Recent advances in super-resolution microscopy have enabled unprecedented visualization of cellular structures, tracking of nanomaterials in biological environments, or the elucidation of specific nano-bio interactions. Yet, dynamic quantification of cargo release from individual nanocarriers remains unexplored. Here, we leverage the high spatial resolution of direct stochastic optical reconstruction microscopy (dSTORM) to monitor protein release at the single-nanocarrier level. Poly(lactic-co-glycolic acid) (PLGA) nanocapsules labelled with Cyanine5 and loaded with bovine serum albumin (BSA) tagged with Alexa Fluor 488 are characterized using dSTORM alongside other characterization techniques. dSTORM allowed us to simultaneously observe changes in nanocarrier size and cargo localization over time. Our results demonstrate a time-dependent increase in nanocapsule diameter and a decrease in nanocarrier concentration. The quantitative analysis of individual nanocarriers reveals single-particle protein release profiles, characterized by an initial burst followed by sustained release, with complete release achieved after 30 days. This study represents the first application of super-resolution microscopy to spatially and temporally resolve protein release from nanocarriers, offering single-molecule sensitivity and nanometric resolution, and capturing heterogeneity that ensemble-averaged techniques overlook. Our approach complements other pharmacokinetic analyses and establishes a robust method to evaluate the cargo release from other nanocarriers by super-resolution microscopy.
