Molecular logistics using cytocleavable polyrotaxanes for the reactivation of enzymes delivered in living cells

The intracellular delivery of enzymes is an essential methodology to extend their therapeutic application. Herein, we have developed dissociable supermolecule-enzyme polyelectrolyte complexes based on reduction-cleavable cationic polyrotaxanes (PRXs) for the reactivation of delivered enzymes. These PRXs are characterized by their supramolecular frameworks of a polymeric chain threading into cyclic molecules, which can form polyelectrolyte complexes with anionic enzymes while retaining their three dimensional structure, although their enzymatic activity is reduced. Upon the addition of a reductant, the PRXs dissociate into their constituent molecules and release the enzymes, resulting in a complete recovery of enzymatic activity. Under the intracellular environment, the PRX-based enzyme complexes showed the highest intracellular enzymatic activity and efficient activation of anticancer prodrugs to induce cytotoxic effects in comparison with the non-dissociable complexes and the commercial cell-penetrating peptide-based reagents. Thus, the intracellularly dissociable supermolecules are an attractive system for delivering therapeutic enzymes into living cells.