Photoactivatable protecting groups (PPGs) are valuable tools in chemical biology which allow for efficient delivery of “caged” biomolecules both in vitro and in vivo. Lower energy two-photon excitation enhances the spatial precision and tissue penetration of payload activation in biological samples while also reducing phototoxicity when compared with higher energy UV light. In view of the importance biological thiols play in drug, peptide, and protein chemistries, nitrodibenzofuran (NDBF) has emerged as a highly effective PPG for thiols, with good uncaging efficiency and two-photon cross-section. In this report, we probe the spatial resolution limits for single cell applications of two-photon NDBF-uncaging in live cells. We functionalized NDBF with a thiol-containing drug, farnesyltransferase inhibitor L-744,832 (NDBF-FTI), where the free thiol is essential for the drug's activity of inhibiting protein farnesylation. Using Madin-Darby Canine Kidney (MDCK) cells expressing GFP-Ras protein, observation of cellular Ras distribution after NDBF-FTI treatment and irradiation (either membrane-bound or cytosolic) provides a clear and straightforward assay for NDBF-uncaging and FTI release. We found that cells exhibiting inhibited Ras farnesylation were largely confined to an irradiated region of interest in 2D-cultured cells. Furthermore, when cultured in 3D Matrigel, we observed single-cell control over NDBF-FTI photolysis and drug delivery with resolution down to 15 µm. These results demonstrate that biomolecule activation in individual cells is possible using two-photon microscopy and highlight the utility of NDBF-protected thiols for biological applications.

Targeted drug delivery in 2- and 3D cultured cells by two-photon activation of a nitrodibenzofuran-caged farnesyltransferase inhibitor

255th National Meeting (2018)

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