PROJECT SUMMARY This renewal application builds on our discovery that the shape of nanoparticle-based constructs—what we define as structural valency—plays a key role in preserving the targeting ligand characteristics of nanoconstructs even when coated by a protein corona. Protein corona formation and its impact on engineered physicochemical properties of nanoconstructs has been somewhat controversial but is generally accepted to affect cell and tissue targeting specificity and downstream biological effects. Using single-particle, live-cell imaging investigations, we found that the protein corona composition of an ensemble of nanoconstructs was superseded by single-particle shape effects. Compared to their spherical counterparts, nanoconstructs with branched structures retained their ligand shell targeting capabilities both on cell membranes and within cells and cellular compartments. Statistically significant differences in the time-resolved dynamics of differently shaped nanoconstructs interacting with cellular components cannot be attributed to very minimal variations in protein corona composition. We aim to determine the mechanism for how branched nanoconstructs maintain their targeting functionality in biological environments and then to modulate the spatial organization of the single-particle protein corona with light during live-cell interactions. We will tune the nanoscale features of the branched nanoconstructs, including overall size, branch length, and tip radii of curvature, to be commensurate with membrane receptor dimerization and curvature-induced endosomal signaling and will image interactions at the single-particle level. The specific aims include (1) Understand how nanoconstruct design and optical stimuli can manipulate the protein corona at the single-particle level; (2) Investigate structural multivalency of membrane-targeting branched nanoconstructs on receptor dimerization; (3) Improve intracellular targeting and delivery using branched nanoconstruct shapes. This work is expected to be of high impact for precision nanomedicine by the intersection of single-nanoparticle advances with mechanistic insight into biological responses. Our ability to resolve nanoparticle-cell interactions at relevant time and length scales for receptor recognition and internalization will benefit knowledge in protein dimerization and nanoscale curvature effects and deepen understanding of endocytosis and immunomodulatory responses—key processes for advancements in vaccine design and drug delivery.