Intracellular sensing of pathologically relevant biomolecules could provide essential information for accurate evaluation of disease staging and progression, yet the poor cellular uptake of water-soluble molecular probes limits their use as protease sensors. In other cases such as extracellular sensing, cellular uptake should be effectively inhibited. Self-assembly of molecular probes into supramolecular nanoprobes presents a potential strategy to alter their interaction mechanisms with cells to promote or reduce their cellular uptake. Here, we report on the design, synthesis, and assembly of peptide-based molecular beacons into supramolecular protease sensors of either spherical or filamentous shapes. We found that positively charged spherical nanobeacons demonstrate much higher cellular uptake efficiency than its monomeric form, thus making them most suitable for intracellular sensing of the lysosomal protease cathepsin B. Our results also suggest that assembly into filamentous nanobeacons significantly reduces their internalization by cancer cells, an important property that can be utilized for probing extracellular protease activities. These studies provide important guiding principles for rational design of supramolecular nanoprobes with tunable cellular uptake characteristics.

We report here on the use of anticancer drug doxorubicin (Dox) to construct a Forster resonance energy transfer (FRET)-based theranostic molecular probe by covalently linking together through a lysine junction a fluorescent drug, a black hole quencher, and a cell-penetrating peptide. We show that upon cleavage by the target lysosomal protease cathepsin B (CatB) the designed drug beacon could release the fluorescent drug serving as an indicator for CatB. Our cell studies suggest that the drug-beacon design can help to circumvent the Dox drug resistance in NCI/ADR-Res ovarian cancer cells, showing significant improvement in cell cytotoxicity compared to the free drug. We believe our design opens up new opportunities to exploit the new functional and structural features of anticancer drugs in addition to their characteristic cytotoxicity.