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Biodegradable silicon nanoneedles delivering nucleic acids intracellularly induce localized in vivo neovascularization

Abstract

The controlled delivery of nucleic acids to selected tissues remains an inefficient process mired by low transfection efficacy, poor scalability because of varying efficiency with cell type and location, and questionable safety as a result of toxicity issues arising from the typical materials and procedures employed. High efficiency and minimal toxicity in vitro has been shown for intracellular delivery of nuclei acids by using nanoneedles, yet extending these characteristics to in vivo delivery has been difficult, as current interfacing strategies rely on complex equipment or active cell internalization through prolonged interfacing. Here, we show that a tunable array of biodegradable nanoneedles fabricated by metal-assisted chemical etching of silicon can access the cytosol to co-deliver DNA and siRNA with an efficiency greater than 90%, and that in vivo the nanoneedles transfect the VEGF-165 gene, inducing sustained neovascularization and a localized sixfold increase in blood perfusion in a target region of the muscle.

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Figure 1: Porous silicon nanoneedles.
Figure 2: Cell interfacing, cytocompatibility and biodegradation.
Figure 3: Intracellular co-delivery of nucleic acids.
Figure 4: Nanoneedles mediate in vivo delivery.
Figure 5: Safety profile of nanoinjection.
Figure 6: Nanoneedles mediate neovascularization.

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Acknowledgements

The authors would like to thank M. Giacca at the International Centre for Genetic Engineering and Biotechnology (ICGEB in Trieste, Italy) for kindly donating the pDNA-expressing VEGF-165, and I. K. Yazdi and R. Palomba for the amplification and purification of the plasmids used in the study. We are grateful to S. Amra for histology-slide preparation, D. Tinkey for surgical procedures, S. Zacchigna for suggestions on PCR protocols, P. Campagnolo for advice on SMA and isolectin staining, and S. T. Gammon for advice on bioluminescence imaging with luminol. This work was financially supported by: the US Department of Defense (W81XWH-12-10414) and the NIH (1R21CA173579-01A1 and 5U54CA143837); C.C. was supported by a Newton International Fellowship and a Marie Curie International Incoming Fellowship; J.O.M. was supported by a NIH pre-doctoral fellowship 5F31CA154119-02. M.M.S. holds an ERC grant ‘Naturale-CG’ and is supported by a Wellcome Trust Senior Investigator Award. E.T. holds grants from the Hearst Foundation and the Cullen Trust Foundation. C.C. and M.M.S. thank the Rosetrees Trust and the Stoneygate Trust for funding.

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C.C., E.T. and M.M.S. designed the research. C.C. and E.T. conceived the nanoneedles; C.C. developed the nanoneedles with contributions from X.L.; C.C. evaluated loading, release, delivery and efficacy with contributions from E.D.R.; C.C. imaged biodegradation and cell interaction with contributions from E.D.R.; C.C. wrote the initial manuscript with contributions from E.D.R. and J.O.M. C.C. performed all electron microscopy analysis. E.D.R. and J.O.M. assessed cytocompatibility; E.D.R. designed and performed animal surgeries, intravital imaging and quantification of vascularization, vasculature pattern, and extravasation and flow rate over time on VEGF treatment, with contributions from J.O.M. J.O.M. evaluated degradation by ICP-AES, fluorescent and bioluminescent imaging and analysis, real-time PCR, and histological evaluation and staining of tissues with contributions from E.D.R. J.S. performed compressive mechanical testing. E.T. and M.M.S. contributed equally to the work, with M.M.S. supervising the in vitro studies and E.T. the in vivo work. All authors discussed and commented on the manuscript.

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Correspondence to M. M. Stevens or E. Tasciotti.

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The authors declare no competing financial interests.

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Chiappini, C., De Rosa, E., Martinez, J. et al. Biodegradable silicon nanoneedles delivering nucleic acids intracellularly induce localized in vivo neovascularization. Nature Mater 14, 532–539 (2015). https://doi.org/10.1038/nmat4249

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