Mitochondria‐Specific Protein Delivery by Protease‐Triggered Release in Plants with Single‐Walled Carbon Nanotubes

ABSTRACT Single‐walled carbon nanotubes have shown the exceptional ability to cross into intact plant cells due to their unique physical properties, including their high aspect ratio, surface area, and stiffness. This has led to the development of carbon nanotube‐based nanocarriers for the delivery of nucleic acid cargo into intact plants and their organelles without significant cytotoxicity. However, they have yet to be demonstrated as viable vehicles for the delivery of protein into plants. In this work, we describe the development of a carbon nanotube‐based system for the protease‐triggered delivery of protein cargoes into the mitochondria of Arabidopsis thaliana plants. Green fluorescent protein containing a cytosolic cleavage specific to a widely expressed plant phytaspase is used as a model cargo to characterize delivery and demonstrated efficient cleavage and release from the nanotube surface within the cytosol. Together with a modified N‐terminal ATPase presequence, the cargo can be directed to the mitochondria and remains detectable for up to 18 h. These results suggest that a protease‐triggered system can be used for triggered release within plants by conjugating to nanomaterials such as carbon nanotubes.