In certain tumor and diseased tissues, reactive oxygen species (ROS), such as H2O2, are produced in higher concentrations than in healthy cells. Drug delivery and release systems that respond selectively...

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The functional activity and differentiation potential of cells is determined by their interaction with surrounding cells. Approaches that allow unbiased characterization of cell states while at the same time providing spatial information are of major value to assess this environmental influence. However, most current techniques are hampered by a trade-off between spatial resolution and cell profiling depth. Here, we developed a photocage-based technology that allows isolation and in-depth analysis of live cells from regions of interest in complex ex vivo systems, including primary human tissues. The use of a highly sensitive 4-nitrophenyl(benzofuran)-cage coupled to a set of nanobodies allowed high-resolution photo-uncaging of different cell types in areas of interest. Single cell RNA-sequencing of spatially defined CD8 + T cells was used to exemplify the feasibility of identifying location-dependent cell states. The technology described here provides a valuable tool for analysis of spatially defined cells in diverse biological systems, including clinical samples.

The functional activity and differentiation potential of cells is determined by their interaction with surrounding cells. Approaches that allow the unbiased characterization of cell states while at the same time providing spatial information are of major value to assess this environmental influence. However, most current techniques are hampered by a trade-off between spatial resolution and cell profiling depth. Here, we developed a photoswitch-based technology that allows the isolation and in-depth analysis of live cells from regions of interest in complex ex vivo systems, including human tissues. The use of a highly sensitive 4-nitrophenyl(benzofuran)-cage coupled to nanobodies allowed photoswitching of cells in areas of interest with low-intensity violet light and without detectable phototoxicity. Single cell RNA sequencing of spatially defined CD8+ T cells was used to exemplify the feasibility of identifying location-dependent cell states at the single cell level. Finally, we demonstrate the efficient labeling and photoswitching of cells in live primary human tumor tissue. The technology described here provides a valuable tool for the analysis of spatially defined cells in diverse biological systems, including clinical samples.

In certain tumor and diseased tissues, reactive oxygen species (ROS), such as H2O2, are produced in higher concentrations than in healthy cells. To date, only few examples of drug delivery and release systems responds selectively to these small but significantly elevated ROS concentrations. In addition, assuring the stability of the polymer-based carrier in “healthy” biological conditions is still a challenge in the field of oxidation-sensitive materials. Here, we present ROS-responsive block copolymer micelles capable of achieving micellar disruption over days in the presence of 2 mM H2O2 and within hours under higher concentrations of H2O2 (60 – 600 mM). At the same time, these micelles are stable for over two weeks in oxidant-free physiological (pH = 7.4, 37°C) and for at least six days in mildly acidic (pH = 5.0 and pH = 6.0, 37°C) conditions. The observed selectivity is programmed into the material using a 4-(methylthio)phenyl ester based logic gate. Here, oxidation of the thioether moiety results in a large increase in ester hydrolytic lability, effectively switching the ester hydrolysis from off to on. The concept represents a step forward to realize signal responsive drug delivery materials capable of selective action in biological environments.