Modifying Cell Membranes with Anionic Polymer Amphiphiles Potentiates Intracellular Delivery of Cationic Peptides

ACS Appl Mater Interfaces. 2020 Nov 11;12(45):50222-50235. doi: 10.1021/acsami.0c13304. Epub 2020 Oct 30.

Abstract

Rapid, facile, and noncovalent cell membrane modification with alkyl-grafted anionic polymers was sought as an approach to enhance intracellular delivery and bioactivity of cationic peptides. We synthesized a library of acrylic acid-based copolymers containing varying amounts of an amine-reactive pentafluorophenyl acrylate monomer followed by postpolymerization modification with a series of alkyl amines to afford precise control over the length and density of aliphatic alkyl side chains. This synthetic strategy enabled systematic investigation of the effect of the polymer structure on membrane binding, potentiation of peptide cell uptake, pH-dependent disruption of lipid bilayers for endosome escape, and intracellular bioavailability. A subset of these polymers exhibited pKa of ∼6.8, which facilitated stable membrane association at physiological pH and rapid, pH-dependent endosomal disruption upon endocytosis as quantified in Galectin-8-YFP reporter cells. Cationic cell penetrating peptide (CPP) uptake was enhanced up to 15-fold in vascular smooth muscle cells in vitro when peptide treatment was preceded by a 30-min pretreatment with lead candidate polymers. We also designed and implemented a new and highly sensitive assay for measuring the intracellular bioavailability of CPPs based on the NanoLuciferase (NanoLuc) technology previously developed for measuring intracellular protein-protein interactions. Using this split luciferase class of assay, polymer pretreatment enhanced intracellular delivery of the CPP-modified HiBiT peptide up to 30-fold relative to CPP-HiBiT without polymer pretreatment (p < 0.05). The overall structural analyses show that polymers containing 50:50 or 70:30 molar ratios of carboxyl groups to alkyl side chains of 6-8 carbons maximized peptide uptake, pH-dependent membrane disruption, and intracellular bioavailability and that this potentiation effect was maximized by pairing with CPPs with high cationic charge density. These results demonstrate a rapid, mild method for polymer modification of cell surfaces to potentiate intracellular delivery, endosome escape, and bioactivity of cationic peptides.

Keywords: RAFT polymers; cell surface modification; drug delivery; endosomal escape; peptides.

MeSH terms

  • Animals
  • Anions / chemical synthesis
  • Anions / chemistry
  • Cations / chemical synthesis
  • Cations / chemistry
  • Cell Membrane / chemistry*
  • Cell-Penetrating Peptides / chemical synthesis
  • Cell-Penetrating Peptides / chemistry*
  • Cells, Cultured
  • HEK293 Cells
  • Humans
  • Hydrogen-Ion Concentration
  • Molecular Structure
  • Particle Size
  • Polymers / chemical synthesis
  • Polymers / chemistry*
  • Rats
  • Surface Properties
  • Surface-Active Agents / chemical synthesis
  • Surface-Active Agents / chemistry*

Substances

  • Anions
  • Cations
  • Cell-Penetrating Peptides
  • Polymers
  • Surface-Active Agents