Relating structure and internalization for ROMP-based protein mimics

Abstract

Elucidating the predominant cellular entry mechanism for protein transduction domains (PTDs) and their synthetic mimics (PTDMs) is a complicated problem that continues to be a significant source of debate in the literature. Several guanidinium-rich homopolymer structures initially designed to mimic oligoarginine, as well as an amphiphilic block copolymer, were end-labeled with FITC. This enabled the monitoring of PTDM internalization into HeLa cells by flow cytometry and confocal imaging. Additionally, their unlabeled counterparts showed improved ability to deliver proteins into cells with added hydrophobic content. In conjunction, pre-incubation with the protein is required, suggesting that the polymers are not just simply interacting with the membrane, but require association with the cargo of interest. However, the mechanism of cellular entry is not dependent on structure within this study, as punctate fluorescence was prevalent within the cells treated with fluorescently labeled samples and protein-polymer complexes. This suggests that the predominant mode of internalization for the presented PTDM structures is endosomal uptake and does not appear to be affected by concentration or structure. The PTDMs reported here provide a well-controlled platform to vary molecular composition for structure activity relationship studies to further our understanding of PTDs, their non-covalent association with cargo, and their cellular internalization pathways.

Protein Mimic Hydrophobicity Affects Intracellular Delivery but not Cargo Binding

“…Their synthetic nature engenders de novo design and the synthesis of optimized and efficacious delivery systems . Through custom‐made synthetic mimics, precise polymeric architectures, like block copolymers, can be obtained and used to build broader structure‐activity relationships . Hydrophobic components, in conjunction with standard guanidinium‐rich domains, have been shown to impart enhanced performance in terms of cellular uptake and delivery for both CPPs and their mimics; CPPMs and PTDMs gain an edge over their natural counterparts because the hydrophobic domain can be systematically optimized thereby maximizing performance .…”

“…Varying the segregation of the hydrophobic domain has also been shown to impact EGFP binding and delivery . Since Tew and coworkers have demonstrated that the likely and prevalent mode of internalization for these ROMP‐based PTDMs is endosomal uptake and given that hydrophobicity initiated fusion into the endosomal membrane is a method of escape, further modification of PTDM hydrophobicity could be a mechanistically advantageous way to improve delivery. Likewise, hydrophobicity can aid in membrane adsorption and subsequent endocytosis thereby justifying its careful optimization …”

“…Figure shows the performance of the PTDMs as a function of EGFP percent positive cells (Figure a) and MFI (Figure b), where P1 and P4 are positive controls. It demonstrates that PTDMs P1‐P4 affected the highest percentage of cells in the population treated with the complexes while P6‐P7 impacted significantly fewer cells.…”

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

“…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.…”

“…These two separate materials systems, one with peptide‐based carriers and the other with synthetic polymers, have established a simple design rule concerning binding and delivery: some minimum cargo binding must occur to facilitate non‐covalent delivery. 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 .…”