Modulation of physicochemical properties of magnetic agarose microspheres by hydrolysis-suppressive sequential crosslinking

Separation and purification of target proteins are essential for drug discovery and development. A magnetic separation has been exploited due to its high efficiency. Despite many studies on chemical crosslinking of a magnetic agarose microsphere (MAM), the study on a crosslinking reaction preventing MAM from hydrolytic degradation during the reaction and the effects of the crosslinks on the physicochemical and electrochemical properties of the crosslinked MAM (CL-MAM) has rarely been reported. In our study, CL-MAM was prepared using a sequential crosslinking by which MAM was crosslinked using epichlorohydrin (ECH) in DMSO after crosslinking the MAM by 1,3-dichloro-2-propanol (DCP) for a short time. Such a crosslinking prevented the hydrolytic degradation of DCP-crosslinked MAM (D-CL-MAM) and DCP and ECH-crosslinked MAM (DE-CL-MAM). MAM had a mean diameter of 110 ± 57.42 µm and its morphology was not affected by the crosslinking. In FT-IR analysis, increase of peak intensity at 1037 cm−1 confirmed newly formed C-O-C bonds in the agarose of CL-MAM. Thermal stability of MAM increased by both DCP crosslinking and subsequent ECH crosslinking of the agarose. While Tg of MAM was not shifted by the crosslinking, the onset temperatures of glass transition and thermal decomposition increased by DCP crosslinking and further increased by DCP and ECH-crosslinking. Stiffness (S) and elastic modulus (E) of MAM were enhanced by DCP crosslinking and subsequent ECH crosslinking, indicating a half of ECH formed crosslinks between agarose mainchains while the other half crosslinked adjacent pyranose rings in the agarose mainchain. Permittivity analysis revealed that α relaxation temperature of the agarose film corresponded to Tg determined by DSC analysis and the relaxation intensity decreased with increasing crosslink density of the agarose. The higher swelling ratio of DE-CL-MAM compared to D-CL-MAM was attributed to greater S and E values of DE-CL-MAM. The hydrolysis-repressive sequential crosslinking of MAM using DCP and ECH can be a feasible approach to modulate physicochemical and electrochemical properties of MAM.