Composition of Intracellular Protein Corona around Nanoparticles during Internalization.

Abstract

It has been well established that the early-stage interactions of nanoparticles with cells are governed by the extracellular protein corona. However, after entering into the cells, the evolving protein corona is the key to subsequent processing of nanoparticles by cells. To identify the protein corona around intracellular nanoparticles, it is essential to maintain its original compositions during cell treatment. Herein, we develop a paraformaldehyde (PFA) cross-linking strategy to stabilize corona compositions when extracting protein coronas from cells, providing original information on protein coronas around intercellular gold nanoparticles (AuNPs). The stability of the protein corona after PFA cross-linking was carefully investigated with several characterization methods, and the results demonstrate that PFA cross-linking successfully prevents the dissociation and exchange of corona proteins. Then the recovered intracellular protein corona around AuNPs from living HepG2 cells with a PFA cross-linking strategy was subjected to nanoHPLC-MS/MS for proteomic analysis. It was found that the compositions of intracellular protein coronas are dominated by cell-derived proteins and undergo significant variation of protein species and amounts over time during internalization. Time-resolved analysis provides relevant proteins involved in nanoparticle cellular uptake and transportation, indicating that AuNPs are endocytosed mainly by a clathrin-mediated uptake mechanism and directed into an endolysosomal pathway toward their final destination. Such proteomic-based results are verified by pharmacological inhibition and TEM imaging analysis. This work provides a universal strategy to study compositions of protein corona around intercellular nanoparticles and could be a footstone to link the formation of protein corona around nanoparticles to their biological function in cells.