Unravelling drug delivery using in vitro Fluorescence Correlation Spectroscopy (FCS)

Therapeutic cargo, such as small molecule drugs, proteins and nucleic acids, can only fulfill their function in an effective way if they reach their biological target site intact and at sufficiently high concentrations. To facilitate and steer this transport process after administration and mitigate limited solubility and/or stability, the therapeutic cargo can be encapsulated into nanocarriers. These nanocarriers need to function in highly complex and crowded biological media and the journey they need to take to deliver their payload is characterized by crossing several (physical) barriers. Due to these complexities, understanding or even predicting the fate of nanocarriers and their cargo during transport remains a formidable challenge. In this Review, we highlight Fluorescence Correlation Spectroscopy (FCS) as a powerful and versatile tool to perform real-time tracking with high spatial resolution of encapsulation, nanoparticle functionalization, cargo/carrier degradation, and drug release kinetics in relevant and complex biological environments, such as, living cells and full blood. By providing a detailed description of the technical background, outline of the technique to study nanocarrier-mediated drug delivery, and an overview of upcoming technological advancements, this Review will show that FCS opens a window to directly observe the drug delivery process with unprecedented detail and under native conditions. FCS-generated insights are expected to become instrumental in fundamentally understanding the functioning of nanomedicine by deciphering their underlying molecular mechanisms, which in turn can serve as input for the rational design of the next generation of drug delivery vehicles.