Capturing the dynamic integrity of carbocyanine fluorophore-based lipid nanoparticles using the FRET technique

Nanoparticles capable of dynamically reporting their structural integrity in real-time are a powerful tool to guide the design of drug delivery technologies. Lipid nanoparticles (LNPs) offer multiple important advantages for drug delivery, including stability, protection of active substances, and sustained release capabilities. However, tracking their structural integrity and dynamic behaviour in complex biological environments remains challenging. Here, we report the development of a Förster resonance energy transfer (FRET)-enabled LNP platform that achieves unprecedented sensitivity and precision in monitoring nanoparticle disintegration. The FRET-based LNPs were prepared using nanoprecipitation, encapsulating high levels of 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) fluorophores as the donor and acceptors, respectively. The resulting LNPs had a mean diameter of 114 ± 19 nm with a distinct FRET signal. An optimal energy transfer efficiency of 0.98 and an emission quantum yield of 0.13 were achieved at 11.1% fluorophore loading in the LNPs, balancing efficient energy transfer and minimal aggregation-induced quenching. Using the FRET reporting, three dissociation stages of FRET LNPs were observed: solvation, indicated by an increased emission intensity; swelling and partial dissolution, evidenced by changes in emission maxima and mean size; and complete dissociation, confirmed by emission solely from DiO and the absence of particles. Testing the nanoparticles in live cells (telomerase-immortalised human corneal epithelial cells, hTCEpi cells) revealed a direct link to the disappearance of the FRET signal with the dissociation of FRET NPs. The nanoparticles initially exhibited a strong extracellular FRET signal, which diminished after cellular internalisation. This suggests that the LNPs disintegrate after entering the cells. These findings establish FRET-based LNPs as a robust tool for real-time nanoparticle tracking, offering insights into their integrity and release mechanisms, with potential applications in advanced drug delivery and diagnostics.