An integrated nanopore-microfluidic platform for low-voltage electroporation of delivering self-amplifying RNA into dendritic cells.

Efficient delivery of large, negatively charged self-amplifying RNA (saRNA) into dendritic cells (DCs) is critical for next-generation cancer vaccines. However, this remains challenging due to the high sensitivity of DCs to chemical carriers and high-voltage electroporation. In this study, an integrated nanopore-electroporation (NEP) microdevice was developed by combining 200 nm track-etched polycarbonate membrane, bidirectional PDMS microfluidic channels, and Pt/ITO electrodes to localize the electric field and induce membrane permeabilization at low voltage (≤30 V). Multiphysics simulations revealed that 200 nm nanopores concentrated the electric field at the cell-membrane interface, generating transmembrane potentials exceeding 3 V. Using DC2.4, the NEP system achieved 75% propidium iodide (PI) uptake at 25 V with 90% viability, confirming controllable nanoscale perforation. Direct delivery of GFP-encoding saRNA achieved approximately 50% transfection efficiency with sustained protein expression for more than 96 h, significantly outperforming mRNA at an equal dose. Long-term viability (>85% at 96 h) and negligible cytotoxicity demonstrated the excellent biocompatibility of the device. This reagent-minimal, modular NEP platform thus provided a high-efficiency, low-toxicity route for saRNA delivery into hard-to-transfect immune cells, offering a versatile engineering framework for DC-based cancer immunotherapy, RNA vaccine development, and broader cell gene-modification applications.