Polymer Chemistry and the Kinetics of Nanoprecipitation

Loaded polymer nanoparticles (NPs) are a cornerstone of modern nanomedicine, underpinning applications that span biosensing, bioimaging, and therapy. A detailed understanding of the NP formation process is essential for reliable up-scaling and for engineering ever more sophisticated nanostructures. In this work, using Förster resonance energy transfer (FRET) and stopped-flow, we examine the mechanism and kinetics of NP formation by nanoprecipitation, focusing on how polymer hydrophobicity modulates the process. We prepared a series of methacrylate polymers with systematically varied hydrophobicities and labeled them with a donor fluorophore (BODIPY) or an acceptor fluorophore (Rhodamine). By performing nanoprecipitation directly in a stopped-flow instrument, we monitored the emergence of FRET to follow particle assembly. The FRET data revealed the implication of three processes at three time scales: Proto-particle formation occurs within submillisecond time scales. Aggregation of polymer chains takes place over a few to several tens of milliseconds, a stage that is highly sensitive to polymer hydrophobicity and also governs the encapsulation of payloads within NPs. A slower reorganization phase unfolds on the order of 100 ms to seconds. Collectively, these findings provide a quantitative, mechanistic framework for controlling nanoprecipitation, enabling precise tuning of NP architecture and facilitating reliable scale-up for large-scale biomedical applications.