Nanoparticle Intervention in Cell Behavior: Summary/Abstract Colloidal nanoparticles (NPs) are of great interest as diagnostics, imaging agents, drug delivery vehicles, and therapeutics. Gold NPs are an important class of these materials due to their brilliant optical and photothermal properties. Most NP-cell studies focus on NP killing or targeting (with surface ligands) various cells, although recent controversies in the literature suggest that targeting does not work. Gene expression profiles of various cells, as a function of NP dose, are commonly reported. Still unanswered is the question of exactly how, at the molecular level, NPs affect cellular behavior and cellular function, especially in the absence of (or biomolecular corona covering up of) targeting ligands. The three Specific Aims below propose three different mechanisms of how NPs can intervene in cell behavior, across a variety of cell types to show generality. In Aim 1, the hypothesis is that NPs sequester biomolecules from the cellular milieu, making these biomolecules less bioavailable to the cell and thereby alter cellular behavior. The experiments in this Aim focus on cellular migration as a function of chemoattractant concentration in three-dimensional matrices; measurements of cellular migration ability as a function of chemoattractant loss via adsorption to NPs will be undertaken. In Aim 2, the hypothesis is that cellular unfolded protein response to NP exposure is directly correlated to the amount of unfolded proteins that the NPs display on their surfaces. The experiments in this Aim focus on a mass spectral footprinting technique to infer protein display on NP/biological samples that are known to upregulate, or not upregulate, the unfolded protein response in cells. In Aim 3, the hypothesis is that NPs bind to exosomes, small extracellular vesicles that are believed to be important in intercellular communication. The experiments in this Aim focus on in vitro measures of exosome binding or bursting to a library of nanoparticles.!