Protein Mimic Hydrophobicity Affects Intracellular Delivery but not Cargo Binding

Abstract

Protein transduction domain mimics (PTDMs) enable cellular uptake of macromolecular cargo such as proteins and nucleic acids. The presence of hydrophobic domains in PTDMs has been shown to enhance cargo uptake, but the role of hydrophobicity in PTDM‐binding of the desired cargo is not fully understood. Herein, block copolymer PTDMs composed of varying hydrophobic monomers were synthesized via ring‐opening metathesis polymerization (ROMP) to probe the effect that increasingly hydrophobic side chains had on binding enhanced green fluorescent protein (EGFP). PTDM‐facilitated cellular uptake of EGFP into Jurkat T cells was performed to assess the correlation between binding, hydrophobicity, and delivery. Binding studies demonstrated that all PTDMs bound EGFP similarly despite a five log difference in monomer Kow (octanol‐water partition coefficient) and that intermediately hydrophobic PTDMs facilitated higher cellular uptake of EGFP. Taken as a whole, hydrophobicity of the PTDM is a better predictor of effective delivery in Jurkat T cells than cargo binding.

ROMP‐ and RAFT‐Based Guanidinium‐Containing Polymers as Scaffolds for Protein Mimic Synthesis

“…On first glance, this observation is surprising since, in general, the addition of a hydrophobic block increases CPPMs‘ delivery of cargo. [39,40,46,52] It is concievable that Poly-2 forms a stable microstructure in buffer that renders it unable to effectively interact with a protein that is large in comparison to siRNA. When taking previous reports into account, however, this is unlikely, as a hydrophobic block almost universally improves CPPM mediated protein uptake in cells regardless of microstructure character or micelle/vesicle size.…”

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

“…Givent hatb inding affinity seemed to be impacted by alterationsi nP TDMh ydrophobics egregation,amore recent study wasu ndertakeni nw hich theP TDMb lock architecture was maintained,b ut theh ydrophobic side chains were varied in an attempttomodulate theirEGFP bindingstrength. [34] Despitesignificant changest ot he PTDM hydrophobicity,a ll bindinga bilities were essentiallyt he same (Figure6a).H owever, thes imilarity in bindingw as notr eflectedi nt he resultso ft he delivery experimentsd oneu sing thes ameP TDMs.T hisr eporti ndicated that bindinga nd delivery aren ot necessarilyc orrelateda nd demonstrated that theoverall delivery landscapeiscomplex.…”

“…Our group has developed and studied PTDMs capable of non‐covalently binding and delivering protein cargo into difficult‐to‐transfect cell lines . Most of this work has centered on chemically optimizing PTDMs through structure–activity relationships, and special attention has been given to optimizing the ratio of hydrophilic to hydrophobic monomers and the overall polymer hydrophobicity (Figure ) . Some of these same studies have featured cursory explorations of how polymer–protein binding impacts intracellular delivery.…”

“…Given that binding affinity seemed to be impacted by alterations in PTDM hydrophobic segregation, a more recent study was undertaken in which the PTDM block architecture was maintained, but the hydrophobic side chains were varied in an attempt to modulate their EGFP binding strength . Despite significant changes to the PTDM hydrophobicity, all binding abilities were essentially the same (Figure a).…”

“…[4,29,[32][33][34][35] Most of this work has centered on chemically optimizing PTDMst hrough structure-activity re-lationships, and special attention has been given to optimizing the ratio of hydrophilic to hydrophobic monomers and the overall polymer hydrophobicity ( Figure 3). [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.…”