Increased hydrophobic block length of PTDMs promotes protein internalization

Abstract

The plasma membrane is a major obstacle in the development and use of biomacromolecules for intracellular therapeutic applications. Protein transduction domains (PTDs) have been used to overcome this barrier, but often require covalent conjugation to their cargo and can be time consuming to synthesize. Synthetic monomers can be designed to mimic the amino acid moieties in PTDs, and their resulting polymers provide a well-controlled platform to vary molecular composition for structure-activity relationship studies. In this paper, a series of polyoxanorbornene-based synthetic mimics, inspired by PTDs, with varying cationic and hydrophobic densities, and the nature of the hydrophobic chain and degree of polymerizations were investigated in vitro to determine their ability to non-covalently transport enhanced green fluorescent protein into HeLa cells, Jurkat T cells, and hTERT mesenchymal stem cells. Polymers with high charge density lead to efficient protein delivery. Similarly, the polymers with the highest hydrophobic content and density proved to be the most efficient at internalization. The observed improvements with increased hydrophobic length and content were consistent across all three cell types, suggesting that these architectural relationships are not cell type specific. However, Jurkat T cells showed distinct variation in uptake between polymers than with the other two cell types. These results provide important design parameters for more effective delivery of biomacromolecules for intracellular delivery applications.

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.…”

“…[40,44,45] The sequence segregation of the hydrophobic moieties, commonly a phenyl or di-phenyl containing oxanorbornene monomer, was also shown to affect uptake efficiency, with gradient and block copolymers outperforming alternating hydrophobic-guanidinium polymers. [39,46] …”

Sequence segregation improves non-covalent protein delivery

“…The synthesis of each monomer and corresponding PTDM was reported by us [27]. Briefly, the monomers were obtained by ring-opening a Diels-Alder anhydride adduct with a desired alcohol to obtain a mono-functional intermediate, followed by EDC coupling with another equivalent of alcohol, the same type or different, to introduce the second functionality.…”

“…25,26 Recently, we investigated the correlation between hydrophobic content and protein delivery efficiency of these PTDMs. 25,27 Now, in this paper, we use constitutional macromolecular isomers 28 to determine the role of hydrophobic/hydrophilic segregation in non-covalent protein delivery.…”

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.…”