Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Photothermal nanofibres enable safe engineering of therapeutic cells

Nature Nanotechnology volume 16, pages 1281–1291 (2021)Cite this article

Subjects

Abstract

Nanoparticle-sensitized photoporation is an upcoming approach for the intracellular delivery of biologics, combining high efficiency and throughput with excellent cell viability. However, as it relies on close contact between nanoparticles and cells, its translation towards clinical applications is hampered by safety and regulatory concerns. Here we show that light-sensitive iron oxide nanoparticles embedded in biocompatible electrospun nanofibres induce membrane permeabilization by photothermal effects without direct cellular contact with the nanoparticles. The photothermal nanofibres have been successfully used to deliver effector molecules, including CRISPR–Cas9 ribonucleoprotein complexes and short interfering RNA, to adherent and suspension cells, including embryonic stem cells and hard-to-transfect T cells, without affecting cell proliferation or phenotype. In vivo experiments furthermore demonstrated successful tumour regression in mice treated with chimeric antibody receptor T cells in which the expression of programmed cell death protein 1 (PD1) is downregulated after nanofibre photoporation with short interfering RNA to PD1. In conclusion, cell membrane permeabilization with photothermal nanofibres is a promising concept towards the safe and more efficient production of engineered cells for therapeutic applications, including stem cell or adoptive T cell therapy.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Concept of intracellular delivery by photothermal nanofibres and characterization of photothermal electrospun nanofibres.
Fig. 2: PEN photoporation enables safe and efficient delivery of macromolecules to cells.
Fig. 3: PEN photoporation for siRNA gene silencing or CRISPR–Cas9-mediated gene knockout in H1299.
Fig. 4: PEN photoporation enables efficient intracellular delivery of macromolecules into hESCs.
Fig. 5: PEN photoporation enables efficient intracellular delivery of siRNA into T cells.
Fig. 6: PEN photoporation also retains T cell effector functions in vivo.

Similar content being viewed by others

Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information. Source data are provided with this paper. Any further related information can be provided by the corresponding author upon reasonable request.

References

  1. Lee, J. et al. Recent advances in genome editing of stem cells for drug discovery and therapeutic application. Pharmacol. Ther. 209, 107501 (2020).

    Article  CAS  Google Scholar 

  2. Vodnala, S. K. et al. T cell stemness and dysfunction in tumors are triggered by a common mechanism. Science 363, eaau0135 (2019).

    Article  CAS  Google Scholar 

  3. Cox, D. B. T., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).

    Article  CAS  Google Scholar 

  4. Zhou, P. H. et al. In vivo discovery of immunotherapy targets in the tumour microenvironment. Nature 506, 52–57 (2014).

    Article  CAS  Google Scholar 

  5. McManus, M. T. et al. Small interfering RNA-mediated gene silencing in T lymphocytes. J. Immunol. 169, 5754–5760 (2002).

    Article  CAS  Google Scholar 

  6. June, C. H., Blazar, B. R. & Riley, J. L. Engineering lymphocyte subsets: tools, trials and tribulations. Nat. Rev. Immunol. 9, 704–716 (2009).

    Article  CAS  Google Scholar 

  7. Peer, D. A daunting task: manipulating leukocyte function with RNAi. Immunol. Rev. 253, 185–197 (2013).

    Article  Google Scholar 

  8. Yin, H. et al. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 15, 541–555 (2014).

    Article  CAS  Google Scholar 

  9. Kaiser J. Gene therapy trials for sickle cell disease halted after two patients develop cancer. Science https://doi.org/10.1126/science.abh1106 (2021).

  10. Stewart, M. P., Langer, R. & Jensen, K. F. Intracellular delivery by membrane disruption: mechanisms, strategies, and concepts. Chem. Rev. 118, 7409–7531 (2018).

    Article  CAS  Google Scholar 

  11. Stewart, M. P. et al. In vitro and ex vivo strategies for intracellular delivery. Nature 538, 183–192 (2016).

    Article  CAS  Google Scholar 

  12. Chakravarty, P., Qian, W., El-Sayed, M. A. & Prausnitz, M. R. Delivery of molecules into cells using carbon nanoparticles activated by femtosecond laser pulses. Nat. Nanotechnol. 5, 607–611 (2010).

    Article  CAS  Google Scholar 

  13. Baumgart, J. et al. Off-resonance plasmonic enhanced femtosecond laser optoporation and transfection of cancer cells. Biomaterials 33, 2345–2350 (2012).

