An efficient nonviral platform for high-throughput and subcellular precision targeted intracellular delivery of nucleic acids in cell culture based on magnetic nanospears is reported. These magnetic nanospears are made of Au/Ni/Si (∼5 μm in length with tip diameters <50 nm) and fabricated by nanosphere lithography and metal deposition. A magnet is used to direct the mechanical motion of a single nanospear, enabling precise control of position and three-dimensional rotation. These nanospears were further functionalized with enhanced green fluorescent protein (eGFP)-expression plasmids via a layer-by-layer approach before release from the underlying silicon substrate. Plasmid functionalized nanospears are guided magnetically to approach target adherent U87 glioblastoma cells, penetrating the cell membrane to enable intracellular delivery of the plasmid cargo. After 24 h, the target cell expresses green fluorescence indicating successful transfection. This nanospear-mediated transfection is readily scalable for the simultaneous manipulation of multiple cells using a rotating magnet. Cell viability >90% and transfection rates >80% were achieved, which exceed conventional nonviral intracellular methods. This approach is compatible with good manufacturing practices, circumventing barriers to the translation and clinical deployment of emerging cellular therapies.

Advances in gene editing are leading to new medical interventions where patients’ own cells are used for stem cell therapies and immunotherapies. One of the key limitations to translating these treatments to the clinic is the need for scalable technologies for engineering cells efficiently and safely. Toward this goal, microfluidic strategies to induce membrane pores and permeability have emerged as promising techniques to deliver biomolecular cargo into cells. As these technologies continue to mature, there is a need to achieve efficient, safe, nontoxic, fast, and economical processing of clinically relevant cell types. We demonstrate an acoustofluidic sonoporation method to deliver plasmids to immortalized and primary human cell types, based on pore formation and permeabilization of cell membranes with acoustic waves. This acoustofluidic-mediated approach achieves fast and efficient intracellular delivery of an enhanced green fluorescent protein-expressing plasmid to cells at a scalable throughput of 200,000 cells/min in a single channel. Analyses of intracellular delivery and nuclear membrane rupture revealed mechanisms underlying acoustofluidic delivery and successful gene expression. Our studies show that acoustofluidic technologies are promising platforms for gene delivery and a useful tool for investigating membrane repair.

While three-dimensional (3D) configurable hierarchical nanostructures have wide ranging applications in electronics, biology, and optics, finding scalable approaches remains a challenge. We report a robust and general strategy called multiple-patterning nanosphere lithography (MP-NSL) for the fabrication of periodic 3D hierarchical nanostructures in a highly scalable and tunable manner. This nanofabrication technique exploits the selected and repeated etching of polymer nanospheres that serve as resists and that can be shaped in parallel for each processing step. The application of MP-NSL enables the fabrication of periodic, vertically aligned Si nanotubes at the wafer scale with nanometer-scale control in three dimensions including outer/inner diameters, heights/hole-depths, and pitches. The MP-NSL method was utilized to construct 3D periodic hierarchical hybrid nanostructures such as multilevel solid/hollow nanotowers where the height and diameter of each level of each structure can be configured precisely as well as 3D concentric plasmonic nanodisk/nanorings with tunable optical properties on a variety of substrates.