Cellular Responses to the Subtle Perturbation of Lipid Ordering in the Plasma Membrane Induced by Intracellular Delivery Agents

Certain polycations have been used to deliver materials into cells. Polyethylenimine (PEI) acts as a nonviral vector by forming complexes with DNA and RNA. Additionally, some cationic peptides, known as cell-penetrating peptides, promote the internalization of materials that are conjugated to them. However, these intracellular delivery agents damage plasma membranes due to their positive charges and are thus used at sufficiently low concentrations to prevent cytotoxicity. Recently, we developed an assay based on nonlinear optical processes, which revealed that the ordering of lipid molecules in the plasma membrane is perturbed at concentrations at which conventional methods cannot detect cytotoxicity. Thus, the present study was devoted to revealing the cellular responses to subtle perturbations in the plasma membrane. Branched and linear PEIs and a 10-mer arginine peptide, which have no payloads, were applied to living cells at a concentration where lipid ordering in the membrane was perturbed, but with almost no detectable cytotoxicity. The perturbed cells were found to repair their plasma membranes similar to those with an ordered lipid alignment when cultured in a growth medium. During recovery, a few enlarged and shrunken cells with peculiar shapes were temporarily observed. However, these cells did not senesce after exposure to subtle plasma membrane stress. The elucidation of these cellular responses, which have been overlooked thus far, is crucial not only to the biomedical application of intracellular delivery agents but also to the fundamental understanding of how cells manage subtle stress.