Abstract
Fluorescence Correlation Spectroscopy (FCS) is a well-established technique to study dynamic processes like molecular diffusion. Alterations of the diffusional behavior of a given molecule, caused by molecular interactions, changes in local viscosity, etc., can be investigated by FCS. Here, we apply FCS to evaluate the stability of different nanoparticles used for drug delivery under in-vivo mimicking conditions. Nanoparticles are a powerful drug-delivery approach with enhanced permeability and precise targeting. Nevertheless, they need to overcome biological barriers such as nasal and pulmonary mucus to deliver their cargo. As mucus viscosity and adhesiveness constitute a major challenge for nanoparticle delivery, studying the stability and mobility of nanoparticles in mucus is crucial. With a fluorescently labelled cargo, FCS allows us to discriminate between properly formed nanoparticles and cargo molecules released in solution, which can be used as a readout of stability. However, we observed that mucus can induce aggregation of the released cargo hampering the possibility of addressing the nanoparticle behavior via their diffusion, as particles and cargo-aggregates are not distinguishable. Hence, we incorporated other photophysical information into the analysis. We show that, when the fluorescence lifetime of the nanoparticles and of the aggregated cargo remains different, Fluorescence Lifetime Imaging Microscopy (FLIM) and Single Particle Tracking (SPT) can be combined to tackle this issue. Our results indicate that tracking single particles/aggregates and extracting their particle-wise lifetime allows us to discriminate between properly formed nanoparticle and mere cargo-aggregates. With this method, nanoparticles movements and stability can be monitored over time independently of the aggregates. In summary, FCS, FLIM and SPT are powerful and complementary techniques and their combination makes it possible to investigate molecular behavior in complex environments.
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