Abstract
Nanoparticles (NPs) inevitably interact with proteins upon exposure to biological fluids, leading to the formation of an adsorption layer known as the "protein corona". This corona imparts NPs with a new biological identity, directly influencing their interactions with living systems and dictating their fates in vivo. Thus, gaining a comprehensive understanding of the dynamic interplay between NPs and proteins in biological fluids is crucial for predicting therapeutic effects and advancing the clinical translation of nanomedicines. Numerous methods have been established to decode the protein corona fingerprints. However, these methods primarily rely on prior isolation of NP-protein complex from the surrounding medium by centrifugation, resulting in the loss of outer-layer proteins that directly interact with the biological system and determine the in vivo fate of NPs. We discuss here separation techniques as well as in situ characterization methods tailored for comprehensively unraveling the inherent complexities of NP-protein interactions, highlighting the challenges of in situ protein corona characterization and its significance for nanomedicine development and clinical translation.
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