Polymeric nanoparticles (NPs) have demonstrated potential for the delivery of bioactive agents to solid tumors, however, despite decades of research, few polymeric NPs have been able to reach the clinic. Conventional designs have taken a 'one-size fits all' approach, yet several factors play critical roles in defining their nano-bio interactions, including size, shape and surface functionality. Whereas larger NPs between 50-200 nm tend to exhibit enhanced circulation times and tumor accumulation, they are limited in their ability to efficiently permeate tissues. In contrast, ultrasmall NPs (<10 nm) demonstrate markedly improved tissue penetrating capabilities. In order to investigate the roles that ultrasmall NP precise size and surface functionality play in their ability to penetrate a solid tumor we prepared poly(amidoamine) (PAMAM) dendrimers, small (<10 nm) highly branched polymers with tightly controlled size distributions. G2 (2.9 nm), G4 (4.5 nm), and G7 (8.1 nm) dendrimers were prepared containing positive, neutral, and negative surface charge and their penetration evaluated in multicellular tumor spheroids (MCTS) and an in vivo tumor xenograft model. Whereas larger G4 and G7 dendrimers were restricted to the MCTS and tumor periphery even after prolonged treatment times, smaller G2 dendrimers displayed more rapid penetration characteristics. Among charged groups, positive dendrimers exhibited significantly improved accumulation compared to their neutral or negative counterparts. Our findings suggest that dendrimer size is the greatest determinant in its tumor permeation, with NP-cell interactions, endocytosis, and particle flexibility not significantly contributing to penetration depth. Moreover, when the anticancer drug doxorubicin (DOX) was conjugated to dendrimers, DOX penetration demonstrated a similar trend dependent on size, resulting in notable differences in the distribution of dead cells within the MCTS. These findings reveal that dendrimer size and surface functionality determine their tumor penetration behaviors, and provide useful insights in the further development of ultrasmall NPs with precisely controlled tumor distributions
