Correlation between Protonation of Tailor-Made Polypiperazines and Endosomal Escape for Cytosolic Protein Delivery

ACS Appl Mater Interfaces. 2021 Aug 4;13(30):35233-35247. doi: 10.1021/acsami.1c00829. Epub 2021 Jul 20.


Responsive polymers, which become protonated at decreasing pH, are considered a milestone in the development of synthetic cell entry vectors. Exact correlations between their properties and their ability to escape the endosome, however, often remain elusive due to hydrophobic interactions or limitations in the design of water-soluble materials with suitable basicity. Here, we present a series of well-defined, hydrophilic polypiperazines, where systematic variation of the amino moiety facilitates an unprecedented fine-tuning of the basicity or pKa value within the physiologically relevant range (pH 6-7.4). Coincubation of HEK 293T cells with various probes, including small fluorophores or functioning proteins, revealed a rapid increase of endosomal release for polymers with pKa values above 6.5 or 7 in serum-free or serum-containing media, respectively. Similarly, cytotoxic effects became severe at increased pKa values (>7). Although the window for effective transport appears narrow, the discovered correlations offer a principal guideline for the design of effective polymers for endosomal escape.

Keywords: basicity; calcein release; endosomolytic polymers; membrane leakage; pH-responsive polymers.

MeSH terms

  • Acrylic Resins / chemical synthesis
  • Acrylic Resins / pharmacology*
  • Acrylic Resins / toxicity
  • Animals
  • Cattle
  • Cell Membrane / drug effects
  • Endosomes / drug effects*
  • Fluoresceins / metabolism
  • Green Fluorescent Proteins / metabolism*
  • HEK293 Cells
  • Hemolysis / drug effects
  • Humans
  • Hydrogen-Ion Concentration
  • Piperazines / chemical synthesis
  • Piperazines / pharmacology*
  • Piperazines / toxicity
  • Ribonuclease, Pancreatic / metabolism*
  • Serum Albumin, Bovine / metabolism*


  • Acrylic Resins
  • Fluoresceins
  • Piperazines
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Serum Albumin, Bovine
  • Ribonuclease, Pancreatic
  • fluorexon