An anionic, endosome-escaping polymer to potentiate intracellular delivery of cationic peptides, biomacromolecules, and nanoparticles

Brian C Evans,Eric A Dailing,Kurt D Hankenson,Matthew Oliver,R Brock Fletcher,Kedar Ghimire,Alvin J Mukalel,Colleen M Brophy,Kameron V Kilchrist,Jeffrey S Isenberg,John W Tierney,Craig L Duvall,Joyce Cheung-Flynn,Charles A Gersbach,Juan M Colazo,Cynthia Lander

doi:10.1038/s41467-019-12906-y

Abstract

Peptides and biologics provide unique opportunities to modulate intracellular targets not druggable by conventional small molecules. Most peptides and biologics are fused with cationic uptake moieties or formulated into nanoparticles to facilitate delivery, but these systems typically lack potency due to low uptake and/or entrapment and degradation in endolysosomal compartments. Because most delivery reagents comprise cationic lipids or polymers, there is a lack of reagents specifically optimized to deliver cationic cargo. Herein, we demonstrate the utility of the cytocompatible polymer poly(propylacrylic acid) (PPAA) to potentiate intracellular delivery of cationic biomacromolecules and nano-formulations. This approach demonstrates superior efficacy over all marketed peptide delivery reagents and enhances delivery of nucleic acids and gene editing ribonucleoproteins (RNPs) formulated with both commercially-available and our own custom-synthesized cationic polymer delivery reagents. These results demonstrate the broad potential of PPAA to serve as a platform reagent for the intracellular delivery of cationic cargo.

Highlights

Peptides and biologics provide unique opportunities to modulate intracellular targets not druggable by conventional small molecules
To investigate the structural dependence of the cationic cell penetrating peptides (CPPs) sequence on PPAA-mediated peptide uptake, we explored a small library of CPP-modified peptides (Table 1, Supplementary Fig. 2)
The first five peptides comprise a MAPKAP Kinase 2 inhibitory peptide (MK2i)[4] modified with different CPP sequences that are among the most commonly utilized CPPs: the cationic, non-amphipathic CPPs TAT, R6, and YARA, the primary amphipathic CPP penetratin, and the secondary amphipathic CPP transportan. These peptides enable studying the effects of CPP sequence on PPAA-mediated peptide uptake, as we hypothesize that cell delivery is significantly influenced by interactions between the cationic CPP sequence and anionic PPAA

Summary

Introduction

Peptides and biologics provide unique opportunities to modulate intracellular targets not druggable by conventional small molecules. This approach demonstrates superior efficacy over all marketed peptide delivery reagents and enhances delivery of nucleic acids and gene editing ribonucleoproteins (RNPs) formulated with both commercially-available and our own custom-synthesized cationic polymer delivery reagents These results demonstrate the broad potential of PPAA to serve as a platform reagent for the intracellular delivery of cationic cargo. Most marketed delivery reagents that seek to unlock the potential of cytosolically active peptides are essentially all polycationic lipids or polymers that can facilitate interactions with inherently negatively charged cellular membranes These cationic reagents are generally cytotoxic[6] and have limited capacity to package non-nucleic acid cargo. Most CPPs lack potency (often require >100 μM concentrations), produce transient effects, and suffer from internalization into and entrapment within vesicles of the endolysosomal trafficking pathway[10] that limit cytosolic bioavailability[11]

Methods

Results

Conclusion