In Situ Analysis for Protein Corona: from Morphology, Composition, Structure to Dynamic Process

The formation of a protein corona (PC) on the surface of nanoparticles (NPs) in biological environments is a critical factor influencing the fate and functionality of NPs in vivo. This biolayer affects NPs' biodistribution, immune recognition, cellular uptake, and therapeutic efficacy, making it essential for the rational design of nanomedicines. However, traditional analytical techniques often disrupt the native state of the PC, particularly its loosely bound soft corona (SC), leading to incomplete characterizations. In situ analysis methods offer a more accurate representation of PC composition, structure, and dynamics by preserving its native biological context. This review highlights critical advances in in situ PC analysis techniques, including methods for composition identification, structural visualization, and monitoring the dynamic evolution of the PC. It emphasizes the importance of real-time, non-disruptive analysis to better understand the nano-bio interface and its implications for nanomedicine design and safety. Additionally, it discusses challenges in current PC analysis methodologies and proposes future research directions to improve in situ characterization accuracy and standardization.