Intracellular delivery of advanced therapeutics, including biologicals and supramolecular agents, is complex because of the natural biological barriers that have evolved to protect the cell. Efficient delivery of therapeutic nucleic acids, proteins, peptides and nanoparticles is crucial for clinical adoption of emerging technologies that can benefit disease treatment through gene and cell therapy. Nanoneedles are arrays of vertical high-aspect-ratio nanostructures that can precisely manipulate complex processes at the cell interface, enabling effective intracellular delivery. This emerging technology has already enabled the development of efficient and non-destructive routes for direct access to intracellular environments and delivery of cell-impermeant payloads. However, successful implementation of this technology requires knowledge of several scientific fields, making it complex to access and adopt by researchers who are not directly involved in developing nanoneedle platforms. This presents an obstacle to the widespread adoption of nanoneedle technologies for drug delivery. This tutorial aims to equip researchers with the knowledge required to develop a nanoinjection workflow. It discusses the selection of nanoneedle devices, approaches for cargo loading and strategies for interfacing to biological systems and summarises an array of bioassays that can be used to evaluate the efficacy of intracellular delivery.
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This work was funded in part by the Australian government (ARC DECRA project number: DE170100021). C.C. acknowledges funding from the European Research Council Starting Grant (ENBION 759577) and CureEB. N.H.V. acknowledges funding from the CSIRO Research Office for a Science Leader Fellowship and from the Alexander von Humboldt Foundation for Fellowship for Experienced Researchers. W.Z. acknowledges funding from Singapore Ministry of Education (MOE) Academic Research Fund Tier 1 (RG145/18 and RG112/20), Singapore National Research Foundation NRF-ISF joint grant (NRF2019-NRF-ISF003-3292) and Nanyang Technological University Start-Up Grant and NTU-NNI Neurotechnology Fellowship. X.X. acknowledges financial support from the National Natural Science Foundation of China (grant nos. 61771498, 51705543 and 31530023). H.M. thanks the Interdisciplinary Graduate School (IGS) Research Scholarship from the Ageing Research Institute for Society and Education at Nanyang Technological University. The work was conducted in part at the Melbourne Centre for Nanofabrication (MCN) in the Victorian Node of the Australian National Fabrication Facility (ANFF).
The authors declare no competing interests.
Peer review information Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.
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Chiappini, C., Chen, Y., Aslanoglou, S. et al. Tutorial: using nanoneedles for intracellular delivery. Nat Protoc 16, 4539–4563 (2021). https://doi.org/10.1038/s41596-021-00600-7
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