Summary While extracellular vesicles (EVs) demonstrate growing potential as innovative therapeutics in diverse medical context (cancer, regenerative medicine, etc.) or as naturally circulant diagnostic / prognostic probes, their physical properties (size, transport, etc.) remains a critical concern. Here, we introduce a pipeline that relies on interferometric light microscopy (ILM) for measuring not only nanoparticle concentration and size distribution but also for analyzing the interactions of these nanoparticles with their environment. The analysis of interference patterns allows for the physical characterization of (bio)nanoparticles not only in aqueous solutions but also in challenging media with relatively high viscosity, particularly pertinent for characterizing gel-based EV-delivery systems. Through exploration of the instrument’s functionality and the use of calibrated NPs of various known sizes, we successfully obtained information about the local viscosity characteristics of a complex fluid embedding EVs. We present a proof-of-concept for characterizing EVs suspended in unconventional media and their interactions with their surroundings. Leveraging the outcomes of this investigation, we not only highlight the advantages of using ILM for characterizing EVs in complex fluid, particularly pertinent for the development of optimized biological carriers for targeted drug delivery and therapeutic applications, but we also validate a new method for measuring viscosity at the nanoscale.

<i>In situ</i>investigation of extracellular vesicles in viscous formulations: interplay of nanoparticle transport and nano rheology through interferometric light microscopy analysis

In situinvestigation of extracellular vesicles in viscous formulations: interplay of nanoparticle transport and nano rheology through interferometric light microscopy analysis