Chitosan based in situ and ex situ magnetic iron oxide nanoparticles for rapid endotoxin removal from protein solutions

Endotoxin removal from protein solutions remains a very challenging task for bioprocess engineers. In this study, we report a facile design and synthesis of chitosan-based iron oxide nanoparticles using two different approaches, namely in situ and ex situ, to address the current issue. In the in situ method, first chitosan hydrogel was prepared using glutaraldehyde (GA) as a cross-linker, and then the iron oxide nanoparticles were allowed to form inside the hydrogel matrix by a co-precipitation process, resulting in chitosan-iron oxide nanocomposites (in situ CHIO), while in the ex situ method, previously synthesized bare magnetic nanoparticles (MNP) were functionalized with chitosan to obtain chitosan coated iron oxide nanoparticles (ex situ CHIO). The characterization of the resulting materials using several physical techniques viz., Fourier Transform Infrared Spectra (FTIR), X-ray Diffraction (XRD), Thermal Gravimetric Analysis (TGA), Vibrating Sample Magnetometry (VSM) and Transmission Electron Microscopy (TEM), revealed that the characteristic features of the materials obtained by the two different approaches were remarkably different. We further explored the opportunity to employ the materials prepared by the two different methods to obtain their effectiveness in the clearance of a toxic bacterial component, namely, endotoxin, which could be removed from protein solutions by making use of the magnetic property of the prepared nanomaterials. A good endotoxin clearance with minimal protein loss was observed when the samples were treated with the iron oxide incorporated chitosan hydrogel nanocomposites as prepared by the in situ method. In comparison to the ex situ functional nanoparticles, the in situ hydrogel nanocomposites retained their high removal efficiency after five cycles, thus providing an excellent cost-effective material for endotoxin removal. The combination of remarkable removal efficiency, minimal protein loss and reusability of the in situ CHIO nanocomposites obtained by mineralization of the hydrogel offered a complete and versatile toolbox for rapid clearance of a biological toxic molecule in clinically relevant matrices.