CONSPECTUS As a semi-permeable barrier that controls the flux of biomolecules in and out the cell, the plasma membrane is critical in cell function and survival. Many proteins interact with the plasma membrane and modulate its physiology. Within this large landscape of membrane-active molecules, researchers have focused significant attention on two specific classes of peptides, antimicrobial peptides (AMPs) and cell penetrating peptides (CPPs) because of their unique properties. In this account, we describe our efforts over the last decade to build and understand synthetic mimics of antimicrobial peptides (SMAMPs). These endeavors represent one specific example of a much larger effort to understand how synthetic molecules interact with and manipulate the plasma membrane. Using both defined molecular weight oligomers and easier to produce, but heterogeneous, polymers, it has been possible to generate scaffolds with biological potency superior to the natural analogs. In one case, a compound has progressed through a phase II clinical trial for pan)staph infections. Modern biophysical assays highlighted the interplay between the synthetic scaffold and lipid composition leading to negative Gaussian curvature, a requirement for both pore formation and endosomal escape. The complexity of this interplay between lipids, bilayer components, and the scaffolds remains to be better resolved, but significant new insight has been provided. It is worthwhile to consider the various aspects of permeation and how these are related to ‘pore formation.’ More recently, our efforts have expanded toward protein transduction domains, or cell penetrating peptide, mimics. The combination of unique molecular scaffolds and guanidinium) rich side chains has produced an array of polymers with robust transduction (and delivery) activity. Being a new area, the fundamental interactions between these new scaffolds and the plasma membrane are just beginning to be understood. Negative Gaussian curvature is important but the detailed relationships between molecular structure, self)assembly with lipids, and translocation require more investigation. It has become clear that the combination of molecular design, biophysical models, and biological evaluation provide a robust approach to the generation and study of novel proteinomimetics.

The impermeability of the plasma membrane towards large, hydrophilic biomolecules is a major obstacle in their use and development against intracellular targets. To overcome such limitations, protein transduction domains (PTDs) have been used as protein carriers, however they often require covalent fusion to the protein for efficient delivery. In an effort to develop more efficient and versatile biological vehicles, a series of PTD-inspired polyoxanorbornene-based synthetic mimics with identical chemical compositions but different hydrophobic/hydrophilic segregation were used to investigate the role of sequence segregation on protein binding and uptake into Jurkat T cells and HEK293Ts. This series was composed of a strongly segregated block copolymer, an intermediately segregated gradient copolymer, and a non-segregated homopolymer. To assess how protein isoelectric point, the study was extended to other proteins (bovine serum albumin, avidin, and streptavidin). Among the series, the block copolymer maximized both protein binding and translocation efficiencies, closely followed by the gradient copolymer, resulting in two protein transporter molecules more efficacious than currently commercially available agents. These two polymers were also used to deliver the biologically active Cre recombinase into a loxP-reporter T cell line. Since exogenous Cre must reach the nucleus and retain its activity to induce gene recombination, this in vitro experiment better exemplifies the broad applicability of this synthetic system. This study shows that increasing segregation between hydrophobic and cationic moieties in these polymeric mimics improves non-covalent protein delivery, providing crucial design parameters for the creation of more potent biological delivery agents for research and biomedical applications.

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.