base to metal ions, which shifted the equilibrium of the reaction to the Schiff base formation side. Dynamic light scattering measurements, high-performance liquid chromatography and transmission electron microscopy confirmed that the zinc ions enhanced the ability of PLPP to form nanogels by crosslinking with the anionic BSA despite the strong electrostatic repulsion between the two molecules.