Despite the evident success of using a multivalent approach to increase efficacy of targeted delivery, a clear understanding of how multiple ligands behave collectively to influence the uptake of nanoparticle cell-targeting agents has not been reached. Although when present in large quantity, multivalent ligands can increase binding avidities to cells, it is also conceivable, that the manner in which these ligands are presented to the cell may have a significant effect on uptake. Here we examine this parameter using a linear dendritic polymer construct that enabled us to pattern the surfaces of nanoparticles with variable sized ligand clusters in different spatial arrangements. We demonstrate for the first time the clear impact of folate presentation on intracellular uptake both in vitro and in vivo. The findings presented here suggest that the nature of ligand presentation on a nanoparticle surface may play an important role in drug targeting; the results suggest potential impact for other targeting moieties and provide a framework for further refinement of future multivalent targeting strategies.Many biological systems interact through multiple simultaneous interactions [1][2][3] . While it is well acknowledged that, on the macroscale, these multivalent interactions strongly influence ligand-receptor binding kinetics and the biological responses they govern, the microscale patterning of ligands on substrates can also have a profound effect on multivalent kinetics and are able to further modulate biological signaling [4] . In particular, ligand-receptor clusters can dramatically increase interactions between cell and substrate; not only the valency, but spatial factors such as branching mode and the localized clustering of groups are important [5][6][7] in influencing binding and downstream signaling processes [8,9] . Still, despite the prevalence of patterned ligand presentation in cell biology and its demonstrated significance on 2D tissue engineering formats [10,11] , it remains largely unappreciated by those who design nanoparticle synthetic systems intended to interact with cells.Traditionally, ligands have been attached in moderate to large quantities to liposomes, inorganic nanoparticles and linear-linear block copolymer micelles [12,13] ; however, typically they are presented in a randomly distributed fashion across the nanoparticle surface from a linear tether, typically polyethylene glycol (PEG), in a structure that does not enable manipulation of ligands in specific groupings with control of cluster group size and spacing.We hypothesized that precise control over the elements promoting and controlling multivalent presentation on targeted therapeutic carriers could enhance the efficacy and specificity of targeted delivery. To test this hypothesis, scaffolds that are able to localize a variable number of ligands within confined regions are required; this requirement cannot be satisfied by traditional material systems used in targeted delivery [14] such as linear-linear block polymers and PEG-fu...