Protein Binding and Release by Polymeric Cell-Penetrating Peptide Mimics.

There is significant potential in exploiting antibody specificity to develop new therapeutic treatments. However, intracellular protein delivery is a paramount challenge because of the difficulty in transporting large, polar molecules across cell membranes. Cell-penetrating peptide mimics (CPPMs) are synthetic polymers that are versatile materials for intracellular delivery of biological molecules, including nucleic acids and proteins, with superior performance compared to their natural counterparts and commercially available peptide-based reagents. Studies have demonstrated that noncovalent complexation with these synthetic carriers is necessary for the delivery of proteins, but the fundamental interactions dominating CPPM-protein complexation are not well understood. Beyond these interactions, the mechanism of release for many noncovalent carriers is not well established. Herein, interactions expected to be critical in CPPM-protein binding and unbinding were explored, including hydrogen bonding, electrostatics, and hydrophobic interactions. Despite the guanidinium-rich functionality of these polymeric carriers, hydrogen bonding was shown not to be a dominant interaction in CPPM-protein binding. Fluorescence quenching assays were used to decouple the effect of electrostatic and hydrophobic interactions between amphiphilic CPPMs and proteins. Furthermore, by conducting competition assays with other proteins, unbinding of protein cargoes from CPPM-protein complexes was demonstrated and provided insight into mechanisms of protein release. This work offers understanding toward the role of carrier and cargo binding and unbinding in intracellular outcomes. In turn, an improved fundamental understanding of noncovalent polymer-protein complexation will enable more effective methods for intracellular protein delivery.

Non-Covalent Carrier Hydrophobicity as a Universal Predictor of Intracellular Protein Activity.

Over the past decade, extensive optimization of polymeric cell-penetrating peptide (CPP) mimics (CPPMs) by our group has generated a substantial library of broadly effective carriers which circumvent the need for covalent conjugation often required by CPPs. In this study, design rules learned from CPPM development were applied to reverse-engineer the first library of simple amphiphilic block copolypeptides for non-covalent protein delivery, namely, poly(alanine-block-arginine), poly(phenylalanine-block-arginine), and poly(tryptophan-block-arginine). This new CPP library was screened for enhanced green fluorescent protein and Cre recombinase delivery alongside a library of CPPMs featuring equivalent side-chain configurations. Due to the added hydrophobicity imparted by the polymer backbone as compared to the polypeptide backbone, side-chain functionality was not a universal predictor of carrier performance. Rather, overall carrier hydrophobicity predicted the top performers for both internalization and activity of protein cargoes, regardless of backbone identity. Furthermore, comparison of protein uptake and function revealed carriers which facilitated high gene recombination despite remarkably low Cre internalization, leading us to formalize the concept of intracellular availability (IA) of the delivered cargo. IA, a measure of cargo activity per quantity of cargo internalized, provides valuable insight into the physical relationship between cellular internalization and bioavailability, which can be affected by bottlenecks such as endosomal escape and cargo release. Importantly, carriers with maximal IA existed within a narrow hydrophobicity window, more hydrophilic than those exhibiting maximal cargo uptake. Hydrophobicity may be used as a scaffold-independent predictor of protein uptake, function, and IA, enabling identification of new, effective carriers which would be overlooked by uptake-based screening methods.

Covalent Labeling-Mass Spectrometry Provides a Molecular Understanding of Noncovalent Polymer-Protein Complexation.

The delivery of functional proteins to the intracellular space offers tremendous advantages for the development of new therapeutics but is limited by the passage of these large polar biomacromolecules through the cell membrane. Noncovalent polymer-protein binding that is driven by strong carrier-cargo interactions, including electrostatics and hydrophobicity, has previously been explored in the context of delivery of functional proteins. Appropriately designed polymer-based carriers can take advantage of the heterogeneous surface of protein cargoes, where multiple types of physical binding interactions with polymers can occur. Traditional methods of assessing polymer-protein binding, including dynamic light scattering, circular dichroism spectroscopy, and fluorescence-based assays, are useful in the study of new polymer-based carriers but face a number of limitations. We implement for the first time the method of covalent labeling-mass spectrometry (CL-MS) to probe intermolecular surface interactions within noncovalent polymer-protein complexes. We demonstrate the utility of CL-MS for establishing binding of an amphiphilic block copolymer to negatively charged and hydrophobic surface patches of a model protein, superfolder green fluorescent protein (sfGFP), using diethylpyrocarbonate as a pseudo-specific labeling reagent. In addition, we utilize this method to explore differences at the intermolecular surface as the ratio of polymer to protein increases, particularly in the context of defining effective protein delivery regimes. By promoting an understanding of the intermolecular interactions in polymer-protein binding and identifying sites where polymers bind to protein surfaces, noncovalent polymer carriers can be more effectively designed for protein delivery applications.