Effect of polyplex surface charge on cellular internalization and intracellular trafficking

Landon Mott, Caleb Akers, Daniel W. Pack

Research output: Contribution to journalArticlepeer-review


Design of non-viral gene delivery vectors requires optimization of various physical properties to maximize transgene expression. In particular, polyplex surface charge plays an important role in particle interactions with biological constituents including serum proteins and the plasma membrane. Ternary polyplexes comprising 25-kDa branched polyethylenimine (PEI), 15-kDa poly(α-glutamic acid) (PGA), and DNA were prepared by electrostatic complexation, and the surface charge was controlled by varying the ratio of polycation and polyanion. Cellular internalization of positively and negatively charged polyplexes was similar, but positively charged polyplexes exhibited superior gene delivery efficiency, suggesting differences in intracellular processing depending on surface charge. Thus, pharmacological inhibitors were used to evaluate the endocytic mechanisms involved in internalization and intracellular trafficking of PGA/PEI/DNA polyplexes of positive (+11 mV) and negative (−11 mV) zeta potential. Positively charged polyplexes were internalized primarily through caveolin-dependent endocytosis and avoided trafficking to lysosomes. Negatively charged polyplexes, however, were internalized to a greater extent by clathrin-dependent endocytosis and were found in acidified endolysosomal compartments, likely leading to degradation and poor gene delivery. Thus, transfection mediated by PGA/PEI/DNA polyplexes, may benefit from the addition of targeting ligands that would facilitate internalization through caveolin-dependent endocytosis while retaining advantages of negatively charged particles, including serum stability and low cytotoxicity.

Original languageEnglish
Article number104465
JournalJournal of Drug Delivery Science and Technology
StatePublished - Jun 2023

Bibliographical note

Funding Information:
Flow cytometry was performed on Attune Cytometer in the lab of Dr. David Feola, University of Kentucky, College of Pharmaceutical Sciences. Dynamic light scattering was performed in the lab of Dr. Eric Grulke, University of Kentucky, College of Engineering. Confocal fluorescence microscopy was performed at the Arts & Sciences Imaging Center, University of Kentucky. TEM was performed in the Electron Microscopy Center, University of Kentucky. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Publisher Copyright:
© 2023 Elsevier B.V.


  • Caveolin
  • Endocytosis
  • Macropinocytosis
  • Polymeric gene delivery
  • Ternary polyplex

ASJC Scopus subject areas

  • Pharmaceutical Science


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