Synthetic crosslinked polymeric nanoparticles are attractive carriers for a wide variety of therapeutic

molecules for both systemic and local delivery applications. Covalent crosslinked nanoparticles are

resilient to disassembly triggered by dilution or interaction with extracellular proteins such as serum

albumin. To be suitable as intracellular drug delivery device, a nanoparticle should disassemble upon

cellular uptake to release a cargo and to be cleared from the body afterwards. For this purpose,

ketals are capable to selectively induce nanoparticle disassembly in response to the acidic pH of 5-6

in endosomes where nanoparticles are typically stored upon endocytosis.

Despite the fact that for several crosslinked nanoparticle systems it has unambiguously been

demonstrated that degradation effectively occurs in response to a trigger, it remains elusive

whether this is also the case in a living system. Therefore, we aim to devise a strategy based on FRET

(Föster Resonance Energy Transfer) which allows for determining whether or not two fluorescent

molecules are within close proximity. FRET has intensively been used for monitoring intracellular

disassembly of non-covalently bound assemblies, however, to the best of our knowledge, the use of

FRET for studying intracellular degradation in vitro and in vivo of synthetic covalently linked

nanostructures is unexplored.