Nanosecond pulsed electric fields induce cell-size-dependent selective permeabilization of urothelial cancer cells.

Plasma membrane integrity is vital for cell viability, yet its controlled disruption enables targeted delivery of therapeutic agents. Here, we examined membrane durability and repair capacity in normal and malignant urothelial cells using short, high-voltage nanosecond pulses. Pulses were applied to monolayer cultures, spheroids, and patient-derived organoids. Plasma membrane permeability was assessed via YO-PRO-1 dye uptake, and mechanical effects of permeabilization were analyzed using atomic force microscopy. Urothelial cancer cells exhibited nearly fourfold higher dye uptake than non-malignant cells, along with more pronounced osmotic swelling and loss of cellular stiffness. Membrane resealing in cancer cells was delayed and exhibited stronger dependence on extracellular Ca²⁺. The higher susceptibility of urothelial cells was correlated with their larger size, which enable them to reach the electroporation threshold at lower electric fields. These findings highlight key differences in membrane vulnerability and repair dynamics, providing foundation for the development of membrane-targeted therapies for urothelial cancer.