    Article  CAS  Google Scholar 

  14. Lukianova-Hleb, E. Y., Ren, X. Y., Zasadzinski, J. A., Wu, X. W. & Lapotko, D. O. Plasmonic nanobubbles enhance efficacy and selectivity of chemotherapy against drug-resistant cancer cells. Adv. Mater. 24, 3831–3837 (2012).

    Article  CAS  Google Scholar 

  15. Heinemann, D. et al. Delivery of proteins to mammalian cells via gold nanoparticle mediated laser transfection. Nanotechnology 25, 245101 (2014).

    Article  Google Scholar 

  16. Lakshmanan, S. et al. Physical energy for drug delivery; poration, concentration and activation. Adv. Drug Deliv. Rev. 71, 98–114 (2014).

    Article  CAS  Google Scholar 

  17. Sengupta, A., Kelly, S. C., Dwivedi, N., Thadhani, N. & Prausnitz, M. R. Efficient intracellular delivery of molecules with high cell viability using nanosecond-pulsed laser-activated carbon nanoparticles. ACS Nano 8, 2889–2899 (2014).

    Article  CAS  Google Scholar 

  18. Xiong, R. H. et al. Laser-assisted photoporation: fundamentals, technological advances and applications. Adv. Phys. X 1, 596–620 (2016).

    CAS  Google Scholar 

  19. Liu, J. et al. Repeated photoporation with graphene quantum dots enables homogeneous labeling of live cells with extrinsic markers for fluorescence microscopy. Light Sci. Appl. 7, 47 (2018).

    Article  Google Scholar 

  20. Soenen, S. J. et al. Cellular toxicity of inorganic nanoparticles: common aspects and guidelines for improved nanotoxicity evaluation. Nano Today 6, 446–465 (2011).

    Article  CAS  Google Scholar 

  21. Soenen, S. J., De Cuyper, M., De Smedt, S. C. & Braeckmans K. in Methods in Enzymology Vol. 509 (ed. Düzgüneş, N.) 195–224 (Academic, 2012).

  22. Soenen, S. J. et al. Cytotoxic effects of gold nanoparticles: a multiparametric study. ACS Nano 6, 5767–5783 (2012).

    Article  CAS  Google Scholar 

  23. Joris, F. et al. Assessing nanoparticle toxicity in cell-based assays: influence of cell culture parameters and optimized models for bridging the in vitro–in vivo gap. Chem. Soc. Rev. 42, 8339–8359 (2013).

    Article  CAS  Google Scholar 

  24. Soenen, S. J. et al. The cellular interactions of PEGylated gold nanoparticles: effect of PEGylation on cellular uptake and cytotoxicity. Part. Part. Syst. Charact. 31, 794–800 (2014).

    Article  CAS  Google Scholar 

  25. Malysheva, A., Ivask, A., Doolette, C. L., Voelcker, N. H. & Lombi, E. Cellular binding, uptake and biotransformation of silver nanoparticles in human T lymphocytes. Nat. Nanotechnol. 16, 926–932 (2021).

    Article  Google Scholar 

  26. Harizaj, A. et al. Cytosolic delivery of gadolinium via photoporation enables improved in vivo magnetic resonance imaging of cancer cells. Biomater. Sci. 9, 4005–4018 (2021).

    Article  CAS  Google Scholar 

  27. Huang, C. et al. Stimuli-responsive electrospun fibers and their applications. Chem. Soc. Rev. 40, 2417–2434 (2011).

    Article  CAS  Google Scholar 

  28. Lv, D., et al. Green electrospun nanofibers and their application in air filtration. Macromol. Mater. Eng. 303, 1800336 (2018).

    Article  Google Scholar 

  29. Yamanaka, S. Pluripotent stem cell-based cell therapy—promise and challenges. Cell Stem Cell 27, 523–531 (2020).

    Article  CAS  Google Scholar 

  30. Bargehr, J. et al. Epicardial cells derived from human embryonic stem cells augment cardiomyocyte-driven heart regeneration. Nat. Biotechnol. 37, 895–906 (2019).

    Article  CAS  Google Scholar 

  31. Pavel-Dinu, M. et al. Gene correction for SCID-X1 in long-term hematopoietic stem cells. Nat. Commun. 10, 1634 (2019).

