Supramolecular Assembly of Stimuli-Responsive Protein-Chromophore Conjugate and Triggered Protein Delivery

Protein delivery has emerged as a powerful therapeutic tool to treat many life-threatening diseases. In this study, we report the synthesis of a protein (Lysozyme = LYS)-supramolecular-structure-directing-unit (SSDU) conjugate using a redox-responsive self-destructive linker, its spontaneous self-assembly in water, intracellular delivery, and selective killing of cancer cells. The LYS surface has a few (on average 6) free amine groups, which were used to attach with the SSDU consisting of a hydrazide-functionalized naphthalene-diimide (NDI) chromophore through a urethane linkage, producing a LYS-NDI conjugate having 3 NDI chromophores on average per protein. LYS-NDI was further labeled with green-emitting FITC to produce LYS-NDI-FITC, which showed spontaneous self-assembly in water, producing near-spherical aggregates of ∼90 nm in size. In such an aggregated state, LYS remained in a dormant state and was protected from enzymatic degradation to a significant extent. In the presence of glutathione (GSH), cleavage of the disulfide bond in the linker produced free thiol, which initiated a cascade reaction through intramolecular nucleophilic attack on the adjacent carbamate linkage, releasing LYS in its active form. The LYS-NDI conjugate showed significantly higher cellular uptake selectively in cancer cells, which produced reactive oxygen species (ROS) leading to cell death. Neither LYS nor NDI showed such prominent cellular uptake or cell killing.