The Role of Cargo Binding Strength in Polymer-Mediated Intracellular Protein Delivery

Delivering proteins into the intracellular environment is a critical step toward probing vital cellular processes for the purposes of ultimately developing new therapeutics. Polymeric carriers are widely used to facilitate protein delivery with guanidinium-rich macromolecules leading the way within this category. Although binding interactions between natural proteins and synthetic polymers have been studied extensively, the relationship between polymer-protein binding and intracellular delivery is seldom explored. Elucidating the role of cargo binding in delivery is a promising direction that is expected to provide new insights that further optimize intracellular protein delivery. Herein, model polymeric carriers called protein transduction domain mimics (PTDMs) were studied for their ability to bind to a variety of protein cargoes, including an antibody, where the proteins encompassed a range of sizes (∼16-151 kDa) and isoelectric points (4.7-11.4). The PTDM-protein complexes were also delivered into Jurkat T cells in an attempt to establish a general correlation between binding ability and delivery outcomes. Binding assays resulted in a vast range of dissociation constants (K_d), which spanned from 3.5 to 4820 nM and indicated a variety of binding strengths between PTDM and protein. More significantly, PTDMs preferentially bound certain types of proteins over others, such as the antibody fragment over the whole antibody. Furthermore, increased PTDM-protein binding affinity did not correlate with protein delivery, suggesting that the successful internalization of complexes is independent of binding equilibrium. Although binding did not correlate with internalization here, the potential for binding affinity to impact other aspects of delivery, like cargo functionality inside the cell, remains an open possibility.