Spatially‐Resolved Organoid Transfection by Porous Silicon‐Mediated Optoporation

Abstract Engineering the spatial organisation of organotypic cultures is pivotal for refining tissue models that are useful for gaining deeper insights into complex, non‐cell autonomous processes. These advanced models are key to improving the understanding of fundamental biological mechanisms and therapeutic strategies. Controlling gene regulation through spatially‐resolved delivery of nucleic acids provides an attractive approach to produce such tissue models. An emerging strategy for spatially‐resolved transfection uses photosensitizing nanoparticles coupled with laser pulses to optoporate cells in culture and locally mediate gene delivery. However, localized optoporation in 3D systems remains challenging. Here we propose a solution to this longstanding hurdle, demonstrating that porous silicon nanoparticles are a safe and bioresorbable photosensitising nanomaterial capable of spatially‐resolved transfection of mRNA in MCF‐7 organoids by near‐infrared two‐photon optoporation. Functionalization with an azobenzene–lysine photo‐switchable moiety enhances the transfection efficiency of the nanoparticles up to 84% in a 2D cell system. Moreover, the nanoparticles enable spatially selective mRNA transfection to MCF‐7 spheroids, demonstrating targeted gene delivery in complex 3D cellular environments. The approach for spatially‐resolved 3D optoporation offers a way forward for the design of tailored spheroids and organoids by spatially selective nucleic acids delivery.