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Tutorial: using nanoneedles for intracellular delivery

Nature Protocols volume 16, pages 4539–4563 (2021)Cite this article

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Abstract

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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Fig. 1: The five key factors determining a nanoinjection workflow.
Fig. 2: Scanning electron microscopy images of nanoneedle devices and their interfacial interactions with adherent cells.
Fig. 3: Key steps for six common nanoneedle fabrication methods and representative scanning electron microscopy (SEM) images.
Fig. 4: Modalities of nanoneedle interfacing.
Fig. 5: Evaluating nanoinjection.
Fig. 6: Characterization of nanoinjection by electron microscopy.

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This paper does not include previously unpublished results. Thus, no data in raw format are made available.

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Acknowledgements

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).

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  1. These authors contributed equally: Ciro Chiappini, Yaping Chen.

Authors and Affiliations

  1. Centre for Craniofacial and Regenerative Biology, King’s College London, London, UK

    Ciro Chiappini

  2. London Centre for Nanotechnology, King’s College London, London, UK

    Ciro Chiappini

  3. Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia

    Yaping Chen, Stella Aslanoglou, Nicolas H. Voelcker & Roey Elnathan

  4. Melbourne Centre for Nanofabrication, Victorian Node of the Australian National Fabrication Facility, Clayton, Victoria, Australia

    Yaping Chen, Stella Aslanoglou, Nicolas H. Voelcker & Roey Elnathan

  5. CSIRO Manufacturing, Clayton, Victoria, Australia

    Stella Aslanoglou & Nicolas H. Voelcker

  6. Center for Advanced Biomaterials for Healthcare, Istituto Italiano di Tecnologia, Naples, Italy

    Anna Mariano, Valentina Mollo & Francesca Santoro

  7. School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore

    Huanwen Mu & Wenting Zhao

  8. Center for Musculoskeletal Regeneration, Orthopedics & Sports Medicine, Houston Methodist Research Institute, Houston, TX, USA

    Enrica De Rosa

  9. State Key Laboratory of Optoelectronic Materials and Technologies, Guangdong Province Key Laboratory of Display Material and Technology, School of Electronics and Information Technology, Sun Yat-sen University, Guangzhou, China

    Gen He & Xi Xie

  10. IRCCS San Raffaele Pisana Hospital, Rome, Italy

    Ennio Tasciotti

  11. San Raffaele University, Rome, Italy

    Ennio Tasciotti

  12. Sclavo Pharma, Siena, Italy

    Ennio Tasciotti

  13. Department of Materials Science and Engineering, Monash University, Clayton, Victoria, Australia

    Nicolas H. Voelcker & Roey Elnathan

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  1. Ciro Chiappini
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Contributions

C.C., X.X., F.S., W.Z., N.H.V. and R.E. were responsible for conceptualization. C.C., W.Z., N.H.V. and R.E. were responsible for funding acquisition. C.C. and R.E. were responsible for project administration. C.C., Y.C., S.A., A.M., V.M., H.M., E.D.R., G.H., X.X., F.S., W.Z. and R.E. were responsible for writing the original draft.C.C., Y.C., S.A., A.M., V.M., H.M., G.H., E.T., X.X., F.S., W.Z., N.H.V. and R.E. were responsible for reviewing and editing the manuscript.

Corresponding authors

Correspondence to Ciro Chiappini, Xi Xie, Francesca Santoro, Wenting Zhao, Nicolas H. Voelcker or Roey Elnathan.

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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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