Reversible Permeabilization of Cell Membranes via Lysenin Channels

Abstract

The selective nature of the cell-membrane hinders the permeability of drugs, fluorescent probes, and other macromolecules into living cells. To introduce foreign molecules as well as preserve cell viability, reversible permeabilization of the cell membrane is required. To achieve this aim, several techniques have been proposed including microinjection, electroporation, optoporation, and lipophilic and peptide carriers. These methods are either highly invasive, inefficient, limited to single-cell, require sophisticated instruments, or need tedious conjugation process. Therefore, we proposed a simple yet efficient method of controlled transport of exogenous molecules into viable cells using the pore-forming toxin lysenin. Lysenin inserts a large conducting pathway into the lipid bilayer membrane containing sphingomyelin, hence allowing cell-impermeable molecules to cross the membrane barrier. In addition, the lysenin pore is irreversibly blocked by biologically inert chitosan molecules. In this way, the lysenin-induced pore formation and the activity act as a nano-valve. In our work, we temporarily permeabilized mammalian cells ATDC5 with lysenin channels and loaded the membrane-impermeable fluorescent dye propidium iodide. The process was blocked by the addition of chitosan and the viability of the cells was assessed by using viable-cell indicators. Similarly, we employed lysenin channels to introduce the cell-impermeant actin marker phalloidin into the cells. These results indicate that lysenin channel can be used as a simple and efficient tool to deliver bioactive molecules into living cells.