Advances in nano-medicine require conceptual understanding of physiological processes. Apoptosis is a fundamental physiological process, which is characterised among other things by an increased permeability of the nuclear envelope (NE). The latter is a tight transport barrier, known to restrict nuclear delivery rate of therapeutic nano-particles. Therefore, an understanding of the underlying mechanism, which leads to the breakdown of the barrier during apoptosis, could stimulate the development of new approaches in gene therapy. We set out to elucidate this mechanism following induction of apoptosis on isolated cell nuclei. We tested the hypothesis if caspases, mediators of apoptosis, trigger the NE leakiness at the level of the nuclear pore complexes (NPCs) using fluorescence techniques. As the permeability barrier inside the NPC channel is thought to be based on hydrophobic-hydrophobic protein interactions we further investigated the NPC channel hydrophobicity using atomic force microscopy (AFM). Caspase-9 was found to induce NE leakiness to large macromolecules. Leakiness was prevented by pretreatment of NPCs with an importin-b mutant, which irreversibly binds and thereby obstructs the NPC channel. Utilizing an ultra sharp, hydrophobic AFM tip as a chemical nano-sensor, which reaches deep into the apoptotic NPC channel, a remarkable decrease of hydrophobic binding sites was detected therein. We conclude that caspase 9 gives rise to NE leakiness by perturbing the hydrophobicty-based barrier inside the NPC channel. This explains the high passive NE permeability in early apoptosis.