    Article  Google Scholar 

  32. Fee, T., Surianarayanan, S., Downs, C., Zhou, Y. & Berry, J. Nanofiber alignment regulates NIH3T3 cell orientation and cytoskeletal gene expression on electrospun PCL+gelatin nanofibers. PLoS ONE 11, e0154806 (2016).

    Article  Google Scholar 

  33. Schmader, K. E. et al. Effects of geriatric evaluation and management on adverse drug reactions and suboptimal prescribing in the frail elderly. Am. J. Med. 116, 394–401 (2004).

    Article  Google Scholar 

  34. DiTommaso, T. et al. Cell engineering with microfluidic squeezing preserves functionality of primary immune cells in vivo. Proc. Natl Acad. Sci. USA 115, E10907–E10914 (2018).

    Article  CAS  Google Scholar 

  35. Zhang, M. et al. The impact of Nucleofection® on the activation state of primary human CD4 T cells. J. Immunol. Methods 408, 123–131 (2014).

    Article  CAS  Google Scholar 

  36. Xiong, R. H. et al. Comparison of gold nanoparticle mediated photoporation: vapor nanobubbles outperform direct heating for delivering macromolecules in live cells. ACS Nano 8, 6288–6296 (2014).

    Article  CAS  Google Scholar 

  37. Xiong, R. H. et al. Cytosolic delivery of nanolabels prevents their asymmetric inheritance and enables extended quantitative in vivo cell imaging. Nano Lett. 16, 5975–5986 (2016).

    Article  CAS  Google Scholar 

  38. Cathcart, R., Schwiers, E. & Ames, B. N. Detection of picomole levels of hydroperoxides using a fluorescent dichlorofluorescein assay. Anal. Biochem. 134, 111–116 (1983).

    Article  CAS  Google Scholar 

  39. Lebel, C. P., Ischiropoulos, H. & Bondy, S. C. Evaluation of the probe 2′,7′-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem. Res. Toxicol. 5, 227–231 (1992).

    Article  CAS  Google Scholar 

  40. Bolea-Fernandez, E., Balcaen, L., Resano, M. & Vanhaecke, F. Overcoming spectral overlap via inductively coupled plasma-tandem mass spectrometry (ICP-MS/MS). A tutorial review. J. Anal. At. Spectrom. 32, 1660–1679 (2017).

    Article  CAS  Google Scholar 

  41. Encina, E. R. & Coronado, E. A. Plasmon coupling in silver nanosphere pairs. J. Phys. Chem. C. 114, 3918–3923 (2010).

    Article  CAS  Google Scholar 

  42. Encina, E. R. & Coronado, E. A. On the far field optical properties of Ag–Au nanosphere pairs. J. Phys. Chem. C 114, 16278–16284 (2010).

    Article  CAS  Google Scholar 

  43. Querry, M. R. Optical Constants. PhD thesis, Univ. Missouri (1985).

  44. Chettiar, U. K. & Engheta, N. Internal homogenization: effective permittivity of a coated sphere. Opt. Express 20, 22976–22986 (2012).

    Article  Google Scholar 

  45. Agari, Y. & Ueda, A. Thermal-conductivity of poly(vinyl chloride) polycaprolactone blends. J. Polym. Sci. B 32, 59–62 (1994).

    Article  CAS  Google Scholar 

  46. Costa, M. et al. A method for genetic modification of human embryonic stem cells using electroporation. Nat. Protoc. 2, 792–796 (2007).

    Article  CAS  Google Scholar 

  47. Helledie, T., Nurcombe, V. & Cool, S. M. A simple and reliable electroporation method for human bone marrow mesenchymal stem cells. Stem Cells Dev. 17, 837–848 (2008).

    Article  CAS  Google Scholar 

  48. Pieters, T. et al. Efficient and user-friendly pluripotin-based derivation of mouse embryonic stem cells. Stem Cell Rev. Rep. 8, 768–778 (2012).

    Article  CAS  Google Scholar 

  49. De Munter, S. et al. Rapid and effective generation of nanobody based CARs using PCR and Gibson assembly. Int. J. Mol. Sci. https://doi.org/10.3390/ijms21030883 (2020).

  50. De Munter, S. et al. Nanobody based dual specific CARs. Int. J. Mol. Sci. https://doi.org/10.3390/ijms19020403 (2018).

Download references

Acknowledgements

This research was supported by the National Natural Science Foundation of China (grant nos 21774060 and 21644004), the European Research Council (ERC Consolidator Grant 648124), the Research Foundation Flanders (FWO, 1500418N, 12Q8718N and 1S62517N), the National Key R&D Program of China (2017YFF0207804), and the Youth Innovation Promotion Association CAS (grant no. 2018491). We acknowledge funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement number 810685 (DelNam project).

