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

Abstract

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), 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.

Associative and Dissociative Processes in Non‐Covalent Polymer‐Mediated Intracellular Protein Delivery

“…Most recently,a na dditional binding-delivery correlation study was conducted, in which the same PTDM was used for all binding and delivery experimentsw ith am ore expansive set of protein cargoes. [36] In that report, changing the protein cargo changed the PTDM binding strength significantly but again resulted in alack of correlation with intracellular internalizationd espite aw iderr ange of binding strengths( K d = 3.5 to 4820 nm). Other key observations were that al ack of binding between the PTDM and the protein cargo was correlated with no delivery and that increasing PTDM binding affinity for cargo by an order of magnitude did not enhanced elivery.…”

“…Some of these same studies have featured cursory explorations of how polymer–protein binding impacts intracellular delivery. However, most recently, we have explicitly attempted to find a correlation between the binding affinity of our PTDMs for protein cargo with their ability to deliver it …”

“…Most recently, an additional binding‐delivery correlation study was conducted, in which the same PTDM was used for all binding and delivery experiments with a more expansive set of protein cargoes . In that report, changing the protein cargo changed the PTDM binding strength significantly but again resulted in a lack of correlation with intracellular internalization despite a wider range of binding strengths ( K d =3.5 to 4820 n m ).…”

“…The body of literature surrounding this topic, albeit small, consistently shows that no binding is associated with no delivery, which suggests that complex formation is critical and practically considered part of the overall mechanism . However, beyond some minimum amount of cargo binding, it appears that increasing binding strength does not enhance delivery . In the aforementioned instances for which K d values were calculated, the studied range was confined from nanomolar up to micromolar.…”

“…[29,[32][33][34][35] Some of these same studies have featured cursory explorations of how polymer-protein binding impacts intracellulard elivery.H owever,m ost recently,w eh ave explicitly attempted to find ac orrelation between the binding affinity of our PTDMs for protein cargo with their ability to deliver it. [36] In one of our earlier reports, the importance of PTDM-protein complexation prior to delivery was explored in as ingleexperiment, though it was not the main focus of the study. [33] In that experiment, aP TDM was used to facilitatet he delivery of enhanced green fluorescent protein (EGFP) into Jurkat Tc ells by using two different methods.…”