Pre- and Postfunctionalization of Dye-Loaded Polymeric Nanoparticles for Preparation of FRET-Based Nanoprobes

Surface modification of nanoparticles (NPs) provides them with desired functions for biological and biomedical applications. A unique feature of polymeric NPs is flexibility in their modification through pre- and postfunctionalization. Here, we explored these two approaches in the case of dye-loaded NPs, formulated by nanoprecipitation from a hydrophobic polymer based on poly(methyl methacrylate) (PMMA). We synthesized two PMMA-conjugates bearing an azide group together with one or three negatively charged carboxylates to ensure surface exposure of the reactive group. In our postfunctionalization approach, we prepared azide-bearing NPs and then monitored by Forster resonance energy transfer (FRET) the in situ click reaction with the fluorescent ligand bearing strained alkyne (DBCO). Remarkably, for polymers with a single charge next to the azide, the reaction was much faster compared with the three-charge analogue. We concluded that the click reaction proceeds though an initial ligand adsorption to the NP surface, so that the single charge provides reduced electrostatic repulsion with the ligand and closer placement of the azide to the surface. In the prefunctionalization approach, the dye ligand was conjugated to the polymer through a cleavable disulfide bond before formulation of NPs. We found that the negative charge next to azide is essential to expose the dye ligand to the NPs' surface, allowing reductive cleavage of the disulfide. The latter enabled preparation of FRET-based probes with a ratiometric response to the reductive stimulus. They were validated in live cells, showing color switching to a reductive environment of endosomes, as monitored by ratiometric fluorescence imaging. The present work provides insights into two types of chemical functionalization of polymeric NPs and the surface reactivity as a function of introduced charged groups, which will help to design effective functional nanoparticle probes for biosensing and bioimaging.