Author information

Authors and Affiliations

  1. Joint Laboratory of Advanced Biomedical Materials (Nanjing Forestry University–Ghent University), International Innovation for Center for Forest Chemicals and Materials, College of Chemical Engineering, Nanjing Forestry University, Nanjing, People’s Republic of China

    Ranhua Xiong, Dawei Hua, Chaobo Huang & Stefaan C. De Smedt

  2. Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium

    Ranhua Xiong, Dawei Hua, Jelter Van Hoeck, Dominika Berdecka, Juan C. Fraire, Laurens Raes, Aranit Harizaj, Félix Sauvage, Koen Raemdonck, Stefaan C. De Smedt & Kevin Braeckmans

  3. Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerp, Belgium

    Dominika Berdecka & Winnok H. De Vos

  4. Department of Human Structure and Repair, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium

    Laurens Léger, Jeffrey Aalders & Jolanda van Hengel

  5. Department of Diagnostic Sciences and Cancer Research Institute Ghent, Ghent University, Ghent, Belgium

    Stijn De Munter

  6. Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium

    Glenn Goetgeluk, Melissa Pille & Bart Vandekerckhove

  7. Department of Chemistry, Atomic and Mass Spectrometry Research Group, Ghent University, Ghent, Belgium

    Joke Belza, Thibaut Van Acker, Eduardo Bolea-Fernandez & Frank Vanhaecke

  8. Department of Modern Mechanics, University of Science and Technology of China, Hefei, People’s Republic of China

    Ting Si

  9. Center for Advanced Light Microscopy, Ghent University, Ghent, Belgium

    Stefaan C. De Smedt & Kevin Braeckmans

Authors
  1. Ranhua Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dawei Hua
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jelter Van Hoeck
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dominika Berdecka
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Laurens Léger
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Stijn De Munter
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Juan C. Fraire
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Laurens Raes
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Aranit Harizaj
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Félix Sauvage
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Glenn Goetgeluk
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Melissa Pille
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Jeffrey Aalders
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Joke Belza
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Thibaut Van Acker
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Eduardo Bolea-Fernandez
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Ting Si
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Frank Vanhaecke
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Winnok H. De Vos
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Bart Vandekerckhove
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Jolanda van Hengel
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Koen Raemdonck
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Chaobo Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Stefaan C. De Smedt
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Kevin Braeckmans
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.X., K.B. and S.C.D.S. conceived the concept of PEN photoporation for contact-free photoporation and designed the experiments. R.X., C.H. and D.H. fabricated and characterized the PEN substrates. R.X. performed the PEN photoporation experiments and analysed the data, and also performed the CFD simulations together with J.C.F. J.B., E.B.-F. and T.V.A. performed and analysed the ICP-MS/MS measurements. L.R. and M.P. prepared the RNPs. L.L. and J.A. were involved in the preparation and PEN photoporation of hESCs. J.V.H., D.B., S.D.M., K.R., G.G., A.H., F.S. and B.V. were involved in the preparation and PEN photoporation of human T cells. K.B., S.C.D.S., C.H., K.R., T.S., F.V., J.V.H., W.H.D.V. and B.V. advised on experiments and data analysis. All authors discussed the experimental results and jointly wrote the manuscript.

Corresponding authors

Correspondence to Ranhua Xiong, Chaobo Huang, Stefaan C. De Smedt or Kevin Braeckmans.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Nanotechnology thanks Massimiliano Caiazzo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Tables 1–3, Notes 1–3 and Figs. 1–19.

Reporting Summary

Supplementary Video 1

A 3D volume rendering of photothermal electrospun nanofibres.

Supplementary Video 2

Numerical simulation of the heat transfer from a single IONP to the surrounding medium via the surrounding nanofibre. Black solid circles in the top right and bottom right indicate the profile of nanoparticle. The time unit in the bottom left is second. The orange dashed lines indicate the boundary of the nanofibre. The simulation conditions were h = 40 nm, N = 1 and I = 0.08 J cm–2.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xiong, R., Hua, D., Van Hoeck, J. et al. Photothermal nanofibres enable safe engineering of therapeutic cells. Nat. Nanotechnol. 16, 1281–1291 (2021). https://doi.org/10.1038/s41565-021-00976-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41565-021-00976-3

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research