Most nanoparticles’ parameters affect their interactions with cells. To date, all the parameters studied are basically static (e.g., size, shape, ligands, charge). This is unfortunate, as proteins have structural dynamics that most nanoparticles do not possess. Here we study single-chain polymeric nanoparticles (SCPNs), whose structure undergoes dynamic changes. We produced multiple sets of particles from identical polymer chains via a supramolecular reshuffling approach that allowed iterative reshuffling between a compact/static and a sparse/dynamic form. These particles are topological isomers as they have identical molecular formulas differing in connectivity, and thus structural dynamics. We show that cell uptake discriminates these SCPN topological isomers. Through different endocytic pathways, the sparse/dynamic isomers are uptaken more, but the compact/static isomers access the cytosol more efficiently as evidenced by a glucocorticoid translocation assay. These results highlight the importance of structural dynamics’ role in cellular interactions.