Sequence segregation improves non-covalent protein delivery

Abstract

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.

Protein Mimic Hydrophobicity Affects Intracellular Delivery but not Cargo Binding

“…The Tew group has previously shown that precise optimization of the hydrophobic domain led to enhancements in siRNA delivery to a non‐trivial target, Jurkat T cells, while the presence of a hydrophobic block boosted EGFP delivery to HeLa cells . 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.…”

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.…”

“…In this sense, it is quite possible that binding affinity could be correlated to the activity or bioavailability of the cargo in the intracellular environment. Questions surrounding cargo function and bioavailability post‐delivery are usually answered by delivering enzymes or other functional proteins and monitoring said function . In a separate body of non‐delivery‐related work, polymer–enzyme binding was studied with respect to turning enzyme function on and off .…”

“…[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.…”

“…In another study,P TDMs with varied architectures wereu sed to deliver EGFP into Jurkat Tc ells, and this time binding and delivery weres tudied together more intensively. [35] This series of PTDMs varied the polymer architecture from random to gradient to block copolymer,i nw hich the hydrophobic and hydrophilicb locks were mixed or segregated to variousd egrees, as seen in Figure 5a.Q uantitative binding studies between the PTDMs and one protein, EGFP,r evealed thatc hanges to the PTDM architecture affected the ability of the PTDM to bind the protein (Figure 5b). PTDMs 1a nd 2, the block copolymera nd the gradient copolymer, respectively,e xhibited single-digit micromolar dissociation constant (K d )v alues, whereas the homopolymers, PTDMs 3a nd 4, exhibited double-digit micromolar K d values.…”