Introducing exogenous biomolecules into individual cells with precise control over space, time, and dosage is crucial for both fundamental and applied biological research. Glass nanopipettes have long been employed to deliver biomolecules into individual cells; yet, their reliance on the electrical charge of the target molecule and the need for penetrating the cellular membrane pose significant limitations. We demonstrate that voltage pulses applied through a glass nanopipette in proximity to the cell membrane induce localized electroporation and generate directional flow, enabling controlled delivery of both charged and neutral biomolecules into subcellular compartments, e.g., the nucleus, without the need for penetrating the cellular membrane. This approach minimizes cell damage and preserves cell viability, even after multiple rounds of injection. Our findings will serve as a reference for the design of novel nanopipette methods, contributing to the newly established field of spatiotemporal analysis of live cells
