High-performance sono/nano-catalytic system: Fe3O4@Pd/CaCO3-DTT core/shell nanostructures,a suitable alternative for traditional reducing agents for antibodies

Herein, a novel heterogeneous nanoscale reducing agent for antibody cleavage, made of iron oxide nanoparticles, silica network, palladium on calcium carbonate (10%), and dithiothreitol (Fe₃O₄@Pd/CaCO₃-DTT), is presented as a substantial alternative for traditional homogeneous analogues. Conventionally, antibody fragmentation is accomplished using reducing agents and proteases that digest or cleave certain portions of the immunoglobulin protein structure to provide active thiol sites for drug tagging aims. Then, dialysis process is needed to separate excess chemical structures and purify the reduced antibody. In this work, we have made an effort to design a suitable heterogeneous tool for protein cleavage and skip the dialysis process for purification of the reduced antibody. In this regard, firstly, various preparation methods including microwave irradiation, reflux and ultrasonication have been precisely compared, and it has been proven that the best result is obtained through 10 min ultrasound (US) irradiation using an US bath with 50 KHz frequency and 200 W L⁻¹ power density. Then, all the necessary structural analyses have been done and thoroughly interpreted for the final product. Afterward, the catalytic performance of Fe₃O₄@Pd/CaCO₃-DTT nanoscale system in the presence of US waves (50 KHz, 200 W) has been monitored using some disulphide derivatives. The NPs could be conveniently separated from the mixture through their substantial paramagnetic property. Thus, dialysis process in which various types of membranes are used is practically jumped after the reduction process. In this work, this is clearly demonstrated that there is a constructive synergistic effect between US waves and prepared Fe₃O₄@Pd/CaCO₃-DTT nanoscale reducing agent. Ultimately, trastuzumab (anti HER-2) antibody has been used to test the performance of the prepared Fe₃O₄@Pd/CaCO₃-DTT NPs in a real protein reduction reaction.