Detaching Photosensitive Nanoparticles from Cells Prevents Them from Remaining in Cells after Photoporation.

Efficient and safe intracellular delivery of macromolecules, like nucleic acids, remains a major challenge in ex vivo cell engineering. While photoporation is a highly attractive method for cytosolic delivery of (macro)molecules, photosensitizers remaining in the photoporated cells may pose safety concerns, especially when the cells are engineered for therapeutic use. In this study we introduce the concept of "detachable photosensitizers" using gold nanoparticles (AuNPs) as photosensitizer. We demonstrate that phenylboronic acid (PBA)-functionalized AuNPs can be anchored to membranes of HeLa cells and Jurkat cells, thereby enabling the efficient intracellular delivery of dextrans and mRNA upon pulsed laser irradiation (photoporation). Subsequently, the PBA-AuNPs can be detached from the cells "on-demand" by adding ATP to the cell medium, as cis-diol groups in ATP displace the boronate ester bonds between the PBA-AuNPs and cell-surface. ICP-MS analysis demonstrated efficient detachment, with the average cell-associated gold mass per viable cell falling below the theoretical mass of a single intact 80 nm AuNP. This "attach-and-detach" strategy offers a robust and effective approach to addressing safety concerns associated with the use of plasmonic nanomaterials as sensitizers for photoporation, and holds significant potential for the safe manufacturing of engineered cells in the context of cell therapy.