Structure-Dependent Modulation of Light-Induced Membrane Permeabilization by Photoresponsive Tetraphenylethene Derivatives Revealed through Multiscale Simulations and Cellular Experiments

Medium-sized therapeutics such as nucleic acids and peptides often exhibit poor membrane permeability, posing a major barrier to efficient intracellular delivery. To address this challenge, we previously developed a light-driven molecular machine based on a tetraphenylethene derivative ( TPE-C8-N ) that enables carrier-free intracellular delivery of biomolecules under light irradiation. However, the molecular mechanism underlying light-induced membrane permeabilization remains unclear. Here, we combined cellular experiments with multiscale molecular dynamics (MD), path-sampling, and umbrella-sampling simulations to establish a structure-dependent strategy for controlling membrane permeabilization induced by photoresponsive TPE derivatives. A series of derivatives, TPE-C n -N ( n = 4, 8, 12, and 16), was synthesized to examine the effect of alkyl chain length on intracellular delivery. Cellular experiments showed that TPE-C8-N localizes to the plasma membrane and exhibits the highest endocytosis-independent delivery efficiency, whereas longer-chain derivatives tend to form intracellular aggregates and rely more strongly on endocytosis-mediated uptake, demonstrating that alkyl chain length enables switching between distinct intracellular delivery pathways. Coarse-grained MD simulations revealed that alkyl chain length governs the self-assembly morphology of TPE molecules on the membrane, with TPE-C8-N forming linear assemblies that induce pronounced membrane curvature. Furthermore, rare-event simulations using ∞RETIS and umbrella sampling showed that TPE-C8-N modestly promotes pore nucleation and accumulates more strongly near the pore than TPE-C16-N, suggesting that multiple TPE-C8-N molecules can cooperatively enhance photoisomerization-induced membrane perturbation. However, TPE-C8-N does not stabilize expanded open pores, supporting transient membrane-defect formation rather than persistent pore stabilization as the primary permeabilization mechanism. These results provide molecular-level and thermodynamic insights into light-induced membrane permeabilization and establish design guidelines for light-driven intracellular delivery systems.