A study of particle behaviour in dynamic extracellular environments using nanoparticles as model nanoscale probes

Quantifying nanoparticle behaviour in complex biological environments is essential for understanding transport and interactions within heterogeneous living systems. Fluorescent labelling is commonly used to visualise nanoparticles; however, surface modification by fluorescent probes can alter nanoparticle interactions and obscure native behaviour. Therefore, accessible label-free methods are needed to characterise nanoparticle dynamics under biologically relevant conditions. In this study, an optical caustics-based microscopy technique was used to track the motion of 100 nm gold nanoparticles in the extracellular space above human mesenchymal stem cell (hMSC) monolayers. Nanoparticle trajectories were analysed using diffusion coefficients, anomalous diffusion exponents, and convex hull metrics to quantify transport behaviour. Results demonstrated that the local extracellular environment significantly influences nanoparticle dynamics. In the presence of fetal bovine serum, nanoparticle diffusion rates changed significantly after one hour of exposure to the hMSC layer. Surface charge, protein content, and particle localisation relative to cells also had a marked effect on mobility. Comparison with idealised Brownian transport predictions showed that the living extracellular environment behaves as a dynamic and evolving transport medium that modifies nanoparticle movement. These findings highlight the sensitivity of nanoscale transport to local biological conditions and establish a label-free framework for investigating nanoparticle dynamics in heterogeneous biological systems.